CN116801910A - Means and methods for producing antibody-linker conjugates - Google Patents

Abstract

The present invention relates to a method for producing antibody-payload conjugates by microbial transglutaminase (MTG). The method includes placing a structure (shown in the N→C direction) (Sp ₁ )-RK-(Sp ₂ )-B-(Sp ₃ ) or (Sp ₁ )-B-(Sp ₂ )-RK- The step of coupling a linker of (Sp ₃ ) to a Gln residue contained in the antibody, wherein (Sp ₁ ) is a chemical spacer or is absent; (Sp ₂ ) is a chemical spacer or is absent; (Sp ₃ ) is Chemical spacer or absent; R is arginine or arginine derivative or arginine mimetic; K is lysine or lysine derivative or lysine mimetic; B is linker or payload ; and wherein the linker is coupled to the Gln residue contained in the antibody via a primary amine contained in the side chain of a lysine residue, a lysine derivative or a lysine mimetic. Further, the present invention relates to antibody-linker conjugates, antibody-drug conjugates and linker constructs comprising RK motifs.

Description

Means and methods for producing antibody-linker conjugates

The present invention relates to methods for the production of antibody-linker conjugates by microbial transglutaminase. The invention further provides antibody-linker conjugates, antibody-drug conjugates, linker constructs and pharmaceutical compositions comprising the antibody-linker conjugate or antibody-drug conjugate of the invention, and uses thereof.

Antibody-drug conjugates (ADCs) typically consist of an antibody and a small molecule drug coupled to the antibody via a chemical linker. After decades of preclinical and clinical studies, a series of ADCs have been approved to treat specific tumor types, such as Vibutux for relapsed Hodgkin lymphoma and systemic anaplastic large cell lymphoma. brentuximab vedotin, ), gemtuzumab ozogamicin for acute myeloid leukemia/> ), ado-trastuzumab emtansine for HER2-positive metastatic breast cancer/> ), inotuzumab ozogamicin for B-cell malignancies/> ) and most recently polatuzumab vedotin-piiq, ). Recently, enfortumab vedotin (/> ), trastuzumab deruxtecan,/> ), sacituzumab govitecan, ), and belantamab mafadotin,/> ) has been approved for listing. For a review of ADCs, see for example (Zhao P et al., 2020, Acta Pharmaceutica Sinica B, 10, 1589-1600). While many ADCs have shown impressive anticancer activity, many patients do not respond to these treatments, experience severe side effects before showing signs of efficacy or relapse after a certain period of time, so there is still a medical need for beneficial drug-like properties. Novel ADC forms that can be produced at reasonable cost in sufficient quantity and quality to support drug development and are suitable as therapeutic agents.

The key step in ADC preparation is the covalent coupling step of the payload to the antibody. Most ADCs currently in clinical development proceed by conjugation to endogenous lysine or cysteine residues of the antibody, with the average degree of modification carefully controlled to yield an average drug-to-antibody ratio (DAR) in the range of 3.5-4.0 . Historically, this ratio has been chosen based on (a) minimizing the amount of unconjugated antibody and (b) using very high DAR to avoid species in the mixture, which are critical in manufacturing and Can be problematic in formulation (Lambert JM and Berkenbilt A., 2018, Annu. Rev. Med. 69, 191-207) and often results in poor pharmacokinetic properties (Lyon RP et al., 2015, Nat Biotechnol, 33, 733-735). Recently, a variety of genetic, chemical, and enzymatic methods have been developed for site-specific conjugation, which can achieve a DAR of 2 (or 4) while avoiding under- or over-modification of the antibody. These methods are summarized by Yamada et al. (reviewed in Kei Yamada and Yuji Ito, 2019, ChemBioChem, 2729-2739).

Enzyme couplings have shown great interest because these coupling reactions are often rapid, site-specific, and can be performed under physiological conditions. Among the available enzymes, microbial transglutaminase (MTG) from the species Streptomyces mobaraensis is increasingly gaining interest as an attractive alternative to conventional chemical protein conjugation of functional moieties, including antibodies. s concern. MTG catalyzes the transamidation reaction under physiological conditions between the "reactive" glutamine of a protein or peptide and the "reactive" lysine residue of the protein or peptide, which can also be a simple low molecular weight primary amine. , such as 5-aminopentyl (Jeger S et al., 2010, Angew. Chem. Int. Ed., 49, 9995-9997).

Jeger et al. described conjugation of antibodies using transglutaminase as enzyme occurring at residue Q295, however, this was only possible when removing the glycan moiety at asparagine residue 297 (N297) with PNGase F coupling, while glycosylated antibodies cannot be coupled efficiently (coupling efficiency is less than 20%) (Jeger S et al., 2010, Angew. Chem. Int, Ed., 49, 9995-9997; Mindt T et al., 2008 Years, Bioconj Chem, 9, 271-278).

Other methods of generating ADC by MTG are based on the use of aglycosylated antibodies, in which residue N297 is replaced by an amino acid residue that cannot undergo glycosylation. However, substitution of N297 for another amino acid may lead to undesired effects as it may affect the overall stability of the entire Fc domain (Subedi GP and Barb AW., 2015, Structure, 23, 1573-1583) and the entire conjugation. The efficacy of things. As a result, increased antibody aggregation and reduced solubility may result, which becomes particularly important for hydrophobic payloads. Further, the glycan present at N297 has important immunomodulatory effects as it triggers effector functions such as antibody-dependent cellular cytotoxicity (ADCC), etc. These immunomodulatory effects will be lost during deglycosylation or any of the other methods mentioned above to obtain non-glycosylated antibodies. Furthermore, any sequence modifications to established antibodies may also lead to regulatory issues, which is problematic because generally accepted and clinically validated antibodies are used as the starting point for ADC coupling.

Recently, Spycher et al. disclosed a transglutaminase-based conjugation method that does not require prior deglycosylation of antibodies for payload conjugation (Spycher et al., WO 2019/057772). The ability to conjugate naturally glycosylated antibodies offers significant advantages in terms of production: in terms of Good Manufacturing Processes (GMP), an enzymatic deglycosylation step is undesirable because a deglycosylating enzyme (e.g., PNGase) must be ensured F) Both the cleaved glycans are removed from the reaction mixture. Furthermore, genetic engineering of the antibody for payload attachment is not required, thus avoiding sequence insertions that could increase immunogenicity and reduce the overall stability of the antibody.

In view of the above, there is still a need in the art for improved methods for generating ADCs with high coupling efficiencies.

Further, there is a need in the art for novel ADCs with improved efficacy and/or pharmacokinetic properties and highly defined drug-to-antibody ratios.

Contents of the invention

The invention is characterized by the description and claims provided herein. In particular, the invention relates inter alia to the following embodiments:

1. A method for producing an antibody-linker conjugate by microbial transglutaminase (MTG), the method comprising adding (as shown in the N→C direction) (Sp ₁ )-RK-(Sp ₂ ) The step of coupling a linker of the -B-(Sp ₃ ) or (Sp ₁ )-B-(Sp ₂ )-RK-(Sp ₃ ) structure to the Gln residue contained in the antibody, wherein,

·(Sp ₁ ) is a chemical spacer or does not exist;

·(Sp ₂ ) is a chemical spacer or does not exist;

·(Sp ₃ ) is a chemical spacer or does not exist;

·R is arginine or arginine derivative or arginine mimetic;

·K is lysine or lysine derivative or lysine mimetic;

·B is the connection part or payload;

And wherein the linker is coupled to the Gln residue contained in the antibody via a primary amine contained in the side chain of a lysine residue, a lysine derivative or a lysine mimetic.

2. The method according to embodiment 1, wherein the chemical spacers (Sp ₁ ), (Sp ₂ ) and (Sp ₃ ) each independently comprise 0 to 12 amino acid residues.

3. The method according to embodiment 1 or 2, wherein the linker contains no more than 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 amino acid residues.

4. The method according to any one of embodiments 1 to 3, wherein the net charge of the linker is neutral or positive.

5. The method according to any one of embodiments 1 to 4, wherein the linker does not comprise negatively charged amino acid residues.

6. The method according to any one of embodiments 1 to 5, wherein the linker comprises the amino acid sequence RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3) or RKR (SEQ ID NO: 3). ID NO:4).

7. The method according to any one of embodiments 1 to 6, wherein the linker comprises the amino acid sequence RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2) or ARK (SEQ ID NO: 3).

8. The method according to any one of embodiments 1 to 7, wherein the linker comprises the amino acid sequence RKAA (SEQ ID NO: 1).

9. The method according to any one of embodiments 1 to 8, wherein B is a connecting moiety.

10. The method of embodiment 9, wherein connection portion B comprises

·Bioorthogonal labeling groups, or

· Non-bioorthogonal entities for cross-linking.

11. The method according to embodiment 10, wherein the bioorthogonal labeling group or the non-bioorthogonal entity for cross-linking consists of or contains at least one molecule or moiety selected from the following Group consisting of:

·–NN≡N or –N ₃ ;

·Lys(N ₃ );

·Tetrazine;

·Alkynes;

·Strained cyclooctyne;

·BCN;

·Strained olefin;

·Photoreactive groups;

·aldehyde;

·Acyl trifluoroborate;

·Protein degradation agent ('PROTAC');

·Cyclopentadiene/spirolocyclopentadiene;

·Thio-selective electrophile;

·-SH; and

·Cysteine.

12. The method according to any one of embodiments 9 to 11, comprising the further step of coupling one or more payloads to connecting portion B.

13. The method of embodiment 12, wherein one or more payloads are coupled to linker B via a click reaction.

14. The method according to any one of embodiments 1 to 8, wherein B is a payload.

15. The method according to any one of embodiments 12 to 14, wherein the payload includes at least one of the following:

·toxin;

·Cytokines;

·Growth factors;

·Radionuclides;

·hormone;

·Antiviral agents;

·Antibacterial agents;

·Fluorescent dyes;

·Immune modulators/immunostimulants;

·Half-life increased part;

·Solubility increased part;

·Polymer-toxin conjugates;

·Nucleic acid;

·Biotin or streptavidin moiety;

·Vitamins;

·Protein degradation agent (‘PROTAC’);

·Target binding moiety; and/or

·Anti-inflammatory agent.

16. The method according to embodiment 15, wherein the toxin is at least one selected from the group consisting of:

·pyrrolobenzodiazepines (e.g., PBD);

·Auristatin (such as MMAE, MMAF);

·Maytansinoid (e.g., maytansinoid, DM1, DM4, DM21);

·Duocarmycin;

·Nicotinamide phosphoribosyltransferase (NAMPT) inhibitor;

·Tubulysin;

· Enedyne (e.g., calicheamicin);

·Anthracycline derivatives (PNU) (e.g., doxorubicin);

·Pyrrolyl kinesin spindle protein (KSP) inhibitor;

· Cryptophycin;

·Drug efflux pump inhibitors;

·Sandromycin;

·Amanitanic acid (e.g., alpha-amanitinine); and

· Camptothecins (eg, exatecans, deruxtecans).

17. The method according to any one of embodiments 14 to 16, wherein the chemical spacer ( _Sp2 ) comprises a self-immolative moiety.

18. The method of embodiment 17, wherein the self-cleaving moiety is directly attached to payload B.

19. The method according to embodiment 17 or 18, wherein the self-cleaving moiety comprises a p-aminobenzylcarbamoyl (PABC) moiety.

20. Method according to any one of embodiments 1 to 19, wherein the antibody is an IgG antibody, in particular an IgG1 antibody.

21. The method according to embodiment 20, wherein the linker-coupled Gln residue is comprised in the Fc domain of the antibody, in particular wherein the linker-coupled Gln residue is a Gln residue of the _CH2 domain of an IgG antibody. Base Q295 (EU number).

22. The method according to embodiment 20, wherein the linker-coupled Gln residue is introduced into the heavy or light chain of the antibody by molecular engineering.

23. The method according to embodiment 22, wherein the Gln residue introduced into the heavy or light chain of the antibody by molecular engineering is N297Q (EU numbering) of the _CH2 domain of aglycosylated IgG antibody.

24. The method of embodiment 22, wherein the Gln residue introduced into the heavy or light chain of the antibody by molecular engineering is comprised in a peptide that has been (a) integrated into the heavy or light chain of the antibody or ( b) Fusion to the N-terminus or C-terminus of the heavy or light chain of the antibody.

25. The method according to embodiment 24, wherein the peptide comprising a Gln residue has been fused to the C-terminus of the heavy chain of the antibody.

26. The method according to any one of embodiments 20 to 22 or 24 to 25, wherein the IgG antibody is a glycosylated IgG antibody, in particular wherein the IgG antibody is at residue N297 of the _CH 2 domain (EU numbering) Glycosylated.

27. The method according to any one of embodiments 1 to 26, wherein the antibody is selected from the group consisting of Brentuximab, Trastuzumab, Gemtuzumab ( Gemtuzumab), Inotuzumab, Avelumab, Cetuximab, Rituximab, Daratumumab, Pa Pertuzumab, Vedolizumab, Ocrelizumab, Tocilizumab, Ustekinumab, Golimumab ), Obinutuzumab, Sacituzumab, Belantuzumab, Polatuzumab, and Enfortumab.

28. The method according to any one of embodiments 1 to 27, wherein the antibody is selected from the group consisting of: velbutuximab, gemtuzumab, trastuzumab, orintuzumab , polotuzumab, ennozumab, sacerituzumab, and belantuzumab.

29. The method according to any one of embodiments 1 to 28, wherein the antibody is polotuzumab or trastuzumab or ennozumab.

30. The method according to any one of embodiments 1 to 29, wherein the linker is coupled to the γ-carboxamide group of a Gln residue comprised in the antibody.

31. The method according to any one of embodiments 1 to 30, wherein the linker is adapted to react with at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90 % or 95% coupling efficiency to glycosylated antibodies.

32. Method according to any one of embodiments 1 to 31, wherein the microbial transglutaminase is derived from a Streptomyces species, in particular Streptomyces mobaraensis.

33. An antibody-linker conjugate prepared by the method according to any one of embodiments 1 to 32.

34. An antibody-linker conjugate, comprising:

a) Antibodies; and

b) Joint, which includes the following structures:

(Sp ₁ )-RK-(Sp ₂ )-B-(Sp ₃ ) or

(Sp ₁ )-B-(Sp ₂ )-RK-(Sp ₃ ); where

·(Sp ₁ ) is a chemical spacer or does not exist;

·(Sp ₂ ) is a chemical spacer or does not exist;

·(Sp ₃ ) is a chemical spacer or does not exist;

·R is arginine or arginine derivative or arginine mimetic;

·K is lysine or lysine derivative or lysine mimetic;

·B is the connection part or payload;

Wherein, the linker is connected via the γ-carboxamide group of the glutamine residue contained in the antibody and the lysine residue, lysine derivative or lysine mimetic contained in the RK motif contained in the linker. Isopeptide bonds formed between primary amines contained in the side chains couple to the antibody.

35. The antibody-linker conjugate according to embodiment 34, wherein the chemical spacers (Sp ₁ ), (Sp ₂ ) and (Sp ₃ ) each independently comprise 0 to 12 amino acid residues.

36. The antibody-linker conjugate according to embodiment 34 or 35, wherein the linker comprises no more than 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 Amino acid residues.

37. The antibody-linker conjugate according to any one of embodiments 34 to 36, wherein the net charge of the linker is neutral or positive.

38. The antibody-linker conjugate according to any one of embodiments 34 to 37, wherein the linker does not comprise negatively charged amino acid residues.

39. The antibody-linker conjugate according to any one of embodiments 34 to 38, wherein the linker comprises an amino acid sequence selected from the group consisting of: RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2 ), ARK (SEQ ID NO:3), and RKR (SEQ ID NO:4).

40. The antibody-linker conjugate according to any one of embodiments 34 to 39, wherein the linker comprises an amino acid sequence selected from the group consisting of: RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2 ), and ARK (SEQ ID NO:3).

41. The antibody-linker conjugate according to any one of embodiments 34 to 40, wherein the linker comprises the amino acid sequence RKAA (SEQ ID NO: 1).

42. The antibody-linker conjugate according to any one of embodiments 34 to 41, wherein B is a linker moiety.

43. The antibody-linker conjugate according to embodiment 42, wherein linker B comprises

·Bioorthogonal labeling groups, or

· Non-bioorthogonal entities for cross-linking.

44. The antibody-linker conjugate according to embodiment 43, wherein the bioorthogonal labeling group or the non-bioorthogonal entity for cross-linking consists of or contains at least one molecule or moiety, the molecule or partially selected from the group consisting of:

·–NN≡N or –N ₃ ;

·Lys(N ₃ );

·Tetrazine;

·Alkynes;

·Strained cyclooctyne;

·BCN;

·Strained olefin;

·Photoreactive groups;

·aldehyde;

·Acyl trifluoroborate;

·Protein degradation agent ('PROTAC');

·Cyclopentadiene/spirocyclopentadiene;

·Thio-selective electrophile;

·-SH; and

·Cysteine.

45. The antibody-linker conjugate according to any one of embodiments 42 to 44, wherein one or more payloads are coupled to linker B.

46. The antibody-linker conjugate according to embodiment 45, wherein one or more payloads are coupled to linker B via a click reaction.

47. The antibody-linker conjugate according to any one of embodiments 34 to 41, wherein B is a payload.

48. The antibody-linker conjugate according to any one of embodiments 45 to 47, wherein the payload includes at least one of the following:

·toxin;

·Cytokines;

·Growth factors;

·Radionuclides;

·hormone;

·Antiviral agents;

·Antibacterial agents;

·Fluorescent dyes;

·Immune modulators/immunostimulants;

·Half-life increased part;

·Solubility increased part;

·Polymer-toxin conjugates;

·Nucleic acid;

·Biotin or streptavidin moiety;

·Vitamins;

·Protein degradation agent (‘PROTAC’);

·Target binding moiety; and/or

·Anti-inflammatory agent.

49. The antibody-linker conjugate according to embodiment 48, wherein the toxin is at least one selected from the group consisting of:

·pyrrolobenzodiazepines (e.g., PBD);

·Auristatin (such as MMAE, MMAF);

Maytansinoids (e.g., maytansinoids, DM1, DM4, DM21);

·Becarcinomycin;

·Nicotinamide phosphoribosyltransferase (NAMPT) inhibitor;

·Microtubulysin;

· Enedyne (e.g., calicheamicin);

·Anthracycline derivatives (PNU) (e.g., doxorubicin);

·Pyrrolyl kinesin spindle protein (KSP) inhibitor;

·Nodulin;

·Drug efflux pump inhibitors;

·Sandromycin;

·Amanitanic acid (e.g., alpha-amanitinine); and

· Camptothecins (e.g., ixotecan, drotecan).

50. The antibody-linker conjugate according to any one of embodiments 47 to 49, wherein the chemical spacer ( _Sp2 ) comprises a self-cleaving moiety.

51. The antibody-linker conjugate according to embodiment 50, wherein the self-cleaving moiety is directly attached to payload B.

52. The antibody-linker conjugate according to embodiment 50 or 51, wherein the self-cleaving moiety comprises a p-aminobenzylcarbamoyl (PABC) moiety.

53. Antibody-linker conjugate according to any one of embodiments 34 to 52, wherein the antibody is an IgG antibody, in particular an IgG1 antibody.

54. Antibody-linker conjugate according to embodiment 53, wherein the linker-coupled Gln residue is comprised in the Fc domain of the antibody, in particular wherein the linker-coupled Gln residue is _CH 2 of an IgG antibody Gln residue Q295 of the domain (EU numbering).

55. The antibody-linker conjugate according to embodiment 53, wherein the linker-coupled Gln residue is introduced into the heavy or light chain of the antibody by molecular engineering.

56. The antibody-linker conjugate according to embodiment 55, wherein the Gln residue introduced into the heavy chain or light chain of the antibody through molecular engineering is N297Q (EU numbering) of the _CH 2 domain of the non-glycosylated IgG antibody. .

57. The antibody-linker conjugate according to embodiment 55, wherein the Gln residue introduced into the heavy or light chain of the antibody by molecular engineering is comprised in a peptide that has been (a) integrated into the heavy or light chain of the antibody. chain or (b) fused to the N-terminus or C-terminus of the heavy or light chain of the antibody.

58. The antibody-linker conjugate according to embodiment 57, wherein a peptide comprising a Gln residue has been fused to the C-terminus of the heavy chain of the antibody.

59. The antibody-linker conjugate according to any one of embodiments 53 to 55 or 57 to 58, wherein the IgG antibody is a glycosylated IgG antibody, in particular wherein the IgG antibody is at a residue of the _CH 2 domain Glycosylation at N297 (EU numbering).

60. The antibody-linker conjugate according to any one of embodiments 34 to 59, wherein the antibody is selected from the group consisting of: velbutuximab, trastuzumab, gemtuzumab, Intuzumab, avelumab, cetuximab, rituximab, daratumumab, pertuzumab, vedolizumab, ocrelizumab, tocilizumab monoclonal antibody, ustekinumab, golimumab, otuzumab, sacerituzumab, belantuzumab, polotuzumab, and ennozumab.

61. The antibody-linker conjugate according to any one of embodiments 34 to 60, wherein the antibody is selected from the group consisting of: velbutuximab, gemtuzumab, trastuzumab, Intolizumab, polotuzumab, ennozumab, sacerituzumab, and belantuzumab.

62. The antibody-linker conjugate according to any one of embodiments 34 to 61, wherein the antibody is polotuzumab or trastuzumab or ennozumab.

63. An antibody-drug conjugate, comprising:

a) IgG antibodies; and

b) A linker comprising drug moiety B, wherein drug moiety B is covalently linked to an amino acid sequence selected from the group consisting of: RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 2) NO:3) or RKR (SEQ ID NO:4);

Wherein, the linker is via the γ-carboxamide group at glutamine residue Q295 (EU numbering) of the antibody's _CH2 domain and the primary amine contained in the side chain of the lysine residue contained in the linker. The isopeptide bond formed between them is coupled to the IgG antibody.

64. The antibody-drug conjugate according to embodiment 63, wherein drug moiety B is linked to the N-terminus or C-terminus of the amino acid sequence comprised in the linker via a self-cleaving moiety.

65. The antibody-drug conjugate of embodiment 64, wherein the self-cleaving moiety includes a p-aminobenzylcarbamoyl (PABC) moiety.

66. The antibody-drug conjugate according to any one of embodiments 63 to 65, wherein the IgG antibody is a glycosylated IgG antibody, in particular wherein the IgG antibody is at residue N297 of the _CH2 domain (EU numbering ) is glycosylated.

67. The antibody-drug conjugate according to any one of embodiments 63 to 66, wherein the IgG antibody is an IgG1 antibody.

68. The antibody-drug conjugate according to any one of embodiments 63 to 67, wherein the IgG antibody is polotuzumab or comprises a heavy chain as set forth in SEQ ID NO: 5 and as set forth in Antibodies to the light chain listed in SEQ ID NO:6.

69. The antibody-drug conjugate according to any one of embodiments 63 to 67, wherein the IgG antibody is trastuzumab or comprises a heavy chain as set forth in SEQ ID NO:7 and as set forth in SEQ ID NO:7 Antibodies to the light chains listed in ID NO:8.

70. The antibody-drug conjugate according to any one of embodiments 63 to 67, wherein the IgG antibody is ennozumab or comprises a heavy chain as set forth in SEQ ID NO: 9 and as set forth in SEQ ID NO: 9 Antibodies to the light chains listed in NO: 10 or 11.

71. The antibody-drug conjugate according to any one of embodiments 63 to 70, wherein the drug is a toxin selected from the group consisting of:

·pyrrolobenzodiazepines (e.g., PBD);

·Auristatin (such as MMAE, MMAF);

Maytansinoids (e.g., maytansinoids, DM1, DM4, DM21);

·Becarcinomycin;

·Nicotinamide phosphoribosyltransferase (NAMPT) inhibitor;

·Microtubulysin;

· Enedyne (e.g., calicheamicin);

·Anthracycline derivatives (PNU) (e.g., doxorubicin);

·Pyrrolyl kinesin spindle protein (KSP) inhibitor;

·Nodulin;

·Drug efflux pump inhibitors;

·Sandromycin;

·Amanitanic acid (e.g., alpha-amanitinine); and

· Camptothecins (e.g., ixotecan, drotecan).

72. The antibody-drug conjugate according to any one of embodiments 63 to 71, wherein the linker has the structure RKAA-PABC-B, especially wherein B is auristatin or maytansinoid, especially wherein, Auristatin is a MMAE, and among them, the maytansinoids are DM1 or maytansinoids.

73. The antibody-drug conjugate according to any one of embodiments 63 to 71, wherein the linker has the structure RKA-PABC-B, especially wherein B is auristatin or maytansinoid, especially wherein, Auristatin is a MMAE, and among them, the maytansinoids are DM1 or maytansinoids.

74. The antibody-drug conjugate according to any one of embodiments 63 to 71, wherein the linker has the structure ARK-PABC-B, especially wherein B is auristatin or maytansinoid, especially wherein, Auristatin is a MMAE, and among them, the maytansinoids are DM1 or maytansinoids.

75. The antibody-drug conjugate according to any one of embodiments 63 to 71, wherein the linker has the structure RKR-PABC-B, especially wherein B is auristatin or maytansinoid, especially wherein, Auristatin is a MMAE, and among them, the maytansinoids are DM1 or maytansinoids.

76. A linker construct comprising the following structure:

(Sp ₁ )-RK-(Sp ₂ )-B-(Sp ₃ ) or

(Sp ₁ )-B-(Sp ₂ )-RK-(Sp ₃ ); where

·(Sp ₁ ) is a chemical spacer or does not exist;

·(Sp ₂ ) is a chemical spacer or does not exist;

·(Sp ₃ ) is a chemical spacer or does not exist;

·R is arginine or arginine derivative or arginine mimetic;

·K is lysine or lysine derivative or lysine mimetic;

·B is the connection part or payload.

77. The linker construct according to embodiment 76, wherein the chemical spacers (Sp ₁ ), (Sp ₂ ) and (Sp ₃ ) each independently comprise 0 to 12 amino acid residues.

78. The linker construct according to embodiment 76 or 77, wherein the linker comprises no more than 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 amino acid residues .

79. The linker construct according to any one of embodiments 76 to 78, wherein the net charge of the linker is neutral or positive.

80. The linker construct according to any one of embodiments 76 to 79, wherein the linker does not comprise negatively charged amino acid residues.

81. The linker construct according to any one of embodiments 76 to 80, wherein the linker comprises the amino acid sequence RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3) or RKR (SEQ ID NO:4).

82. The linker construct according to any one of embodiments 76 to 81, wherein B is a connecting moiety.

83. The linker construct of embodiment 82, wherein linker B comprises

·Bioorthogonal labeling groups, or

· Non-bioorthogonal entities for cross-linking.

84. The linker construct of embodiment 83, wherein the bioorthogonal labeling group or non-bioorthogonal entity for cross-linking consists of or includes at least one molecule or moiety selected from Free group consisting of:

·–NN≡N or –N ₃ ;

·Lys(N ₃ );

·Tetrazine;

·Alkynes;

·Strained cyclooctyne;

·BCN;

·Strained olefin;

·Photoreactive groups;

·aldehyde;

·Acyl trifluoroborate;

·Protein degradation agent ('PROTAC');

·Cyclopentadiene/spirocyclopentadiene;

·Thio-selective electrophile;

·-SH; and

·Cysteine.

85. Linker construct according to any one of embodiments 76 to 84, wherein the linker construct consists of or comprises structure RKAA-B, in particular wherein B is Lys( _N3 ) or cysteine acid.

86. Linker construct according to any one of embodiments 76 to 84, wherein the linker construct consists of or comprises structure RKA-B, in particular wherein B is Lys( _N3 ) or cysteine acid.

87. Linker construct according to any one of embodiments 76 to 84, wherein the linker construct consists of or comprises structure ARK-B, in particular wherein B is Lys( _N3 ) or cysteine acid.

88. The linker construct according to any one of embodiments 76 to 84, wherein the linker construct consists of or comprises structure B-RKR, in particular wherein B is Lys( _N3 ) or cysteine acid.

89. The linker construct according to any one of embodiments 76 to 81, wherein B is a payload.

90. The linker construct of embodiment 89, wherein the payload includes at least one of:

·toxin;

·Cytokines;

·Growth factors;

·Radionuclides;

·hormone;

·Antiviral agents;

·Antibacterial agents;

·Fluorescent dyes;

·Immune modulators/immunostimulants;

·Half-life increased part;

·Solubility increased part;

·Polymer-toxin conjugates;

·Nucleic acid;

·Biotin or streptavidin moiety;

·Vitamins;

·Protein degradation agent (‘PROTAC’);

·Target binding moiety; and/or

·Anti-inflammatory agent.

91. The linker construct according to embodiment 90, wherein the toxin is at least one selected from the group consisting of:

·pyrrolobenzodiazepines (e.g., PBD);

·Auristatin (such as MMAE, MMAF);

Maytansinoids (e.g., maytansinoids, DM1, DM4, DM21);

·Becarcinomycin;

·Nicotinamide phosphoribosyltransferase (NAMPT) inhibitor;

·Microtubulysin;

· Enedyne (e.g., calicheamicin);

·Anthracycline derivatives (PNU) (e.g., doxorubicin);

·Pyrrolyl kinesin spindle protein (KSP) inhibitor;

·Nodulin;

·Drug efflux pump inhibitors;

·Sandromycin;

·Amanitanic acid (e.g., alpha-amanitinine); and

· Camptothecins (e.g., ixotecan, drotecan).

92. The linker construct according to any one of embodiments 89 to 91, wherein the chemical spacer ( _Sp2 ) comprises a self-cleaving moiety.

93. The linker construct of embodiment 92, wherein the self-cleaving moiety is directly attached to payload B.

94. The linker construct according to embodiment 92 or 93, wherein the self-cleaving moiety comprises a p-aminobenzylcarbamoyl (PABC) moiety.

95. The linker construct according to any one of embodiments 89 to 94, wherein the linker construct consists of or comprises the structure RKAA-PABC-B, in particular wherein B is auristatin or the like. Maytansinoids, especially wherein auristatin is MMAE, and wherein the maytansinoids are DM1 or maytansinoids.

96. The linker construct according to any one of embodiments 89 to 94, wherein the linker construct consists of or comprises the structure RKA-PABC-B, in particular wherein B is auristatin or the like. Maytansinoids, especially wherein auristatin is MMAE, and wherein the maytansinoids are DM1 or maytansinoids.

97. The linker construct according to any one of embodiments 89 to 94, wherein the linker construct consists of or comprises the structure ARK-PABC-B, in particular wherein B is auristatin or the like. Maytansinoids, especially wherein auristatin is MMAE, and wherein the maytansinoids are DM1 or maytansinoids.

98. The linker construct according to any one of embodiments 89 to 94, wherein the linker construct consists of or comprises the structure B-PABC-RKR, in particular wherein B is auristatin or the like. Maytansinoids, especially wherein auristatin is MMAE, and wherein the maytansinoids are DM1 or maytansinoids.

99. Use of the linker construct according to any one of embodiments 76 to 98 for the production of antibody-linker conjugates by microbial transglutaminase.

100. Use according to embodiment 99, wherein the antibody is an IgG antibody, in particular an IgGl antibody.

101. The method according to embodiment 65 or 66, wherein the antibody is polotuzumab or trastuzumab or ennozumab.

102. A pharmaceutical composition comprising:

a) An antibody-linker conjugate according to any one of embodiments 33 to 62, in particular wherein the antibody-linker conjugate comprises at least one payload;

or

b) an antibody-drug conjugate according to any one of embodiments 63 to 75; and

Pharmaceutical compositions contain at least one pharmaceutically acceptable ingredient.

103. The pharmaceutical composition according to embodiment 102, comprising at least one additional therapeutically active agent.

104. Antibody-linker conjugate according to any one of embodiments 33 to 62, in particular wherein the antibody-linker conjugate comprises at least one payload, an antibody-linker conjugate according to any one of embodiments 63 to 75- Drug conjugates, or pharmaceutical compositions according to embodiment 102 or 103, for use in therapy and/or diagnosis.

105. Antibody-linker conjugate according to any one of embodiments 33 to 62, in particular wherein the antibody-linker conjugate comprises at least one payload, an antibody according to any one of embodiments 63 to 75 -Drug conjugate, or pharmaceutical composition according to embodiment 102 or 103, for treatment

●Patients suffering from neoplastic diseases, neurological diseases, autoimmune diseases, inflammatory diseases or infectious diseases,

●Patients at risk of developing neoplastic, neurological, autoimmune, inflammatory or infectious diseases, and/or

●Patients diagnosed with neoplastic diseases, neurological diseases, autoimmune diseases, inflammatory diseases or infectious diseases.

106. The antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition for use according to embodiment 105, wherein the antibody-linker conjugate or antibody-drug conjugate comprised in the pharmaceutical composition Polotuzumab is included, and wherein the neoplastic disease is a B cell-related cancer.

107. Antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition for use according to embodiment 106, wherein the B cell associated cancer is non-Hodgkin's lymphoma, in particular wherein the B cell associated cancer It is diffuse large B-cell lymphoma.

108. The antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition for use according to embodiment 106 or 107, wherein the antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition Administered in combination with bendamustine and/or rituximab.

109. The antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition for use according to embodiment 105, wherein the antibody-linker conjugate or antibody-drug conjugate comprised in the pharmaceutical composition Trastuzumab is included, and wherein the neoplastic disease is a HER2-positive cancer, in particular HER2-positive breast cancer, gastric cancer, ovarian cancer or lung cancer.

110. The antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition for use according to embodiment 109, wherein the antibody-linker conjugate, antibody drug conjugate or pharmaceutical composition is in combination with Lappa Co-administration of lapatinib, capecitabine and/or taxanes.

111. The antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition for use according to embodiment 105, wherein the antibody-linker conjugate or antibody-drug conjugate comprised in the pharmaceutical composition Ennosumab or ennosumab variants are included, and wherein the neoplastic disease is nectin-4 positive cancer, in particular nectin-4 positive pancreatic cancer, lung cancer, bladder cancer or breast cancer.

112. The antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition for use according to embodiment 111, wherein the antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition is combined with Combination administration of cisplatin and/or pembrolizumab.

113. Antibody-linker conjugate according to any one of embodiments 33 to 62, in particular wherein the antibody-linker conjugate comprises at least one payload, antibody-drug according to any one of embodiments 63 to 75 Use of the conjugate or pharmaceutical composition according to embodiment 102 or 103 for the manufacture of a medicament for treatment

●Patients suffering from neoplastic diseases, neurological diseases, autoimmune diseases, inflammatory diseases or infectious diseases,

●Patients at risk of developing neoplastic, neurological, autoimmune, inflammatory or infectious diseases, and/or

●Patients diagnosed with neoplastic diseases, neurological diseases, autoimmune diseases, inflammatory diseases or infectious diseases.

114. A method of treating or preventing a neoplastic disease, the method comprising administering to a patient in need thereof an antibody-linker conjugate according to any one of embodiments 33 to 62, particularly wherein the antibody-linker conjugate comprises At least one payload, an antibody-drug conjugate according to any one of embodiments 63 to 75, or a pharmaceutical composition according to embodiments 102 or 103.

Thus, in one embodiment, the present invention relates to a method for producing an antibody-linker conjugate by microbial transglutaminase (MTG), the method comprising adding (as shown in the N→C direction) ( The linker of Sp ₁ )-RK-(Sp ₂ )-B-(Sp ₃ ) or (Sp ₁ )-B-(Sp ₂ )-RK-(Sp ₃ ) structure is coupled to the Gln residue contained in the antibody. steps, where,

·(Sp ₁ ) is a chemical spacer or does not exist;

·(Sp ₂ ) is a chemical spacer or does not exist;

·(Sp ₃ ) is a chemical spacer or does not exist;

·R is arginine or arginine derivative or arginine mimetic;

·K is lysine or lysine derivative or lysine mimetic;

·B is the connection part or payload;

And wherein the linker is coupled to the Gln residue contained in the antibody via a primary amine contained in the side chain of a lysine residue, a lysine derivative or a lysine mimetic.

That is, the present invention is based at least in part on the surprising discovery that linkers containing the peptide motif RK (arginyl-lysyl) can be efficiently coupled to glycosylated antibodies. In patent application WO 2019/057772, it was demonstrated that peptide-based linkers can be efficiently coupled to glutamine residues of glycosylated antibodies via lysine residues in the linker. However, it has now surprisingly been shown that the extended motif RK provides further improved coupling efficiency.

The inventors have shown that lysine-containing linkers without RK motifs result in coupling efficiencies of 27% to 77% when attached directly to drug molecules (see Table 4). As provided herein, linkers containing RK motifs were coupled to glycosylated antibodies with efficiencies of at least 82% and, in some cases, up to 100% (see Tables 3 and 5). Therefore, for MTG-based coupling to glycosylated antibodies, especially when the payload is directly coupled to the glycosylated antibody in a one-step reaction, linkers containing RK motifs are particularly preferred over other lysine-based linkers.

It is preferred in the present invention that the linker includes the structure (Sp ₁ )-RK-(Sp ₂ )-B-(Sp ₃ ) or (Sp ₁ )-B-(Sp ₂ )-RK-(Sp ₃ ), wherein , the linker couples to a glutamine residue in the antibody via a primary amine contained in residue K contained in the RK motif of the linker. In certain embodiments, residue K is a lysine residue. However, in certain embodiments, residue K may also be a lysine mimetic or lysine derivative, provided that the lysine mimetic or lysine derivative includes a primary amine in its amino acid side chain .

Thus, in certain embodiments, residue K may be a lysine mimetic. As used herein, the term "lysine mimetic" refers to a compound that has a structure different from lysine, but has similar characteristics to lysine, and therefore can be used without significantly altering the function of the peptide or protein. and/or structural context for replacing lysine in a peptide or protein. In certain embodiments, a lysine mimetic may differ from lysine in the length or composition of the aliphatic chain connecting the primary amine to the alpha-carbon atom. Thus, in certain embodiments, the lysine mimetic may be ornithine or 2,7-diaminoheptanoic acid. In certain embodiments, the lysine mimetic can be a beta-amino acid, such as beta-homolysine.

In certain embodiments, residue K can be a lysine derivative. As used herein, the term "lysine derivative" refers to lysine or a lysine mimetic in which one or more functional groups contained in the lysine or lysine mimetic are modified or substituted. In the present invention, it is preferred that the amino group in the side chain of the lysine derivative is unmodified so that it can be used for coupling with glutamine residues in the protein. In embodiments where residue K is located at the C-terminal position of the linker, K may be a lysine derivative in which the α-carboxyl group is modified or substituted. In certain embodiments, the alpha-carboxyl group of the lysine mimetic can be amidated.

The linker also includes residue R, which together with residue K forms the RK motif of the linker. In certain embodiments, residue R is an arginine residue. However, in certain embodiments, residue R may also be an arginine mimetic or arginine derivative.

Thus, in certain embodiments, residue R may be an arginine mimetic. As used herein, the term "arginine mimetic" refers to a compound that has a structure different from arginine but has similar characteristics to arginine and, therefore, can be used without significantly altering the function and function of the peptide or protein. / or structure used to replace arginine in peptides or proteins. Arginine mimetics may differ from arginine in the length or composition of the aliphatic chain connecting the guanidine group to the alpha-carbon atom. Alternatively or additionally, the arginine mimetic may differ from arginine in the guanidine group itself. That is, the arginine mimetic may contain functional groups with similar physicochemical properties as the guanidine group. In certain embodiments, the arginine mimetic can be homoarginine, 2-amino-3-guanidine-propionic acid, beta-ureidoalanine, or citrulline.

In certain embodiments, residue R can be an arginine derivative. As used herein, the term "arginine derivative" refers to arginine or an arginine mimetic in which one or more functional groups contained in the arginine or arginine mimetic are modified or substituted. Arginine derivatives may be arginine or arginine mimetics in which the guanidine group is substituted or modified. In certain embodiments, the arginine derivative can be omega-methylarginine. In embodiments in which residue R is located at the N-terminal position of the linker, R may be an arginine derivative in which the α-amino group is modified or substituted. In certain embodiments, the alpha-amino group of the arginine mimetic can be acetylated.

It will be appreciated that the RK motif preferably consists of the amino acids arginine and lysine. However, the arginine or lysine residue, or both, may be replaced by mimetics or derivatives as disclosed above. In certain embodiments, the RK motif can be composed of the amino acids arginine and ornithine. In certain embodiments, the RK motif can consist of the amino acids arginine and 2,7-diaminoheptanoic acid. In certain embodiments, the RK motif can consist of the amino acids homoarginine and lysine. In certain embodiments, the RK motif can consist of the amino acids 2-amino-3-guanidino-propionic acid and lysine. In certain embodiments, the RK motif can consist of the amino acids homoarginine and ornithine. In certain embodiments, the RK motif can consist of the amino acids homoarginine and 2,7-diaminoheptanoic acid. In certain embodiments, the RK motif can consist of the amino acids 2-amino-3-guanidino-propionic acid and ornithine. In certain embodiments, the RK motif can consist of the amino acids 2-amino-3-guanidino-propionic acid and 2,7-diaminoheptanoic acid.

In the present invention, the RK motif is embedded in the structure (Sp ₁ )-RK-(Sp ₂ )-B-(Sp ₃ ) or (Sp ₁ )-B-(Sp ₂ )-RK-(Sp ₃ ). That is, the linker may include one or more chemical spacers (Sp). The term "chemical spacer" as used herein describes a chemical residue covalently attached to a linker and/or a chemical moiety interposed between two chemical residues of a linker.

In a specific embodiment, the invention relates to a method according to the invention, wherein the chemical spacers (Sp ₁ ), (Sp ₂ ) and (Sp ₃ ) each independently comprise from 0 to 12 amino acid residues.

That is, in certain embodiments, chemical spacers (Sp ₁ ), (Sp ₂ ), and/or (Sp ₃ ) may or may not be present. In embodiments where (Sp ₁ ), (Sp ₂ ) and/or (Sp ₃ ) are present, (Sp ₁ ), (Sp ₂ ) and/or (Sp ₃ ) may comprise one or more amino acid residues. In such embodiments, each of (Sp ₁ ), (Sp ₂ ), and/or (Sp ₃ ) may comprise 0 to 12 amino acid residues. It must be noted that the chemical spacers (Sp ₁ ), (Sp ₂ ) and/or (Sp ₃ ) may also include non-amino acid residues, as will be disclosed in more detail below.

The "amino acid residues" contained in the chemical spacers (Sp ₁ ), (Sp ₂ ) and/or (Sp ₃ ) may be amino acids, amino acid mimetics or amino acid derivatives. It will be understood that the term amino acid includes not only alpha-amino acids, but also other amino acids, such as beta-amino acids, gamma-amino acids or delta-amino acids. The alpha-amino acid residues may be present in the chemical spacers (Sp ₁ ), (Sp ₂ ) and/or (Sp ₃ ) in their L-form or D-form. In embodiments where (Sp ₁ ), (Sp ₂ ) and/or (Sp ₃ ) comprise a chiral β-amino acid, a γ-amino acid or a δ-amino acid, the chiral β-amino acid, γ-amino acid or δ-amino acid may It exists in S-form or R-form. Thus, in the broadest sense, the term "amino acid residue" as used herein may refer to any organic compound containing an amino group ( _-NH2 ) and a carboxyl group (-COOH). Thus, whenever reference is made to an "amino acid" or "amino acid residue" in this disclosure, it will be understood that the term amino acid residue may also include amino acid mimetics or derivatives.

Further, it should be understood that the term amino acid residue is not limited to the known group of proteinogenic amino acids, namely alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, Glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine acids, but also covers non-classical and unnatural amino acids. As used herein, a "non-canonical amino acid" can be any amino acid that is not part of the protein's amino acid group, but can be obtained from natural sources. However, it must be noted that some non-canonical amino acids may also be found in naturally occurring peptides and/or proteins.

As used herein, a "non-natural amino acid" or "synthetic amino acid" may be any molecule that falls within the general definition of an amino acid (i.e., includes amino and carboxyl groups) but is not found in nature. Therefore, unnatural amino acids are preferably obtained by chemical synthesis. It is understood that in some cases the distinction between non-classical amino acids and unnatural amino acids may be uncertain. For example, an amino acid defined as an unnatural amino acid may be identified in nature at a later point in time and thus be reclassified as a non-canonical amino acid.

Examples of non-classical amino acids or unnatural amino acids may be, but are not limited to, D-amino acids (such as D-alanine, D-arginine, D-methionine), high-amino acids (such as homoserine, homoserine, etc.) amino acids, homocysteine, α-aminoadipic acid), N-methylated amino acids (such as sarcosine, N-Me-leucine), α-methyl amino acids (such as α-methyl -histidine, α-aminoisobutyric acid), β-amino acids (such as β-alanine, D-3-aminoisobutyric acid, L-β-homoalanine), γ-amino acids (such as , γ-aminobutyric acid), alanine mimetics or derivatives (such as β-cyclopropylalanine, phenylglycine, dehydroalanine, β-cyanoalanine, β-(3 -pyridyl)-alanine, β-(1,2,4-triazol-1-yl)-alanine, β-(1-piperazinyl)-alanine), phenylalanine analog or derivatives (such as, 4-iodophenylalanine, pentafluoro-phenylalanine, naphthyl-alanine, 4-aminophenylalanine), arginine mimetics or derivatives (such as, β-ureidoalanine, ω-methylarginine), lysine mimetics or derivatives (such as, (3-(3-methyl-3H-diazeridan-3-yl)propanine) Amino)carbonyl-l-lysine, Nε,Nε,Nε-trimethyllysine), histidine mimetics or derivatives (such as, 2,5-diiodohistidine, 1-methyl amino acids), tyrosine mimetics or derivatives (such as 3-aminotyrosine, thyronine, 3,5-dinitrotyrosine, 3-hydroxy-methyl-tyrosine, O -phospho-L-tyrosine), tryptophan mimetics or derivatives (such as, 5-hydroxy-tryptophan, 1-methyltryptophan), serine mimetics or derivatives (such as, β-( 2-Thienyl)-serine, β-(3,4-dihydroxyphenyl)-serine, O-phosphoserine), threonine mimetics or derivatives (such as isothreonine, O-phosphothreonine acid), proline mimetics or derivatives (such as hydroxyproline, 3,4-dehydro-proline, pyroglutamic acid, thiaproline, cis-octahydroindole-2 -carboxylic acid), leucine and isoleucine mimetics or derivatives (such as isoleucine, norleucine, 4,5-dehydroleucine, (4S)-4-hydroxy-L -Isoleucine), valine mimetics or derivatives (such as norvaline, γ-hydroxyvaline), citrulline mimetics or derivatives (such as thiocitrulline, homocitrulline acid), cysteine mimetics or derivatives (such as, penicillamine, selenocysteine, butyrine-sulfoxime), methionine mimetics or derivatives (such as, S- Methylmethionine, L-methionine sulfone, L-methionine sulfoxide, L-methionine sulfoxime, selenomethionine), aspartic acid mimetic or derivative (such as, DL-threo-β-hydroxyaspartic acid, L-aspartic acid β-methyl ester), glutamate mimetics or derivatives (such as, γ-methyleneglutamic acid, γ- Carboxyglutamic acid, γ-hydroxyglutamic acid, L-glutamic acid 5-methyl ester, L-2-aminopimelic acid), asparagine mimetics or derivatives (such as, L-threo-3-hydroxy Asparagine, N,N-dimethyl-L-asparagine, L-2-amino-2-carboxyethanesulfonamide, 5-diazo-4-oxo-L-norvaline), glutamine Aminoamide mimetics or derivatives (such as, 4-F-(2S,4R)-fluoroglutamine, γ-glutamylformamide, theanine, L-glutamic acid γ-monoxalate), Amino acids containing cyclic moieties (such as 4-aminopiperidine-4-carboxylic acid, azetidine-2-carboxylic acid, pipecolic acid, 1-aminocyclopentacarboxylic acid, spinacine) , or amino acids containing bioorthogonal moieties (such as, propargylglycine, α-allylglycine, L-azido-homoalanine, p-benzoyl-1-phenylalanine, p- 2-Fluoroacetyl-1-phenylalanine, (S)-2-amino-3-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl) Propionic acid).

In addition to the above-mentioned α-amino acids, the chemical spacers (Sp ₁ ), (Sp ₂ ) and/or (Sp ₃ ) may comprise one or more β-amino acids, γ-amino acids, δ-amino acids or ε-amino acids. Thus, in certain embodiments, the linker may be a peptide mimetic. Peptidomimetics may not exclusively comprise the classical peptide bond formed between two α-amino acids, but may additionally or alternatively comprise one or more amide bonds between the α-amino acid and the β-amino acid, the γ-amino acid, respectively. - between amino acids, δ-amino acids or ε-amino acids, or between two β-amino acids, γ-amino acids, δ-amino acids, δ-amino acids or ε-amino acids. Therefore, in any instance of the invention where the linker is described as a peptide, it will be understood that the linker may also be a peptidomimetic and thereby not exclusively consist of alpha-amino acids, but may comprise one or more beta-amino acids, Gamma-amino acids, delta-amino acids or epsilon-amino acids or molecules not classified as amino acids. Examples of β-amino acids, γ-amino acids, δ-amino acids or ε-amino acids that may be included in the linkers of the invention include, but are not limited to, β-alanine, γ-aminobutyric acid, 4-amino-3-hydroxy- 5-phenylpentanoic acid, 4-amino-3-hydroxy-6-methylheptanoic acid, 6-aminocaproic acid, and statins.

Furthermore, the chemical spacers (Sp ₁ ), (Sp ₂ ) and/or (Sp ₃ ) may include amino acid derivatives and/or amino acid mimetics. In embodiments where (Sp ₁ ), (Sp ₂ ) and/or (Sp ₃ ) comprise one or more amino acid derivatives, it is preferred that the amino acid derivatives have free amino and carboxyl groups such that they can form peptide or isopeptide bonds. . In embodiments where (Sp ₁ ), (Sp ₂ ) and/or (Sp ₃ ) comprise one or more amino acid derivatives, the amino acid mimetics may have free amino and carboxyl groups such that they may form peptides or isopeptides key. However, in certain embodiments, amino acid mimetics or derivatives may have substituted amino groups that do not prevent peptide bond formation. Examples of such amino acid mimetics or derivatives may be N-methylated amino acids, such as sarcosine or N-Me-leucine.

In embodiments where the amino acid residue comprised in (Sp ₁ ) or (Sp ₃ ) is a terminal amino acid residue, the terminal amino acid residue may comprise a modified, protected or substituted N-terminal amino or C-terminal carboxyl.

Further, an amino acid mimetic or derivative may be an amino acid containing a derivatized amino group, such as proline, or a mimetic of other cyclic amino acids such as azetidine-2-carboxylic acid, pipecolic acid, or stingine. products or derivatives. Furthermore, the amino acid mimetic may also include other functional groups that replace the amino and/or carboxyl groups of the standard amino acids, which allows the amino acid mimetic to form alternative bonds with adjacent amino acids, amino acid derivatives and/or amino acid mimetics, and form peptide mimetics. .

The term "amino acid mimetic" as used herein refers to a compound that has a structure that is different from a specific amino acid, but acts in a manner similar to the specific amino acid, and therefore can be used to replace the specific amino acid. An amino acid mimetic is said to function in a manner similar to a specific amino acid if it satisfies, at least to some extent, similar structural and/or functional characteristics to the amino acid it mimics. The term "amino acid derivative" refers to an amino acid as defined herein in which one or more functional groups contained in the amino acid are modified or substituted. The amino acid derivatives may preferably be derivatives of proteinogenic amino acids or non-classical amino acids. Any functional group of the amino acid derivative can be substituted or modified.

In embodiments where the linker contains one or more terminal amino acid residues, the terminal amino acid residues may be protected. For example, in embodiments where (Sp ₁ ) includes an N-terminal amino acid residue, the N-terminal amino group may be protected. For example, in certain embodiments, the N-terminal amino acid residue included in the spacer (Sp ₁ ) can be acetylated. In other embodiments, the R residue contained in the RK motif may be the N-terminal amino acid of the linker. In such embodiments, the N-terminal amino group of arginine, arginine mimetics or arginine derivatives may be protected, for example, by acetylation. In certain embodiments, linker B or payload B may be amino acid or amino acid based. In such embodiments, the N-terminal amino group of the amino acid-based payload or linker B may be protected, for example, by acetylation.

Likewise, in embodiments in which ( _Sp3 ) contains a C-terminal amino acid residue, the C-terminal carboxyl group may be protected. For example, in certain embodiments, the C-terminal amino acid residue in the spacer ( _Sp3 ) can be amidated. In other embodiments, the K residue contained in the RK motif may be the C-terminal amino acid of the linker. In such embodiments, the C-terminal carboxyl group of lysine, lysine mimetic or lysine derivative may be protected, for example, by amidation. In certain embodiments, linker B or payload B may be amino acid or amino acid based. In such embodiments, the C-terminal carboxyl group of the amino acid-based payload or linker B may be protected, for example, by amidation.

In certain embodiments, each of the chemical spacers (Sp ₁ ), (Sp ₂ ), and/or (Sp ₃ ) can comprise from 0 to 12 amino acid residues, including amino acid derivatives and amino acid mimetics. That is, in certain embodiments, (Sp ₁ ) may comprise 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues, (Sp ₂ ) may include 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues, and (Sp ₃ ) may include 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino residues.

In a particular embodiment, the invention relates to a method according to the invention, wherein the linkers comprise no more than 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 Amino acid residues.

That is, in certain embodiments, the linker may include 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acid residues, including amino acid mimetics and amino acid derivatives. It will be understood that when B is an amino acid-based linker or payload, the amino acid residues (including amino acid mimetics and amino acid derivatives) contained in the linker are preferably in the RK motif, chemical spacer (Sp ₁ ), Amino acid residues included in (Sp ₂ ) and/or (Sp ₃ ), and in certain embodiments also included in B. In embodiments where the linker contains only two amino acid residues, these two amino acid residues are contained in the RK motif. In such embodiments, (Sp ₁ ), (Sp ₂ ) and/or (Sp ₃ ) are absent or do not comprise any amino acid, amino acid mimetic or amino acid derivative.

In certain embodiments, the linker may comprise from 2 to 25 amino acid residues, including amino acid mimetics and amino acid derivatives. In other embodiments, the linker may comprise from 2 to 20 amino acid residues, including amino acid mimetics and amino acid derivatives. In other embodiments, the linker may comprise from 2 to 15 amino acid residues, including amino acid mimetics and amino acid derivatives. In other embodiments, the linker may comprise from 2 to 10 amino acid residues, including amino acid mimetics and amino acid derivatives. In other embodiments, the linker may comprise from 3 to 10 amino acid residues, including amino acid mimetics and amino acid derivatives. In other embodiments, the linker may contain 3 to 8 amino acid residues, including amino acid mimetics and amino acid derivatives. In other embodiments, the linker may contain 3 to 6 amino acid residues, including amino acid mimetics and amino acid derivatives.

In a specific embodiment, the invention relates to a method according to the invention, wherein the net charge of the linker is neutral or positive.

In certain embodiments, the linker is a peptide linker (or a peptide mimetic disclosed herein). That is, the chemical spacers (Sp ₁ ), (Sp ₂ ) and/or (Sp ₃ ), if present, consist exclusively of amino acids, amino acid mimetics or amino acid derivatives. The net charge of a peptide is usually calculated at neutral pH (7.0). In the simplest method, the net charge is calculated by adding the number of positively charged amino acid residues (Arg and Lys and optionally His) and the number of negatively charged amino acid residues (Asp and Glu). Determine the difference between these two groups. In cases where the linker includes a non-canonical amino acid or an amino acid derivative in which the charged functional groups are modified or substituted, those skilled in the art are aware of methods for determining the charge of the non-canonical amino acid or amino acid derivative at neutral pH.

In certain embodiments, the payload, linker B, or any non-amino acid moiety contained in (Sp ₁ ), (Sp ₂ ), and/or (Sp ₃ ) may also contribute to the net charge of the linker. However, those skilled in the art know that the preferred method is to calculate the net charge of the entire linker (including any non-amino acid moieties) at neutral pH (7.0).

In certain embodiments, the net charge of a linker is calculated based only on the amino acid residues contained in the linker (including amino acid mimetics and amino acid derivatives). Thus, in a specific embodiment, the invention relates to a method according to the invention, wherein the net charge of the amino acid residues comprised in the linker is neutral or positive.

In a specific embodiment, the invention relates to a method according to the invention, wherein the linker does not comprise negatively charged amino acid residues.

That is, the linker may be free of negatively charged amino acid residues (including amino acid mimetics and amino acid derivatives). Negatively charged amino acid residues are amino acids, amino acid mimetics or amino acid derivatives that carry a negative charge at neutral pH (7.0). Typical negatively charged amino acids are glutamic acid and aspartic acid. However, negatively charged non-classical amino acids, amino acid mimetics and amino acid derivatives are known in the art.

In a specific embodiment, the invention relates to a method according to the invention, wherein the linker comprises at least one positively charged amino acid residue outside the RK motif. That is, (Sp ₁ ), (Sp ₂ ) and/or (Sp ₃ ) comprise at least one positively charged amino acid. In certain embodiments, (Sp ₁ ), (Sp ₂ ) and/or (Sp ₃ ) comprise at least one histidine residue.

In addition to or instead of amino acid residues (including amino acid mimetics and derivatives), chemical spacers (Sp ₁ ), (Sp ₂ ) and/or (Sp ₃ ) may include or consist of non-amino acid moieties.

That is, in certain embodiments, chemical spacers (Sp ₁ ), (Sp ₂ ), and/or (Sp ₃ ) may not consist entirely of amino acids, amino acid mimetics, or amino acid derivatives. That is, the chemical spacers (Sp ₁ ), (Sp ₂ ) and/or (Sp ₃ ) may include non-amino acid components, or may consist solely of non-amino acid components. In certain embodiments, chemical spacers (Sp ₁ ), (Sp ₂ ), and/or (Sp ₃ ) can include amino acid and non-amino acid components.

For example, without limitation, each of the chemical spacers (Sp ₁ ), (Sp ₂ ), and/or (Sp ₃ ) may include a carbon-containing backbone of 1 to 200 atoms, optionally at one or more atoms A substituted carbon-containing backbone of at least 10 atoms (eg, 10 to 100 atoms or 20 to 100 atoms), optionally wherein the carbon-containing backbone is a linear hydrocarbon or contains cyclic groups, symmetric or asymmetric branches Hydrocarbons, monosaccharides, disaccharides, linear or branched (asymmetrically branched or symmetrically branched) oligosaccharides, other natural linear or branched (asymmetrically branched or symmetrically branched) oligomers, or more generally Any dimer, trimer or higher oligomer (linear, asymmetric branching or symmetric branching) resulting from any chain growth or step growth polymerization process.

(Sp ₁ ), (Sp ₂ ) and/or (Sp ₃ ) may be any linear, branched and/or cyclic C _2-30 alkyl, C _2-30 alkenyl, C _2-30 alkyne group, C _2-30 heteroalkyl group, C _2-30 -heteroalkenyl group, C _2-30 heteroalkynyl group, optionally, one or more homocyclic aromatic compound groups or heterocyclic compound groups can be inserted therein Group; especially any linear or branched C _2-5 alkyl, C _5-10 alkyl, C _11-20 alkyl, -OC _1-5 alkyl, -OC _5-10 alkyl, -OC _11-20 Alkyl, or (CH ₂ -CH ₂ -O-) _1-24 or (CH ₂ ) _x1 -(CH ₂ -O-CH ₂ ) _1-24 -(CH ₂ ) _x2 -yl (where, x1 and x2 are independently an integer selected from 0 to 20), amino acid, oligopeptide, glycan, sulfate, phosphate or carboxylate. In some embodiments, (Sp ₁ ), (Sp ₂ ), and/or (Sp ₃ ) may include C _2-6 alkyl.

In certain embodiments, chemical spacers (Sp ₁ ), (Sp ₂ ), and/or (Sp ₃ ) may include one or more polyethylene glycol (PEG) moieties or similar condensation polymers, such as, poly( carboxybetaine methacrylate) (pCBMA), polyoxazoline, polyglycerol, polyvinylpyrrolidone, or poly(hydroxyethyl methacrylate) (pHEMA). Polyethylene glycol (PEG) is a polyether compound with many applications ranging from industrial manufacturing to medicine. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE), depending on its molecular weight. The structure of PEG is usually represented as H-(O-CH ₂ -CH ₂ ) _n -OH. Those skilled in the art are aware of methods of functionalizing condensation polymers so that they can be coupled to amino acid residues or payloads.

The inventors have shown that linkers containing a PEG moiety can couple to glycosylated antibodies as efficiently as similar linkers without a PEG moiety. For example, the linkers ARK-PEG ₂ -PABC-MMAE (Figure 14) and ARK-PEG ₂ -(NH)-(CH ₃ )-S-C4-maytansine (Figure 15) were combined at 92% and 90% ( (compared to 94% of ARK-PABC-MMAE) coupled to glycosylated polotuzumab. In another example, the linkers ARK-PEG ₂ -PABC-MMAE (Figure 14) and ARK-PEG ₂ -(NH)-(CH ₃ )-S-C4-maytansine (Figure 15) were synthesized in 99% (with (compared to 100% efficiency of ARK-PABC-MMAE) conjugated to glycosylated trastuzumab.

Therefore, in a specific embodiment, the invention relates to a method according to the invention, wherein the linker comprises one or more PEG moieties. In certain embodiments, the PEG moiety may be included in chemical spacers (Sp ₁ ), (Sp ₂ ), and/or (Sp ₃ ). In certain embodiments, each PEG moiety included in the linker can comprise 2 to 20 ethylene glycol monomers, 2 to 15 ethylene glycol monomers, 2 to 10 ethylene glycol monomers, or 2 to 5 ethylene glycol monomers. In certain embodiments, a PEG moiety is included in ( _Sp2 ) to link the linker moiety or payload directly to the RK motif. In certain embodiments, a PEG moiety is included in (Sp ₂ ) to link the linker moiety or payload to the amino acid residue included in (Sp ₂ ). In certain embodiments, a PEG moiety is included in ( _Sp2 ) to link the RK motif to the self-cleaving moiety, which in turn is linked to the payload. In certain embodiments, a PEG moiety is included in (Sp ₂ ) to link the amino acid residue included in (Sp ₂ ) to a self-cleaving moiety, which in turn is linked to the payload.

In certain embodiments, chemical spacers (Sp ₁ ), (Sp ₂ ), and/or (Sp ₃ ) can include dextran. The term "glucan" as used herein refers to complex branched glucans consisting of chains of varying lengths, which may have a weight ranging from 3 kDa to 2000 kDa. Straight chains usually consist of α-1,6 glycosidic bonds between glucose molecules, while branched chains start from α-1,3 bonds. Glucan can be synthesized from sucrose, for example by lactic acid bacteria. In the context of the present invention, the dextran used as a carrier may preferably have a molecular weight of about 15 kDa to 1500 kDa.

In certain embodiments, chemical spacers (Sp ₁ ), (Sp ₂ ), and/or (Sp ₃ ) can include oligonucleotides. The term "oligonucleotide" as used herein refers to oligomers or polymers of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), as well as non-naturally occurring oligonucleotides. Owing to greater stability, oligonucleotides are preferably polymers of DNA.

In certain embodiments, chemical spacers (Sp ₁ ), (Sp ₂ ) and/or (Sp ₃ ), if present, consist solely of amino acid residues (including amino acid mimetics and derivatives) and PEG moieties. In certain embodiments, chemical spacers (Sp ₁ ), (Sp ₂ ) and/or (Sp ₃ ), if present, consist solely of amino acid residues (including amino acid mimetics and derivatives). In certain embodiments, all amino acid residues contained in the chemical spacers (Sp ₁ ), (Sp ₂ ), and/or (Sp ₃ ) are alpha-L-amino acids. That is, in certain embodiments, the linker, which does not include the payload or linker B, consists solely of amino acid residues. In certain embodiments, the linker, which does not include the payload or linker B, consists solely of alpha-L-amino acid residues. Such peptide-based linkers may contain protecting groups at the N-terminus and/or C-terminus. That is, the N-terminal amino group may be acetylated and/or the C-terminal carboxyl group may be amidated.

It must be noted that the chemical spacers (Sp ₁ ), (Sp ₂ ) and/or (Sp ₃ ) can have the same structure. However, it is preferred that each of the chemical spacers (Sp ₁ ), (Sp ₂ ) and/or (Sp ₃ ) has a different structure, and/or that not all chemical spacers (Sp ₁ ), (Sp ₂ ) and/or (Sp ₃ ) both exist simultaneously. That is, in certain embodiments, only one or two of the chemical spacers (Sp ₁ ), (Sp ₂ ), and/or (Sp ₃ ) may be present in the linker.

In certain embodiments, the RK motif can be directly linked to one or more small hydrophobic amino acid residues. For example, in certain embodiments, an RK motif can be directly linked to one or more alanine residues.

That is, in a specific embodiment, the invention relates to a method according to the invention, wherein the linker comprises the amino acid sequence RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO:4) or RK-Val-Cit (SEQ ID NO:54). It will be appreciated that the RK motif used to couple the linker to the glutamine residue of the antibody may be comprised in the amino acid sequence RKAA, RKA, ARK, RKR or RK-Val-Cit.

In a specific embodiment, the invention relates to a method according to the invention, wherein the linker comprises the amino acid sequence RKAA (SEQ ID NO:1), RKA (SEQ ID NO:2) or ARK (SEQ ID NO:3).

In a specific embodiment, the invention relates to a method according to the invention, wherein the linker comprises the amino acid sequence RKAA (SEQ ID NO: 1).

In a specific embodiment, the invention relates to a method according to the invention, wherein the linker comprises the amino acid sequence RK-Val-Cit (SEQ ID NO: 54).

In the present invention, it is preferred that the linker is coupled to the antibody via a primary amine in the side chain of residue K contained in the RK motif. Therefore, it is preferred that the chemical spacers (Sp ₁ ), (Sp ₂ ) and/or (Sp ₃ ) do not contain additional lysine residues, lysine mimetics or lysine derivatives, which can be Used as an additional amine donor in transglutaminase-based coupling reactions. In other embodiments, any free N-terminal amino group contained in the linker can be substituted (eg, acetylated) such that it is unable to serve as a substrate for microbial transglutaminase.

The joint of the invention also includes at least one connection part or payload B. Linkers of the invention can be used to couple payloads directly to antibodies in a one-step coupling process. In other embodiments, a linker comprising one or more linking moieties can be coupled to the antibody in a first step, and then one or more payloads can be linked to the antibody-linker conjugate in a second step. Table 1 below illustrates the two terms used in this article:

Table 1: One-step coupling and two-step coupling

In certain embodiments, a linker may include one or more connecting portions B. Thus, in a specific embodiment, the invention relates to a method according to the invention, wherein B is a connecting moiety.

As used herein, "linking moiety" generally refers to a molecule that is at least bifunctional. In the present invention, the linking moiety includes a first functional group that allows coupling of the linking moiety to the linker of the invention and a second functional group that can be used to couple additional molecules to the linker before or after coupling of the linker to the antibody. In certain embodiments, the linking moieties of the present invention are amino acids, amino acid mimetics, or amino acid derivatives. In such embodiments, the linking moiety is preferably attached to the linker via its amino group, while functional groups contained in the amino acid side chains can be used to couple additional molecules to the linker. Alternatively, the linking moiety can be attached to the linker via its carboxyl group, and functional groups contained in the amino acid side chains can be used to couple additional molecules to the linker.

In a particular embodiment, the invention relates to a method according to the invention, wherein connection part B comprises

·Bioorthogonal labeling groups, or

· Non-bioorthogonal entities for cross-linking.

The term "bioorthogonal labeling group" has been established by Sletten and Bertozzi (ABioorthogonal Quadricyclane Ligation, J Am Chem Soc 2011, 133(44), 17570-17573) to designate chemical reactions that can lead to Reactive groups that occur within living systems without interfering with natural biochemical processes. A "non-bioorthogonal entity for cross-linking" may be any molecule that includes or consists of a first functional group, wherein the first functional group can be chemically or enzymatically cross-linked to a payload that includes a compatible second functional group . Even where the cross-linking reaction is a non-bioorthogonal reaction, it is preferred that the reaction does not introduce additional modifications to the antibody beyond cross-linking of the payload to the linker. In view of the above, linking moiety B may consist of a "bioorthogonal labeling group" or a "non-bioorthogonal entity", or may include a "bioorthogonal labeling group" and a "non-bioorthogonal entity". For example, in the case of a linking moiety Lys( _N3 ), both the entire Lys( _N3 ) and the azide group may be considered a bioorthogonal labeling group in the present invention. Lys(N ₃ ) refers to 6-azido-L-lysine, which can also be abbreviated as K(N ₃ ).

In a particular embodiment, the invention relates to a method according to the invention, wherein the bioorthogonal labeling group or the non-bioorthogonal entity for cross-linking consists of or contains at least one molecule or moiety, The molecule or moiety is selected from the group consisting of:

·–NN≡N or –N ₃ ;

·Lys(N ₃ );

·Tetrazine;

·Alkynes;

·Strained cyclooctyne;

·BCN;

·Strained olefin;

·Photoreactive groups;

·aldehyde;

·Acyl trifluoroborate;

·Protein degradation agent ('PROTAC');

·Cyclopentadiene/spirocyclopentadiene;

·Thio-selective electrophile;

·-SH; and

·Cysteine.

Bioorthogonal labeling groups or non-bioorthogonal entities included in the linker for cross-linking can, for example, participate in any of the binding reactions shown in Table 2:

Table 2

Linking moiety B may be or comprise what is referred to in Table 2 as "binding partner 1" or "binding partner 2".

In certain embodiments, linking moiety B can be cysteine, a cysteine mimetic, or a cysteine derivative with a free sulfhydryl group.

The free sulfhydryl groups of such Cys residues (or mimetics or derivatives) can be coupled to payload constructs containing thio-selective electrophiles such as maleimides. Toxin constructs containing maleimide moieties have been frequently used and are also approved by medical authorities, such as Adcetris. Thus, toxin constructs including MMAE toxins can be coupled to the free sulfhydryl groups of Cys residues in the linkers of the invention.

It must be noted that other thio-selective electrophiles such as 3-arylpropionitrile (APN) or phosphonamidates can also be used instead of maleimide in the method of the invention.

Providing Cys residues in linkers according to the invention therefore has the advantage of allowing the use of readily available toxin-maleimide constructs for the generation of antibody-payload conjugates or, more generally, the full utilization of Cys-Maleimide constructs. Leimide combines the advantages of chemistry. Also, off-the-shelf antibodies can be used, which do not have to be deglycosylated. In specific embodiments, the Cys residue may be C-terminal or intrachain in an amino acid-based linker.

In another embodiment, linking moiety B may comprise an azide group. The person skilled in the art is aware of molecules containing an azide group that can be incorporated into the linker according to the invention, such as 6-azido-lysine (Lys(N ₃ )) or 4-azido-homoalanine (Xaa(N ₃ )). Linkers containing azide groups can be used as substrates in different bioorthogonal reactions, such as strain-promoted azide-alkyne cycloaddition (SPAAC), copper-catalyzed azide-alkyne cycloaddition ( CuAAC) or Staudinger connection. For example, in certain embodiments, a payload including a cyclooctyne derivative (such as DBCO, DIBO, BCN, or BARAC) can be coupled via SPAAC to a linker including an azide group.

In another embodiment, linking moiety B may include a tetrazine group. The person skilled in the art is aware of tetrazine-containing molecules, preferably amino acid derivatives containing tetrazine groups, which can be incorporated into the linkers according to the invention. Tetrazine-containing linker moieties can be used as substrates in bioorthogonal tetrazine ligations. For example, in certain embodiments, a payload comprising cyclopropene, norbornene, a norbornene derivative, or a cyclooctyne group such as bicyclo[6.1.0]nonyne (BCN) can be coupled to Linkers containing tetrazine groups.

In certain embodiments, linking moiety B may include a cyclic diene, such as a cyclopentadiene derivative. Amant et al., Tuning the Diels-Alder Reaction for Bioconjugation to Maleimide Drug-Linkers 2018, 29, 7, 22406-2414; and Amant et al. , A Reactive Antibody Platform for One-Step Production of Antibody-Drug Conjugates through a Diels-Alder Reaction with Maleimide (a reactive antibody platform for one-step production of antibody-drug conjugates through a Diels-Alder reaction with maleimide), Bioconjugate Chem; 2019, 30, 9, 2340-2348 Potential cyclopentadiene derivatives that can be linked to maleimide-containing payload molecules have been described.

In certain embodiments, linking moiety B may include a photoreactive group. As used herein, the term "photoreactive group" refers to a chemical group that responds to the application of an external energy source to produce a reactive species that covalently bonds to an adjacent chemical structure (e.g., extractable hydrogen) . Examples of photoreactive groups are, but are not limited to, aryl azides such as phenyl azide, o-hydroxyphenyl azide, m-hydroxyphenyl azide, tetrafluorophenyl azide, o-nitrophenyl azide, m-nitrophenyl azide, or azido-methylcoumarin, bisaziridine, psoralen or benzophenone.

In a particular embodiment, the invention relates to a method according to the invention, comprising the further step of coupling one or more payloads to the connection part B.

Instead of coupling a linker containing one or more payloads directly to the antibody in a one-step process, in certain embodiments the present invention involves a two-step process, wherein a linker containing at least one linker moiety B is coupled in a first step The antibody is coupled, and one or more payloads can then be coupled to linker B in a second step.

The term "payload" as used herein means any naturally occurring or synthetically generated molecule, including small molecular weight molecules or chemically synthesized chemical entities as well as large molecules that require production by host cell fermentation or that can also be chemically synthesized and confer new functions to the antibody or biological entity. It will be appreciated that the payload may include further structures or functional groups that allow the payload to interface with the connecting moiety contained in the linker or other portions of the linker such as chemical spacers (Sp ₁ ) and/or (Sp ₃ ) or RK motif) coupling.

In the two-step conjugation method, the payload can be linked to the linking moiety by any suitable method known in the art. Preferably, the payload may be linked to any of the bioorthogonal labeling groups disclosed herein or to non-bioorthogonal entities for cross-linking. That is, the payload preferably includes functional groups compatible with the bioorthogonal labeling group contained in at least one linker B or the non-bioorthogonal entity used for cross-linking.

Several bioorthogonal reactions that can be used to link payloads to bioorthogonal labeling groups included in linking moiety B are known in the art. For example, a number of chemical ligation strategies have been developed that meet the requirement of bioorthogonality, including between azides and cyclooctyne (also known as copper-free click chemistry, Baskin et al. (“Copper-free click chemistry for dynamic in vivo imaging”)). "Copper-free click chemistry for dynamic in vivo imaging", Proceedings of the National Academy of Sciences.104(43):16793-7)), between nitrone and cyclooctyne (Ning et al. (" Protein Modification by Strain-Promoted Alkyne-Nitrone Cycloaddition", Angewandte Chemie International Edition. 49 (17): 3065)) 1,3-dipolar cycloaddition; aldehyde and ketones to form oximes/hydrazones (Yarema et al. ("Metabolic Delivery of Ketone Groups to Sialic Acid Residues. Application To Cell Surface Glycoform Engineering)", Biochemistry Journal, 273(47):31168-79); Tetrazine Ligation (Blackman et al. ("The Tetrazine Ligation: Fast Bioconjugation based on Inverse-Required Diels-Alder reactivity") electron-demand Diels-Alder Reactivity)", Journal of the American Chemical, 130(41): 13518-9)); isonitrile-based click reaction ( ("Exploring isonitrile-based click chemistry for ligation with biomolecules", Organic & Biomolecular Chemistry, 9(21):7303)); and more recently, tetracycloalkane ligation (Sletten and Bertozzi (JACS, "A Bioorthogonal Quadricyclane Ligation", J Am Chem Soc, 2011, 133(44), 17570-17573)); Copper(I)-catalyzed azide-alkynes Cycloaddition (CuAAC, Kolb & Sharpless ("The growing impact of click chemistry on drug discovery", Drug Discov Today.8(24):1128-1137)), strain-promoted stacking Nitride-alkyne cycloaddition (SPAAC, Agard et al. ("A Comparative Study of Bioorthogonal Reactions with Azides", ACS Chem. Biol. 1:644-648) ), or strain-promoted alkyne-nitroketone cycloaddition (SPANC), MacKenzie et al. cycloadditions involving nitrones andalkynes-rapid tunable reactions for bioorthogonal labeling)", Curr Opin ChemBiol. 21:81-8)). All such documents are incorporated herein by reference in order to provide full public disclosure and avoid lengthy repetition.

It should be understood that after the linker has been coupled to the Gln residue of the antibody by microbial transglutaminase, the payload is preferably a bioorthogonal labeling group contained in the linker of the invention or a non-bioorthogonal labeling group for cross-linking. Entity coupling. However, the invention also encompasses antibody-linker conjugates, wherein in a first step one or more payloads are coupled to a linker comprising at least one linking moiety B, and wherein in a second step, by microbial transduction Glutaminase couples the resulting linker-payload construct to the antibody.

In a particular embodiment, the invention relates to a method according to the invention, wherein one or more payloads are coupled to linking moiety B via a click reaction.

That is, one or more payloads may be connected to connection portion B in a click reaction, particularly any click reaction disclosed herein.

In a particularly preferred embodiment, at least one payload can be coupled to the linker B comprised in the linker via a thiol-maleimide coupling. That is, in certain embodiments, the payload may include a maleimide group, and linker B may be a molecule including a thiol group, such as, but not limited to, a cysteine residue or a cysteine residue. Amino acid mimetics, such as homocysteine. However, B can also be a non-amino acid molecule containing a free sulfhydryl group. In another embodiment, the payload may include free thiol groups and linker B may include maleimide groups.

In another particularly preferred embodiment, at least one payload can be coupled via strain-promoted azide-alkyne cycloaddition (SPAAC) to the linking moiety B comprised in the linker. That is, in certain embodiments, the payload may comprise an alkynyl group (such as, but not limited to, a cyclooctynyl group), and the linker B may be a molecule containing an azide group (such as, but not limited to, in The lysine derivative Lys( _N3 ) disclosed herein. However, B can also be a non-amino acid molecule containing a free azide group. In another embodiment, the payload can include an alkynyl group (such as, cyclooctyl alkynyl group), and linking moiety B may include an azide group.

In addition to a click reaction between the linker moiety in the linker and the functional group in the payload, the payload can be covalently bound to the linker moiety by any enzymatic or non-enzymatic reaction known in the art.

Preferably the payload is attached to the connecting moiety via a covalent bond. However, in certain embodiments, the payload may be linked to the linking moiety via a strong non-covalent bond. That is, in certain embodiments, linking moiety B may include a biotin moiety such as, but not limited to, the lysine derivative biocytin. In such embodiments, a payload comprising a streptavidin moiety can be linked to a linker comprising a biotin moiety.

In a specific embodiment, the invention relates to a method according to the invention, wherein B is the payload.

In certain embodiments, the payload may already be part of the linker, allowing the payload to be coupled to the antibody in a one-step process. In such embodiments, the linker is preferably coupled to the linker by chemical synthesis. The payload is preferably coupled to a chemical spacer contained in the linker or directly to the RK motif. In embodiments where the payload is coupled to an amino acid residue (including amino acid mimetics and derivatives), the payload can be coupled to the C-terminal carboxyl group or the N-terminal amino group of the amino acid residue. Alternatively, the payload can be coupled to functional groups contained in the side chains of the amino acid residues. Those skilled in the art are aware of methods of functionalizing payloads so that they can be coupled to carboxyl, amino or amino acid side chains.

Further, those skilled in the art are aware of methods of coupling payloads to amino acid-based linkers by chemical synthesis. For example, an amine-containing payload, or a thiol-containing payload (e.g., a maytansinoid analog), or a hydroxyl-containing payload (e.g., an SN-38 analog) can be attached to an amino acid-based The C-terminus of the adapter. However, those skilled in the art are aware of further reactions and reactive groups that can be used to couple payloads to the N-terminus, C-terminus or side chains of amino acids or amino acid derivatives by chemical synthesis. Typical reactions that can be used to couple payloads to amino acid-based linkers via chemical synthesis include, but are not limited to: peptide coupling, activated ester coupling (NHS ester, PFP ester), click reaction (CuAAC, SPAAC), Michael Addition (thiol-maleimide coupling). Conjugation of payloads to peptides has been extensively described in the prior art, for example by Costoplus et al. (Designed to provide improved Bystander killing peptide-cleavable self-cleaving maytansinoid antibody-drug conjugates) Peptide-Cleavable Self-immolative Maytansinoid Antibody-Drug Conjugates Designed To Provide ImprovedBystander Killing), ACS Med Chem Lett. 2019Sep 27;10(10):1393-1399); Sonzini et al. (Using host-guest chemistry to improve antibody-drug binding) Improved PhysicalStability of an Antibody-Drug Conjugate Using Host-Guest Chemistry, BioconjugChem.2020Jan 15;31(1):123-129); Bodero et al. (RGD and heterogeneous drugs for tumor targeting Synthesis and biological evaluation of RGDand isoDGR peptidomimetic-α-amanitin conjugates for tumor-targeting, Beilstein J.Org.Chem.2018,14,407 -415); Nunes et al (Use of a next generation maleimide in combination with THIOMAB ^™ antibody technology provides highly stable, potent and nearly homogeneous THIOMAB ^™ antibody-drug conjugates (TDC)) (Use of a next generation maleimide in combination with THIOMAB ^TM antibody technology delivers a highly stable, potent and near homogeneous ^{THIOMAB TM} antibody-drug conjugate (TDC), RSC Adv., 2017, 7, 24828-24832); Doronina et al. Enhanced activity of monomethylauristatin F through monoclonal antibody delivery: effects of linker technology on efficacy and toxicity, BioconjugChem.2006, Jan-Feb; 17(1) :114-24); Nakada et al. (Novel antibody drug conjugates containing exatecanderivative-based cytotoxic payloads), Bioorg Med Chem Lett. 2016Mar 15; 26(6):1542-1545); and Dickgiesser et al. (Site-Specific Conjugation of Native Antibodies Using EngineeredMicrobial Transglutaminases), Bioconjug Chem. 2020 , Mar 12, doi:10.1021/acs.bioconjchem.0c00061).

It will be appreciated that the payload may be coupled to the N-terminus or C-terminus of the peptide-based or peptide-containing linker according to the invention. In certain embodiments, the payload can be coupled directly to the N-terminal amino or C-terminal carboxyl group of the peptide or amino acid residue (see, eg, Figure 22).

Those skilled in the art are aware of reactive groups suitable for coupling payloads to amino acid residues. For example, an amine-containing payload can be coupled to the C-terminal carboxyl group of an amino acid residue via an amide bond (Figure 22). Alternatively, payloads containing thiol or hydroxyl groups can be coupled to the C-terminal carboxyl group of the amino acid via a thioester or ester bond, respectively. Payloads containing carboxylic acid groups can be coupled to the N-terminal amino group of the amino acid residue via an amide bond.

In certain embodiments, the payload may be indirectly coupled to the N-terminus or C-terminus of the peptide or amino acid residue contained in the linker according to the invention. The person skilled in the art is aware of linker molecules that can be used to couple the payload to the N-terminal amino group or the C-terminal carboxyl group of the amino acid residues contained in the linker according to the invention.

In certain embodiments, a hydroxyl-containing payload can be coupled to the N-terminus of an amino acid residue via a linker molecule. For example, a hydroxyl-containing payload can be coupled to the N-terminal amino group via a urethane linker molecule (Figure 24).

In certain embodiments, a thiol group-containing payload can be coupled to the N-terminus of an amino acid residue via a linker molecule. For example, payloads containing thiol groups can be coupled to the N-terminal amino group via a thiocarbamate linker molecule (Figure 28). Alternatively, a payload containing thiol groups can be coupled to the N-terminal amino group via an alkyl linker molecule containing carboxyl and thiol groups. In certain embodiments, the alkyl linker molecule can be a 3-mercaptopropionic acid linker molecule, wherein the payload forms a disulfide bond with a thiol group contained in the 3-mercaptopropionic acid linker molecule (Figure 29).

In certain embodiments, a payload containing an amide group can be coupled to the N-terminus of an amino acid residue via a linker molecule. For example, a payload containing an amine group can be coupled to an N-terminal amino group via a dicarboxylic acid linker molecule, wherein the dicarboxylic acid linker forms an amide bond with the payload and the amino group of the N-terminal amino acid residue. Examples of dicarboxylic acids that can be used as linker molecules in the present invention are, but are not limited to, succinic acid or pimelic acid (see Figures 9 and 30).

Alternative linker molecules have been described in the art for indirect coupling of the payload to the N-terminus of the amino acid residues contained in the linker according to the invention, or are suitable for coupling the payload directly to the linker according to the invention. The linker contains amino acid residues at the C-terminus of the linker molecule and is encompassed by the present invention.

In a particular embodiment, the invention relates to a method according to the invention, wherein the payload comprises at least one of:

·toxin;

·Cytokines;

·Growth factors;

·Radionuclides;

·hormone;

·Antiviral agents;

·Antibacterial agents;

·Fluorescent dyes;

·Immune modulators/immunostimulants;

·Half-life increased part;

·Solubility increased part;

·Polymer-toxin conjugates;

·Nucleic acid;

·Biotin or streptavidin moiety;

·Vitamins;

·Protein degradation agent (‘PROTAC’);

·Target binding moiety; and/or

·Anti-inflammatory agent.

Any of the payloads disclosed herein can be coupled directly to a linker used in the one-step conjugation process disclosed herein, or can be linked to a linker moiety included in an antibody-linker conjugate that Objects are produced as part of the two-step process disclosed herein.

In certain embodiments, the payload may be a cytokine. The term "cytokine" as used herein refers to any secreted polypeptide that affects other cellular functions and modulates interactions between cells in immune or inflammatory responses. Cytokines include, but are not limited to, monokines, lymphokines, and chemokines, regardless of which cell produces them. For example, monokine is often said to be produced and secreted by monocytes, however, monokine is produced by many other cells (such as natural killer cells, fibroblasts, basophils, neutrophils, endothelial cells , brain astrocytes, bone marrow stromal cells, epidermal keratinocytes and B lymphocytes). Lymphokines are generally thought to be produced by lymphocytes. Examples of cytokines include, but are not limited to, interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor alpha (TNFα), and tumor necrosis factor beta (TNFβ).

In certain embodiments, the payload may be an anti-inflammatory agent. As used herein, the term "anti-inflammatory agent" refers to those classes of agents whose primary mode of action and use is in the field of treatment of inflammation, as well as any other agent from another therapeutic class that has useful anti-inflammatory effects. Such anti-inflammatory drugs include, but are not limited to, nonsteroidal anti-inflammatory drugs (NSAIDs), disease-modifying antirheumatic drugs (DMARDs), macrolide antibiotics, and statins. Preferably, NSAIDs include, but are not limited to, salicylates (e.g., aspirin), arylpropionic acids (e.g., ibuprofen), anthranilic acids (e.g., mefenamicacid), pyrazoles (eg, phenylbutazone), cycloacetate (indomethicin), and oxicam (eg, piroxicam). Preferably, anti-inflammatory agents used in the methods of the invention include sulindac, diclofenac, tenoxicam, ketorolac, naproxen, nabumetone, diflunasal, ketone Profen, arlypropionic acid, tenidap, hydroxychloroquine, sulfasalazine, celecoxib, rofecoxib, meloxicam ), etoricoxib, valdecoxib, methotrexate, etanercept, infliximab, adalimumab, atorvastatin, fluvastatin, lovastatin, Vastatin, simvastatin, clarithromycin, azithromycin, roxithromycin, erythromycin, ibuprofen, dexibuprofen (dexibuprofen), flurbiprofen, fenoprofen, fenbufen, benoprofen benoxaprofen, dexketoprofen, tolfenamic acid, nimesulide and oxaprozin.

In certain embodiments, the anti-inflammatory agent can be an anti-inflammatory cytokine, which when combined with a target-specific antibody can ameliorate inflammation caused, for example, by an autoimmune disease. Cytokines with anti-inflammatory activity may be, but are not limited to, IL-1RA, IL-4, IL-6, IL-10, IL-11, IL-13 or TGF-β.

In certain embodiments, the payload may be a growth factor. The term "growth factor" as used herein refers to naturally occurring substances capable of stimulating cell growth, proliferation, cell differentiation and/or cell maturation. Growth factors exist in the form of proteins or steroid hormones. Growth factors are important in regulating various cellular processes. Growth factors often act as signaling molecules between cells. However, their ability to promote cell growth, proliferation, cell differentiation, and cell maturation varies among growth factors. A non-limiting list of examples of growth factors includes: basic fibroblast growth factor, adrenomedullary protein, angiopoietin, autocrine motility factor, bone morphogenetic protein, brain-derived neurotrophic factor, epidermal growth factor, epithelial growth factor Growth factors, fibroblast growth factor, glial cell line-derived neurotrophic factor, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, growth differentiation factor-9, hepatocyte growth factor, hepatoma Derived growth factors, insulin growth factor, insulin-like growth factor, migration-stimulating factor, myostatin, nerve growth factor and other neurotrophic factors, platelet-derived growth factor, transforming growth factor alpha, transforming growth factor beta, tumor necrosis factor - α, vascular endothelial growth factor, placental growth factor, fetal bovine growth factor, and cytokines (e.g., IL-1-cofactor of IL-3 and IL-6, IL-2-t-cell growth factor, IL-3, IL-4, IL-5, IL-6 and IL-7).

In certain embodiments, the payload may be a hormone. The term "hormone" as used in this article refers to chemicals released by cells or glands in one part of the body that send messages that affect cells in other parts of the organism. Examples of hormones useful in the present invention are, but are not limited to, melatonin (MT), serotonin (5-HT), thyroxine (T4), triiodothyronine (T3), epinephrine, or Adrenaline (EPI), norepinephrine or noradrenaline (NRE), dopamine (DPM or DA), antimullerian hormone or mullerian inhibitory hormone (AMH), adiponectin (Acrp30), adrenocorticotropic hormone or corticotropin (ACTH), angiotensinogen and angiotensin (AGT), antidiuretic hormone or vasopressin (ADH), atrial natriuretic Peptides or atrial propeptide (ANP), calcitonin (CT), cholecystokinin (CCK), corticotropin-releasing hormone (CRH), erythropoietin (EPO), follicle-stimulating hormone (FSH), gastrointestinal GRP, ghrelin, glucagon (GCG), gonadotropin-releasing hormone (GnRH), growth hormone-releasing hormone (GHRH), human chorionic gonadotropin (hCG), human placenta Prolactin (HPL), growth hormone (GH or hGH), inhibin, insulin (INS), insulin-like growth factor or growth hormone (IGF), leptin (LEP), luteinizing hormone (LH), melanocyte-stimulating hormone Hormones (MSH or α-MSH), orexin (orexin), oxytocin (OXT), parathyroid hormone (PTH), prolactin (PRL), relaxin (RLN), secretin (SCT), somatostatin (SRIF), thrombopoietin (TPO), thyroid-stimulating hormone or thyroid hormone (TSH), thyrotropin-releasing hormone (TRH), cortisol, aldosterone, testosterone, dehydroepiandrosterone (DHEA), androstene Dione, dihydrotestosterone (DHT), estrone, estriol (E3), progesterone, calcitriol, calcifediol, prostaglandins (PG), leukotrienes (LT), prostacyclin ( PGI2), thromboxane (TXA2), prolactin-releasing hormone (PRH), liptropine (PRH), brain natriuretic peptide (BNP), neuropeptide Y (NPY), histamine, endothelin, pancreatic polypeptide, kidney hormones and enkephalins.

In certain embodiments, the payload may be an antiviral agent. The term "antiviral agent" as used herein refers to an agent (compound or biological) that is effective in inhibiting the formation and/or replication of viruses in mammals. This includes agents that interfere with host or viral machinery required for virus formation and/or replication in mammals. Antiviral agents include, for example, ribavirin, amantadine, VX-497 (merimepodib, Vertex Pharmaceuticals), VX-498 (VertexPharmaceuticals), Levovirin, Viramidine , histamine dihydrochloride (Ceplene) (histamine dihydrochloride), XTL-001 and XTL-002 (XTL Biopharmaceuticals).

In certain embodiments, the payload may be an antibacterial agent. As used herein, the term "antimicrobial agent" refers to an agent that: (i) inhibits, reduces, or prevents the growth of bacteria, (ii) inhibits or reduces the ability of bacteria to cause infection in a subject, or (iii) inhibits or any substance, compound, combination of substances, or combination of compounds that reduces the ability of bacteria to reproduce or maintain infection in the environment. The term "antimicrobial agent" also refers to compounds capable of reducing the infectivity or virulence of bacteria.

In certain embodiments, the payload may be an immunomodulator. The term "immunomodulator" as used herein in combination therapy refers to a substance that acts to suppress, mask, or enhance the host's immune system. Examples of immunomodulatory agents include, but are not limited to, proteinaceous agents such as cytokines, peptide mimetics, and antibodies (e.g., human, humanized, chimeric, monoclonal, polyclonal, Fv, ScFv, Fab or F(ab)2 fragments or epitope-binding fragments), nucleic acid molecules (eg, antisense nucleic acid molecules, iRNA, and triple helix nucleic acid molecules), small molecules, organic compounds, and inorganic compounds. In particular, immunomodulators include, but are not limited to: methotrexate, leflunomide, cyclophosphamide, Cytoxan, Imuran, Cyclosporine A, minocycline, azathioprine, antibiotics (such as FK506 (tacrolimus)), methylprednisolone (MP), corticosteroids, steroids , mycophenolate mofetil, rapamycin (sirolimus), mizoribine, deoxyspergualin, brequinar, malononitrileamide ( malononitriloaminde) (eg, leflunamide), T cell receptor modulators, and cytokine receptor modulators.

In certain embodiments, the immunomodulatory agent may be an immunostimulatory agent. The term "immunostimulatory agent" as used herein preferably refers to any substance or substances capable of triggering an immune response (eg, an immune response against a specific pathogen). Immune cell activating compounds include Toll-like receptor (TLR) agonists. Such agonists include pathogen-associated molecular patterns (PAMPs), e.g., compositions that mimic infection (such as bacterial-derived immunomodulators (also known as danger signals)) and damage-associated molecular patterns (e.g., compositions that mimic stress or damaged cells). things). TLR agonists include nucleic acids or lipid compositions (eg, monophosphoryl lipid A (MPLA)). In one example, the TLR agonist includes a TLR9 agonist, such as cytosine guanosine oligonucleotide (CpG-ODN), poly(ethyleneimine) (PEI)-condensed oligonucleotide (ODN) (such as , PEI-CpG-ODN- or double-stranded deoxyribonucleic acid (DNA)). In another example, TLR agonists include TLR3 agonists, such as polyinosine-polycytidylic acid (poly(I:C)), PEI-poly(I:C), polyadenylic acid-polyuridylic acid (poly(A:U)), PEI-poly(A:U) or double-stranded ribonucleic acid (RNA). Other exemplary vaccine immunostimulatory compounds include lipopolysaccharide (LPS), chemokines/cytokines, fungal beta-glucans (such as lentin), imiquimod, CRX-527, and OM-174.

In certain embodiments, the payload may be a half-life increasing moiety or a solubility increasing moiety. Half-life increasing moieties are, for example, PEG-moieties (polyethylene glycol moieties; PEGylation), other polymer moieties, PAS moieties (oligomeric peptides composed of proline, alanine and serine; PAS acylation) or serum albumin Protein binding agents. Solubility-increasing moieties are, for example, PEG-moieties (PEGylation) or PAS moieties (PAS-acylation).

In certain embodiments, the payload may be a polymer-toxin conjugate. Polymer-toxin conjugates are polymers capable of carrying many payload molecules. Such conjugates are sometimes called fleximers, such as those marketed by Mersana Therapeutics. The polymer-toxin conjugate may comprise any of the toxins disclosed herein.

In certain embodiments, the payload may be nucleotides. An example of a nucleic acid payload is MCT-485, a very small non-coding double-stranded RNA with oncolytic and immune-activating properties developed by MultiCell Technologies, Inc.

In certain embodiments, the payload may be a fluorescent dye. The term "fluorescent dye" as used herein refers to a dye that absorbs light at a first wavelength and emits at a second wavelength that is longer than the first wavelength. In certain embodiments, the fluorescent dye is a near-infrared fluorescent dye that emits light at a wavelength between 650 nm and 900 nm. In this region, tissue autofluorescence is lower, and less fluorescence extinction enhances deep tissue penetration with minimal background interference. Thus, near-infrared fluorescence imaging can be used to visualize tissue to which the antibody payload conjugates of the invention bind during surgery. "Near-infrared fluorescent dyes" are known in the art and are commercially available. In certain embodiments, the near-infrared fluorescent dye can be IRDye 800CW, Cy7, Cy7.5, NIR CF750/770/790, DyLight 800, or Alexa Fluor 750.

In certain embodiments, the payload may contain radionuclides. The term "radionuclide" as used herein refers to medically useful radionuclides, including, for example, positively charged ions of radioactive metals such as Y, In, Tb, Ac, Cu, Lu, Tc, Re, Co , Fe, etc., such as ⁹⁰ Y, ¹¹¹ In, ⁶⁷ Cu, ⁷⁷ Lu, ⁹⁹ Tc, ¹⁶¹ Tb, ²²⁵ Ac, etc. Radionuclides may be contained in chelating agents such as DOTA or NODA-GA. Further, the radionuclide may be a therapeutic radionuclide or a radionuclide that may be used as a contrast agent in imaging techniques, as described below. Radionuclides or molecules containing radionuclides are known in the art and are commercially available.

In certain embodiments, the payload may be vitamins. The vitamin may be selected from the group consisting of folates (including folic acid, folate and vitamin B9).

In a specific embodiment, the invention relates to a method according to the invention, wherein the toxin is selected from at least one of the group consisting of:

·pyrrolobenzodiazepines (e.g., PBD);

·Auristatin (such as MMAE, MMAF);

Maytansinoids (e.g., maytansinoids, DM1, DM4, DM21);

·Becarcinomycin;

·Nicotinamide phosphoribosyltransferase (NAMPT) inhibitor;

·Microtubulysin;

· Enedyne (e.g., calicheamicin);

·Anthracycline derivatives (PNU) (e.g., doxorubicin);

·Pyrrolyl kinesin spindle protein (KSP) inhibitor;

·Nodulin;

·Drug efflux pump inhibitors;

·Sandromycin;

·Amanitanic acid (e.g., alpha-amanitinine); and

· Camptothecins (e.g., ixotecan, drotecan).

That is, antibody-linker conjugates produced using the methods of the invention preferably contain a toxin payload. The term "toxin" as used herein refers to any compound produced by and toxic to a living cell or organism. Thus, toxins may be, for example, small molecules, peptides or proteins. Specific examples are neurotoxins, necrotoxins, hemotoxins and cytotoxins. In certain embodiments, the toxin is a toxin used to treat neoplastic diseases. That is, the toxin can be conjugated to an antibody using the methods of the invention and delivered to or into malignant cells due to the target specificity of the antibody.

In certain embodiments, the toxin may be auristatin. As used herein, the term "auristatin" refers to a family of antimitotic agents. Auristatin derivatives are also included within the definition of the term "auratstatin". Examples of auristatin include, but are not limited to, synthetic analogs of auristatin E (AE), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and dolastatin .

In certain embodiments, the toxin may be a maytansinoid. In the context of the present invention, the term "maytansinoid" refers to a class of highly cytotoxic drugs originally isolated from the African shrub Maytenus ovatus as well as the additional maytansinol and natural maytansinols. C-3 ester of maytansinol (U.S. Patent No. 4,151,042); synthesis of C-3 ester analogs of maytansinol (Kupchan et al., J. Med. Chem. 21:31-37, 1978; Higashide et al., Nature 270:721- 722, 1977; Kawai et al., Chem. Farm. Bull. 32:3441-3451; and U.S. Patent No. 5,416,064); C-3 esters of simple carboxylic acids (U.S. Patent Nos. 4,248,870; 4,265,814; 4,308,268; 4,308,269; 4,309,428; 4,317,821 ; 4,322,348; and 4,331,598); and C-3 esters and derivatives of N-methyl-L-alanine (U.S. Patent Nos. 4,137,230; 4,260,608; and Kawai et al., Chem. Pharm Bull. 12:3441, 1984). Exemplary maytansinoids that may be used in the methods of the invention or may be included in the antibody-payload conjugates of the invention are maytansinoids, DM1, DM3, DM4 and/or DM21.

In certain embodiments, the toxin may be duocarmycin. Suitable becomycins may be, for example, becomycin A, becomycin Bl, becomycin B2, becomycin CI, becomycin C2, becomycin D, becomycin SA , beclomycin MA and CC-1065. The term "bizelesin" is understood to mean also synthetic analogues of becanthromycin, such as adozelesin, bizelesin, carzelesin, KW-2189 and CBI-TMI.

In certain embodiments, the toxin can be a NAMPT inhibitor. As used herein, the terms "NAMPT inhibitor" and "nicotinamide phosphoribosyltransferase inhibitor" refer to inhibitors that reduce NAMPT activity. The term "NAMPT inhibitor" may also include prodrugs of NAMPT inhibitors. Examples of NAMPT inhibitors include, but are not limited to, FK866 (also known as APO866), GPP 78 hydrochloride, ST 118804, STF31, pyridylcyanoguanidine (also known as CH-828), GMX-1778, and P7C3. Additional NAMPT inhibitors are known in the art and may be suitable for use in the compositions and methods described herein. See, for example, PCT Publication WO2015/054060, US Patent Nos. 8,211,912 and 9,676,721, the entire contents of which are incorporated herein by reference. In some embodiments, the NAMPT inhibitor is FK866. In some embodiments, the NAMPT inhibitor is GMX-1778.

In certain embodiments, the toxin may be tubulysin. Tubulysins are cytotoxic peptides consisting of 9 members (A-I). Tubulysin A has potential application as an anticancer agent. It arrests cells in the G2/M phase. Tubulysin A inhibits polymerization more effectively than vinblastine and induces depolymerization of detached microtubules. Tubulysin A has potent cytostatic effects on various tumor cell lines with IC50s in the picomolar range. Another tubulysin that may be used in the methods of the invention may be tubulysin E.

In certain embodiments, the toxin may be an enediyne. The term "enediyne" as used herein refers to a class of bacterial natural products characterized by nine- and ten-membered rings containing two triple bonds separated by a double bond (see, e.g., K.C. Nicolaou; A.L. Smith; E.W. Yue (1993), "Chemistry and biology of natural and designed enediynes", PNAS 90(13):5881-5888; the entire contents of which are incorporated herein by reference). Some enedynes are capable of Bergman cyclization, and the resulting diradicals (1,4-dehydrobenzene derivatives) are able to abstract hydrogen atoms from the sugar backbone of DNA, which leads to DNA strand cleavage (see e.g. S. Walker; R .Landovitz; W.D.Ding; G.A.Ellestad; D.Kahne (1992) "Cleavage behavior of calicheamicin gamma 1 and calicheamicin T", Proc Nat1 Acad Sci U.S.A.89 (10 ): 4608-12; the entire contents of which are incorporated herein by reference). Their reactivity with DNA confers many enediyne antibiotic properties, and some enediynes are clinically studied as anticancer antibiotics. Non-limiting examples of enedynes are dynemicin, neocarziostatin, calicheamicin, esperramicin (see, e.g., Adrian L. Smith and K.C. Bicolau, "The Enedizyne Antibiotics", J. Med. Chem., 1996, 39(11), pp 2103-2117; and Donald Borders, "The Enedizyne Antibiotics as Antitumor Drugs" antitumor agents", Informa Healthcare; 1st Edition (Nov. 23, 1994, ISBN-10:0824789385; the entire contents of which are incorporated herein by reference). In certain embodiments, the toxin may be calicheamicin.

In certain embodiments, the toxin may be doxorubicin. "Adriamycin" as used herein refers to a member of the anthracycline family derived from the Streptomyces bacterium Streptomyces peucetius var.caesius, and includes doxorubicin, daunorubicin epirubicin, epirubicin and idabicin.

In certain embodiments, the toxin may be a kinesin spindle protein inhibitor. The term "kinesin spindle protein inhibitor" refers to compounds that inhibit the kinesin spindle protein, which is involved in the assembly of bipolar spindles during cell division. Inhibitors of kinesin spindle proteins are being studied for the treatment of cancer. Examples of kinesin spindle protein inhibitors include ispinesib. Further, the term "kinesin spindle protein inhibitor" includes GlaxoSmithKline's SB715992 or SB743921 and CombinatoRx's pentamidine/chlorpromazine.

In certain embodiments, the toxin may be nomonocin as described in US 20180078656 A1, which is incorporated by reference.

In certain embodiments, the toxin may be sandromycin. Sandomycin is a depsipeptide that was first isolated from Nocardioides sp. (ATCC 39419) and has been proven to have cytotoxic and anti-tumor activities.

In certain embodiments, the toxin may be amanitaxin. Amanitaxins (including α-amanitine, β-amanitin and amanitin) are cyclic peptides composed of 8 amino acids. They can be isolated from the Amanita phalloides mushroom or can be prepared from building blocks by synthesis. Amanitaxin specifically inhibits DNA-dependent RNA polymerase II in mammalian cells and thereby affects cellular transcription and protein biosynthesis. Inhibition of transcription in cells results in the cessation of growth and proliferation. Although not covalently bound, the complex between amanitin and RNA polymerase II is very tight (KD = 3 nM). The dissociation of amanitin from the enzyme is a very slow process, making the affected cells less likely to recover. When transcriptional repression in a cell persists for too long, the cell undergoes programmed cell death (apoptosis). In a preferred embodiment, the term "Amatoxin" as used herein means, for example, as described in WO 2010/115630, WO 2010/115629, WO 2012/119787, WO 2012/041504 and WO 2014/135282 Described alpha-amanitin or a variant thereof.

In certain embodiments, the toxin may be camptothecin. The term "camptothecin" as used herein refers to camptothecin or a camptothecin derivative that functions as a topoisomerase I inhibitor. Exemplary camptothecins include, for example, topotecan, exatecan, deruxtecan, irinotecan, DX-8951f, SN38, BN 80915, Leto lurtotecan, 9-nitrocamptothecin and aminocamptothecin. A variety of camptothecins have been described, including those used in the treatment of human cancer patients. Several camptothecins are described, for example, in Kehrer et al., Anticancer Drugs, 12(2):89-105, (2001) or Li et al., ACSMed. Chem. Lett. 2019, 10, 10, 1386-1392).

In the sense of the present invention, toxins can also be inhibitors of drug efflux transporters. Antibody-payload conjugates containing a toxin and an inhibitor of a drug efflux transporter may have the advantage that when internalized into the cell, the inhibitor of the drug efflux transporter prevents the efflux of the toxin from the cell. In the present invention, the drug efflux transporter may be P-glycoprotein. Some common P-glycoprotein drug inhibitors include: amiodarone, clarithromycin, cyclosporine, colchicine, diltiazem, erythromycin, felodipine, ketoconazole, lansoprazole, Ogilvy Prazole and other proton pump inhibitors, nifedipine, paroxetine, reserpine, saquinavir, sertraline, quinidine, tamoxifen, verapamil, and duloxetine. Elacridar and CP 100356 are other common P-gp inhibitors. Zosuquidar and tariqidar were also developed with this in mind. Finally, valspodar and reversan are other examples of such agents.

It should be understood that payload B as defined herein is not only understood as the actual payload itself, but as payload molecules. A payload molecule as used herein may include additional structures, for example, to facilitate coupling of the payload via chemical synthesis with linker B or RK motifs or chemical spacers.

That is, in certain embodiments, the actual payload may be contained within the payload molecule attached to the linker of the invention. Payload molecules can have the following structures:

X-(spacer)-payload,

where payload represents an actual payload, such as one of the compounds disclosed herein, and process), and where (spacer) represents a chemical spacer that sterically separates the actual payload from the reactive group X. However, it should be understood that in certain embodiments, the reactive group X may be a spacer or part of the actual payload. For example, the spacer may comprise a peptide or amino acid residue, wherein the reactive group X may be the amino group of the N-terminal amino acid residue contained in the spacer. In certain embodiments, spacers may be absent. In embodiments where spacers are not present, the functional group may be included in the actual payload. In certain embodiments, spacers may be used to attach functional groups of interest (i.e., functional groups that are compatible with the functional groups contained in the linker moiety) to the actual payload. In certain embodiments, reactive group X can be a maleimide group or a cyclooctyne group, such as, but not limited to, a DBCO or BCN group.

In a specific embodiment, the invention relates to a method according to the invention, wherein the chemical spacer (Sp ₂ ) comprises a self-cleaving moiety.

That is, the linker can contain a self-cleaving moiety to facilitate the release of the payload in the target cell or tissue. The self-cleaving moiety can be included in any part of the linker. However, the self-cleaving moiety is preferably contained in a chemical spacer ( _Sp2 ) that separates the payload from the RK motif. Alternatively, the self-cleaving moiety may be contained in a (spacer) contained in the payload molecule as defined above.

As used herein, the term "self-immolative moiety" refers to at least a bifunctional molecule that can be contained in a linker and that spontaneously degrades after an initial reaction has occurred, thereby releasing the payload. The initial reaction may be hydrolysis of the covalent bond between the self-cleaving moiety and the amino acid residue. In certain embodiments, the covalent bond between the self-cleaving moiety and the amino acid residue may be an amide bond formed between the α-carboxyl group of the amino acid and the amine group contained in the self-cleaving moiety, and the initial reaction may be by a peptide Catalyzed by enzymes or proteases. However, the present invention includes other chemical substances.

In a specific embodiment, the invention relates to a method according to the invention, wherein the self-cleaving moiety is directly attached to the payload B.

More preferably, the self-cleaving moiety is directly attached to the payload B such that upon degradation of the self-cleaving moiety the payload is released. In certain embodiments, the self-cleaving moiety is located between the payload and the RK motif contained in the linker. That is, the self-cleaving moiety can be coupled to the N-terminus of residue R or the C-terminus of residue K. Alternatively, the self-cleaving moiety may be located between the payload and the amino acid residue contained in the chemical spacer ( _Sp2 ), preferably at the N-terminus or C-terminus of the amino acid residue. Further, the self-cleaving moiety may be positioned between the payload and the non-amino acid residues contained in the chemical spacer ( _Sp2 ) by any method known in the art.

It should be understood that the choice of self-cleaving moieties depends inter alia on the functional groups available in the payload molecule.

In a particular embodiment, the invention relates to a method according to the invention, wherein the self-cleaving moiety comprises a p-aminobenzylcarbamoyl (PABC) moiety.

That is, in certain embodiments, the linker may comprise a self-cleaving moiety, a p-aminobenzylcarbamoyl (PABC) moiety. PABC includes a free amine group suitable for coupling to the C-terminus of an amino acid residue or peptide, and a carbamoyl group, through which the PABC can be coupled to a payload, particularly a payload containing an amine. However, those skilled in the art are aware of methods of functionalizing the payload so that it contains amine groups. The self-cleaving moiety PABC is preferably located between the payload and the amino acid residues contained in the linker. The amino acid residue is preferably the residue K contained in the RK motif or the amino acid contained in the chemical spacer (Sp ₂ ). In certain embodiments, the self-cleaving moiety PABC is located between the payload and an alanine residue in the inclusion of a chemical spacer ( _Sp2 ). In certain embodiments, the self-cleaving moiety can be located between the payload and the peptidase cleavage site. In certain embodiments, the self-cleaving moiety may be located between the payload and the cathepsin cleavage site. That is, the self-cleaving moiety can be located between the payload and a motif known to be cleaved by cathepsins.

The term "cathepsin" as used herein refers to a family of proteases. The term cathepsin includes cathepsin A, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin F, cathepsin G, cathepsin H, cathepsin K, cathepsin L1, cathepsin L2, cathepsin O, cathepsin S, cathepsin W and cathepsin Z. In specific embodiments, the cleavable moiety may be a motif specifically hydrolyzed by cathepsin B, such as valine-alanine, valine-citrulline, or alanine-alanine. Salomon et al. "Optimizing LysosomalActivation of Antibody-Drug Conjugates (ADCs) by Incorporation of NovelCleavable Dipeptide Linkers", Mol Pharm , 2019, 16(12), pages 4817-4825 disclose other motifs that can be specifically hydrolyzed by peptidases.

A typical dipeptide structure used in ADC linkers is the valine-citrulline motif, as provided for example in Brentuximab Vedotin; and in Dubowchik and Firestone “Cathepsin B-labile dipeptide linkers for internalization of doxorubicin from immunization Cathepsin B-labile dipeptide linkers for lysosomal release of doxorubicin from internalizing immunoconjugates: model studies of enzymatic drug release and antigen-specific in in vitro anticancer activity)"; Bioconjug Chem; 2002; 13(4); pp. 855-69. This linker can be cleaved by cathepsin B to release the actual payload at the site of disease. The same applies to the valine-alanine motif, which is provided for example in SGN-CD33A.

Thus, in certain embodiments, the linker may include the structure (Sp ₁ )-RK-(Sp ₂ )-Val-Cit-(self-cleaving moiety)-payload. In certain embodiments, the linker may include the structure (Sp ₁ )-RK-(Sp ₂ )-Val-Cit-payload. In certain embodiments, the linker may include the structure (Sp ₁ )-RK-(Sp ₂ )-Val-Cit-PABC-payload.

In certain embodiments, the linker can comprise the structure RK-Val-Cit (Seq ID NO:54). In certain embodiments, the linker may include or consist of the structure RK-Val-Cit-(self-cleaving moiety)-payload. In certain embodiments, the linker may include or consist of the structure RK-Val-Cit-PABC-payload. In certain embodiments, the linker may include or consist of the structure RK-Val-Cit-PABC-MMAE. In certain embodiments, the linker can include or consist of the structure RK-Val-Cit-PABC-Maytansine.

It must be noted that the peptide cleavage site can also be a motif cleavable by other peptidases such as caspase 3, endoasparaginase (Legumain) or neutrophil elastase, or as Dal Corso et al., Innovative Linker Strategies for Tumor-Targeted Drug Conjugates, Chemistry 25(65), pp. 14740-14757.

However, it is important to note that cells contain a broad range of cellular peptidases, and other less conserved amino acid motifs can also be efficiently cleaved by peptidases. Thus, in certain embodiments, the linker may comprise the structure (Sp ₁ )-RK-(Sp ₂ )-PABC-payload, wherein (Sp ₂ ) is absent or consists of amino acid residues.

In certain embodiments, the linker can include the structure (Sp ₁ )-RK-(Sp ₂ )-PABC-payload, wherein (Sp ₂ ) includes a PEG moiety between the PABC moiety and (Sp ₂ ) or between the most C-terminal amino acid residues contained in the RK motif.

In certain embodiments, a linker comprising a self-cleaving moiety PABC is coupled to an amine-containing payload (especially a primary or secondary amine-containing payload). In certain embodiments, the amine-containing payload is ausristatin, such as MMAE. In certain embodiments, the amine-containing payload is a maytansinoid, such as maytansine.

It must be noted that the payload can be coupled to the self-cleaving PABC moiety via additional linker molecules. For example, an amine-containing payload can be coupled to the PABC moiety through a p-nitrophenol (PNP) group. Su et al., Bioconjugate Chem. 2018, 29, 4, 1155-1167 and Dokter et al., Mol Cancer Ther. 2014 Nov; 13(11): 2618-29 have disclosed that allow the inclusion of reactive groups other than amines. The payload is coupled to other linker molecules in the PABC moiety. For example, payloads containing alcohol or phenol groups can be coupled to PABC via an ethylenediamine (EDA) linker (see Figures 18 and 19).

In a particular embodiment, the invention relates to a method according to the invention, wherein the self-cleaving moiety comprises a methylamino group. It has been previously demonstrated that methylamino groups can be used as self-cleaving moieties in peptide-based linkers for ADCs (Costoplus et al., ACS Med. Chem. Lett., 2019, 10, 10, 1393-1399 and Li et al., ACS Med. . Chem. Lott. 2019, 10, 10, 1386-1392).

In particular, a self-cleaving moiety containing a methylamino group can be coupled to the C-terminus of an amino acid residue via an amide bond formed between the α-carboxyl group of the amino acid residue and the amine contained in the methylamino group. The amino acid residue may be an amino acid residue contained in (Sp ₂ ) or residue K contained in an RK motif. Methyl groups contained in the methylamine group can be coupled to the payload via ether or thioether linkages. Therefore, when the payload includes hydroxyl or thiol groups, it may be preferable to use methylamino groups as self-cleaving groups. In certain embodiments, the hydroxyl-containing payload may be a camptothecin (such as ixotecan derivative Dxd) or an anthracycline (such as PNU-159682). In certain embodiments, the thiol-containing payload may be a maytansinoid (such as DM1, DM4, or DM21).

The linker containing a methylamino group may contain the molecular structure C-(NH)-(CH ₃ )-OC or C-(NH)-(CH ₃ )-SC. Exemplary linkers including methylamino groups are shown in Figures 15, 17, and 21.

It will be appreciated that it is preferred to couple the payload to the C-terminal carboxyl group of the amino acid residue using PABC and a self-cleaving moiety containing a methylamino group.

Other self-cleavable moieties that can be used to couple payloads to the C-terminal carboxyl group of amino acid residues include p-aminobenzyl ethanol (PABE), which is used to couple phenol-containing payloads to the C-terminal carboxyl group of amino acid residues. Linker (Zhang et al., Bioconjugate Chem. 2018, 29, 6, 1852–1858) or p-methylaniline for coupling payloads containing tertiary amine or heteroaryl moieties to the C-terminal carboxyl group of an amino acid residue (PMA) linker (Staben et al., Nature Chemistry Vol. 8, pp. 1112-1119 (2016)) (see Figure 20 and Figure 23, respectively). Non-limiting examples of payloads containing phenolic groups are beclomycin GA or pyrrolobenzodiazepine PBD. A non-limiting example of a tertiary amine-containing payload is becomycin GA.

However, the payload can also be coupled to the N-terminal amino group via a self-cleaving moiety. For example, the payload can be coupled to the N-terminal amino group of the amino acid residue via a self-cleaving moiety comprising an ortho-hydroxyl protected aryl sulfate. For example, o-hydroxyl protected aryl sulfates (OHPAS) can be used to couple a phenolic payload (such as a PBD) to the N-terminal amino group of an amino acid residue (see Figure 27). The OHPAS moiety preferably contains a carboxyl group, via which the OHPAS moiety can be coupled directly to the N-terminal amino group of the amino acid residue. Alternatively, the OHPAS moiety can be coupled to the N-terminal amino group of the amino acid residue through a functionalized PEG linker, such as, but not limited to, a functionalized (PEG) ₂ linker. Preferably, one end of the PEG linker is functionalized with an amino group to allow coupling to the carboxyl group contained in the OHPAS moiety and the other end is functionalized with a carboxyl group to allow coupling to the N-terminal amino group of the amino acid residue (Park et al., Bioconjugate Chem. 2019, 30, 7, 1957-1968) (see Figure 26).

Alternatively or in addition, a linker molecule can be positioned between the sulfate group of the OHPAS and the payload to allow coupling of non-phenolic payloads to the OHPAS. For example, p-hydroxybenzyl (PHB) linker molecules can be used to allow payloads containing primary or secondary amines to be coupled to the OHPAS moiety through carbamate formation (see Figures 31 and 32). Payloads containing tertiary amines can be coupled to OHPAS-containing linkers by forming quaternary ammonium (see Figures 33 and 34). Further, p-hydroxybenzylethylenediamine (PHB-EDA) linker molecules can be used to couple hydroxyl-containing payloads to OHPAS moieties via carbamate formation (Park et al., Bioconjugate Chem. 2019, 30, 7, 1957-1968) (see Figure 25).

In certain embodiments, the payload can be coupled via a cleavable moiety to the amino acid residues contained in the linker. As used herein, a "cleavable moiety" is a chemical unit that can be separated from the actual payload by enzymatic or non-enzymatic hydrolysis. In certain embodiments, the cleavable moiety may be an amino acid motif that is hydrolyzable by a peptidase or protease.

In other embodiments, the cleavable moiety contained in the linker may be a carbohydrate moiety. In such embodiments, the cleavable moiety may be a moiety that is cleavable by a glucosidase enzyme. Thus, in certain embodiments, a cleavable moiety may be a moiety that is cleavable by a beta-glucuronidase or beta-galactosidase enzyme.

In other embodiments, the cleavable moiety contained in the linker may be a phosphate moiety. In such embodiments, the cleavable moiety may be a moiety that is cleavable by a phosphatase. Thus, in certain embodiments, the cleavable moiety may be a moiety that is cleaved by a beta-lysosomal acid pyrophosphatase or acid phosphatase.

Examples of other cleavable moieties that can be used to release payloads from linker molecules have been described by Bargh et al., Cleavable linkers in antibody-drug conjugates, Chem Soc Rev. 2019 8 Nov 12;48(16):4361-4374 Description. In certain embodiments, a linker may include a structure (cleavable moiety)-(self-cleaving moiety)-payload. In such embodiments, the self-cleaving moiety may degrade and release the payload when the cleavable moiety is cleaved.

In a particular embodiment, the invention relates to a method according to the invention, wherein the joint is Figure 1, Figure 2, Figure 3, Figure 8, Figure 9, Figure 14, Figure 15, Figure 17, Figure 18, Figure 19, In the joint shown in Figure 20, Figure 21, Figure 22, Figure 23, Figure 24, Figure 25, Figure 26, Figure 27, Figure 28, Figure 29, Figure 30, Figure 31, Figure 32, Figure 33 or Figure 34 of any kind.

In certain embodiments, a joint may include two or more connecting portions and/or payloads B. That is, in certain embodiments, the linker may include the structure

a)(Sp ₁ )-RK-(Sp ₂ )-B ₁ -(Sp ₃ )-B ₂ -(Sp ₄ ),

b)(Sp ₄ )-B ₂ -(Sp ₁ )-RK-(Sp ₂ )-B ₁ -(Sp ₃ ),

c)(Sp ₁ )-B ₁ -(Sp ₂ )-RK-(Sp ₃ )-B ₂ -(Sp ₄ ), or

d)(Sp ₄ )-B ₂ -(Sp ₁ )-B ₁ -(Sp ₂ )-RK-(Sp ₃ ).

In such embodiments, the chemical spacers (Sp ₁ ), (Sp ₂ ), (Sp ₃ ) and RK motifs may have the same properties as defined above. Furthermore, parts B ₁ and B ₂ may be any of the connection parts and/or payloads defined above. Further, the chemical spacer (Sp ₄ ) may have the same characteristics as the chemical spacer (Sp ₁ ), (Sp ₂ ) or (Sp ₃ ), or may not be present.

Therefore, in a specific embodiment, the invention relates to a method according to the invention, wherein the linker comprises a second connection part or payload B ₂ , in particular wherein B ₂ is via a chemical spacer (Sp ₁ ) or (Sp ₃ ) to the connector.

That is, the payload or connection portion B ₂ can be connected to the chemical spacer (Sp ₁ ) or (Sp ₃ ), or directly to the payload or connection portion B ₁ . Payload or linker B ₂ may include any functional group suitable for coupling B ₂ to a functional group contained in (Sp ₁ ), (Sp ₃ ) or B ₁ .

In certain embodiments, payload or linker B ₂ may include an amino group through which B ₂ is linked to (Sp ₃ ) or B ₁ . That is, B ₂ can be connected to (Sp ₃ ) or the carboxyl group contained in B ₁ through the amino group. In certain embodiments, the carboxyl group contained in (Sp ₃ ) may be a carboxyl group contained in the C-terminal amino acid residue of the chemical spacer (Sp ₃ ). In certain embodiments, the carboxyl group contained in B ₁ may be an alpha-carboxyl group of the amino acid-based payload or linker moiety. In certain embodiments, B ₂ can be coupled to a carboxyl group contained in (Sp ₃ ) or B ₁ via a linker molecule. In certain embodiments, the linker molecule can include a self-cleaving moiety.

In certain embodiments, payload or linking moiety B ₂ may include a carboxyl group through which B ₂ is connected to (Sp ₁ ) or B ₁ . That is, B ₂ can be connected to (Sp ₁ ) or the amine group contained in B ₁ through the carboxyl group. In certain embodiments, the amine group contained in (Sp ₁ ) may be the amine group contained in the N-terminal amino acid residue of the chemical spacer (Sp ₁ ). In certain embodiments, the amine group contained in B ₁ may be an alpha-amino group of the amino acid-based payload or linker moiety. In certain embodiments, B ₂ can be coupled to the amine group contained in (Sp ₁ ) or B ₁ via a linker molecule. In certain embodiments, the linker molecule can include a self-cleaving moiety.

However, it must be noted that _B2 may also include other functional groups besides amine or carboxyl groups. In such embodiments, B ₂ can be coupled to (Sp ₁ ), (Sp ₃ ) or B ₁ by any method known in the art, either directly or via a linker or self-cleaving group.

In certain embodiments, payload or linker B ₂ can be coupled to an amino acid side chain contained in (Sp ₁ ) or (Sp ₃ ). That is, B ₂ can be linked to the functional group of the amino acid side chain contained in (Sp ₁ ) or (Sp ₃ ) via a compatible functional group.

In certain embodiments, the (Sp ₁ ), (Sp ₂ ), (Sp ₃ ), and RK motifs consist solely of amino acids, amino acid mimetics, and/or amino acid derivatives. In certain embodiments, B ₁ and/or B ₂ also comprise an amino acid backbone. In such embodiments, the linker may be a linear peptide or a peptide mimetic. In embodiments where B ₁ is an amino acid, an amino acid mimetic, or an amino acid derivative, the linker can have the structure (Sp ₁ )-RK-(Sp ₂ )-B ₁ , wherein (Sp ₁ )-RK-(Sp ₂ ) -B ₁ is a linear peptide or peptidomimetic. In embodiments where B ₁ is an amino acid, an amino acid mimetic, or an amino acid derivative, the linker can have the structure (Sp ₁ )-RK-(Sp ₂ )-B ₁ -(Sp ₃ ), wherein (Sp ₁ )-RK -(Sp ₂ )-B ₁ -(Sp ₃ ) is a linear peptide or peptidomimetic. In embodiments where B ₁ is an amino acid, an amino acid mimetic, or an amino acid derivative, the linker can have the structure RK-(Sp ₂ )-B ₁ -(Sp ₃ ), wherein RK-(Sp ₂ )-B ₁ -( Sp ₃ ) is a linear peptide or peptidomimetic. In embodiments where B ₁ is an amino acid, an amino acid mimetic, or an amino acid derivative, the linker can have the structure RK-(Sp ₂ )-B ₁ , wherein RK-(Sp ₂ )-B ₁ is a linear peptide or peptide mimetic. . In embodiments where B ₁ is an amino acid, an amino acid mimetic or an amino acid derivative, the linker can have the structure RK-B ₁ -(Sp ₃ ), wherein RK-B ₁ -(Sp ₃ ) is a linear peptide or peptide mimetic . In embodiments where B ₁ is an amino acid, an amino acid mimetic, or an amino acid derivative, the linker may have the structure RK-B ₁ , wherein RK-B ₁ is a linear peptide or peptide mimetic.

In embodiments where B ₁ and B ₂ are amino acids, amino acid mimetics or amino acid derivatives, the linker may have the structure (Sp ₁ )-RK-(Sp ₂ )-B ₁ -(Sp ₃ )-B ₂ -(Sp ₄ ), wherein (Sp ₁ )-RK-(Sp ₂ )-B ₁ -(Sp ₃ )-B ₂ -(Sp ₄ ) is a linear peptide or peptide mimetic. In other embodiments where B ₁ and B ₂ are amino acids, amino acid mimetics or amino acid derivatives, the linker can have the structure (Sp ₄ )-B ₂ -(Sp ₁ )-RK-(Sp ₂ )-B ₁ -( Sp ₃ ), wherein (Sp ₄ )-B ₂ -(Sp ₁ )-RK-(Sp ₂ )-B ₁ -(Sp ₃ ) is a linear peptide or peptide mimetic. In other embodiments where B ₁ and B ₂ are amino acids, amino acid mimetics or amino acid derivatives, the linker can have the structure (Sp ₄ )-B ₂ -(Sp ₁ )-B ₁ -(Sp ₂ )-RK-( Sp ₃ ), wherein (Sp ₄ )-B ₂ -(Sp ₁ )-B ₁ -(Sp ₂ )-RK-(Sp ₃ ) is a linear peptide or peptide mimetic.

In embodiments where B ₁ is not an amino acid, an amino acid mimetic, or an amino acid derivative, the linker can have the structure (Sp ₁ )-RK-(Sp ₂ )-B ₁ -(Sp ₃ ), wherein (Sp ₁ )-RK - (Sp ₂ ) is a linear peptide or peptidomimetic, and B ₁ is attached to the C-terminal carboxyl group contained in (Sp ₂ ). In embodiments where B ₁ is not an amino acid, an amino acid mimetic, or an amino acid derivative, the linker can have the structure (Sp ₁ )-B ₁ -(Sp ₂ )-RK-(Sp ₃ ), wherein (Sp ₂ )-RK - (Sp ₃ ) is a linear peptide or peptidomimetic, and B ₁ is linked to the N-terminal amine group contained in (Sp ₂ ). However, it is important to note that _B1 does not necessarily have to be directly coupled to the peptide or peptidomimetic. Instead, B ₁ can be coupled to a peptide or peptidomimetic via a linker molecule and/or a self-cleaving moiety.

In embodiments where B ₁ is an amino acid, amino acid mimetic or amino acid derivative and B ₂ is not an amino acid, amino acid mimetic or amino acid derivative, the linker may have the structure (Sp ₁ )-RK-(Sp ₂ )-B ₁ - (Sp ₃ )-B ₂ -(Sp ₄ ), (Sp ₄ )-B ₂ -(Sp ₁ )-RK-(Sp ₂ )-B ₁ -(Sp ₃ ), (Sp ₁ )-B ₁ -( Sp ₂ )-RK-(Sp ₃ )-B ₂ -(Sp ₄ ) or (Sp ₄ )-B ₂ -(Sp ₁ )-B ₁ -(Sp ₂ )-RK-(Sp ₃ ), where, ( Sp ₁ )-RK-(Sp ₂ )-B ₁ -(Sp ₃ ) or (Sp ₁ )-B ₁ -(Sp ₂ )-RK-(Sp ₃ ) is a linear peptide or peptide mimetic and B ₂ coupled to the C-terminal carboxyl group contained in (Sp ₃ ), B ₁ or RK or coupled to the N-terminal amino group of (Sp ₁ ), B ₁ or RK.

In such embodiments, antibody-payload conjugates can be generated where, for example, the ratio of antibody to payload is 2 or 4, for example, one or two payloads are coupled to each Q295 residue.

In a specific embodiment, the invention relates to a method according to the invention, wherein B ₁ and B ₂ are the same as or different from each other.

That is, the payloads or connecting portions B ₁ and B ₂ may be identical (i.e. have the same chemical structure) or may be structurally different. In some embodiments, B ₁ and B ₂ are both payloads or both connection parts. In embodiments where B ₁ and B ₂ are both payloads, payloads B ₁ and B ₂ may be the same or different payloads. In embodiments where B ₁ and B ₂ are both connecting parts, connecting parts B ₁ and B ₂ may be the same or different connecting parts. In some embodiments, B ₁ may be the connection part and B ₂ may be the payload, or vice versa.

It should be understood that not all payloads or connecting parts can function as intra-chain payloads or connecting parts at position B ₁ , for example, because they do not have the same functionality as (Sp ₂ ) or RK on one side and (Sp on the other side). ₃ ), (Sp ₁ ) or B ₂ is a functional group that forms a covalent bond. Thus, preferably, in embodiments in which _Bi is an intrachain payload or linker moiety, _Bi is a divalent or multivalent molecule. For example, _B1 can be an amino acid, an amino acid mimetic, or an amino acid derivative. In such embodiments, B ₁ can be coupled to (Sp ₂ ) or the C-terminal carboxyl group of RK via its amino group, and to (Sp ₃ ) or the N-terminal amino group of B ₂ via its carboxyl group. Alternatively, B ₁ can be coupled via its carboxyl group to (Sp ₂ ) or the N-terminal amino group of RK, and via its amino group to the C-terminal carboxyl group of (Sp ₁ ) or B ₂ .

In certain embodiments, the joint may include two connecting portions B ₁ and B ₂ .

That is, in certain embodiments, the invention encompasses linkers comprising two bioorthogonal labeling groups and/or non-bioorthogonal entities. For example, a linker according to the invention may comprise an azide-containing linking moiety (such as Lys( _N3 ) or Xaa( _N3 )) and a sulfhydryl-containing linking moiety (such as cysteine). In certain embodiments, linkers according to the present invention may comprise an azide-containing linking moiety (such as, Lys( _N3 ) or Xaa( _N3 )) and a tetrazine-containing linking moiety (such as, tetrazine-modified amino acids). In certain embodiments, linkers according to the invention may comprise a sulfhydryl-containing linking moiety (such as cysteine) and a tetrazine-containing linking moiety (such as a tetrazine-modified amino acid). Linkers containing two different bioorthogonal labeling groups and/or non-bioorthogonal entities have the advantage that they can accept two different payloads, thereby producing an antibody-payload containing more than one payload conjugate.

In this way, an antibody payload ratio of 2+2 can be obtained. The use of a secondary payload could allow the development of an entirely new class of antibody-payload conjugates that surpass current treatments in terms of efficacy and potency.

Such embodiments may specifically allow targeting of two different structures in the cell (eg DNA and microtubules). Because some cancers are resistant to a type of drug, such as microbutule toxins, DNA toxins can still kill cancer cells.

According to another embodiment, two drugs may be used, both of which are fully effective only if released at the same time and in the same tissue. This could lead to reduced off-target toxicity in cases where the antibody is partially degraded in healthy tissue or where one drug is lost prematurely.

Furthermore, dual-labeled probes can be used for non-invasive imaging and treatment or intra/post-operative imaging/surgery. In such embodiments, tumor patients can be selected by non-invasive imaging. The tumor can then be surgically removed using other imaging agents, such as fluorescent dyes, which help the surgeon or robot identify any cancerous tissue during surgery.

In certain embodiments, one of B ₁ and B ₂ can be a linking moiety containing a sulfhydryl group (such as cysteine), and the other of B ₁ and B ₂ can be a linking moiety containing an azide moiety (Such as, Lys(N ₃ )). In such embodiments, two different payloads can be coupled to the linker, one via thiomaleimide coupling and the other via a SPAAC reaction.

In some embodiments, a linker may contain two payloads. Linkers containing only the payload but no linker moiety can be conjugated to antibodies in a one-step process.

It should be understood that in embodiments where B ₁ and B ₂ are both payloads, B ₁ and B ₂ may be structurally the same or different. In certain embodiments, linkers including one or more payloads can be chemically synthesized. Alternatively, one or more payloads may be coupled to a linker moiety contained in the linker by any method disclosed herein before the linker is coupled to the antibody.

In certain embodiments, the linkers of the invention can allow coupling of two different payloads to residue Q295 of the _CH2 domain of an antibody. The use of a secondary payload allows the development of an entirely new class of antibody-payload conjugates that surpass current treatments in terms of efficacy and potency. New application areas are also envisioned, such as dual-type imaging for imaging and therapy or intra-/post-operative procedures (see Azhdarinia A et al., Dual-Label Strategies for Nuclear and Fluorescent Molecular Imaging: Review and Analysis for Nuclear and FluorescenceMolecular Imaging: A Review and Analysis), Mol Imaging Biol. 2012Jun; 14(3): 261-276). For example, dual-labeled antibodies containing molecular imaging agents for preoperative positron emission tomography (PET) and near-infrared fluorescent (NIRF) dyes for guiding demarcation of surgical margins could greatly enhance cancer diagnosis, staging, and resection ( See Houghton JL. et al., Site-specifically labeled CA19.9-targeted immunoconjugates for thePET, NIRF, and multimodal PET/NIRF imaging of pancreatic cancer. multimodal PET/NIRF imaging of pancreatic cancer), Proc Natl AcadSci US A. 2015Dec 29;112(52):15850-5). PET and NIRF optical imaging offer complementary clinical applications, enabling non-invasive whole-body imaging to localize disease and identify tumor margins during surgery, respectively. However, the generation of such dually labeled probes is currently difficult due to the lack of suitable site-specific methods; chemically linking two different probes results in nearly impossible analysis due to random coupling of the probes. and reproducibility.

Furthermore, in Levengood M et al. (Orthogonal Cysteine Protection Enables Homogeneous Multi-Drug Antibody–Drug Conjugates), Angewandte Chemie, Vol. 56, Issue 3, January 16, 2017), a dual-drug-labeled antibody with two different auristatin toxins attached (with different physiochemical properties and complementary anti-cancer activities) was used in a The auristatin component of the ADC alone was active in refractory cell lines and xenograft models. This suggests that dual-labeled ADCs can address cancer heterogeneity and drug resistance more effectively than a single conventional ADC alone. Since one mechanism of resistance to ADCs involves active pumping of the cytotoxic moiety from cancer cells, another dual-drug application could include additional and simultaneous delivery of drugs that specifically block the efflux mechanism of the cytotoxic drug. Therefore, this dual-labeled ADC can help overcome cancer resistance to ADCs more effectively than traditional ADCs.

The term "antibody" is used herein in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, so long as they exhibit required biological activity. The terms "antibody" and "antibodies" broadly include naturally occurring antibody forms (eg, IgG, IgA, IgM, IgE).

The antibody is preferably a monoclonal antibody. Antibodies can be of human origin, but can also be derived from mice, rats, goats, donkeys, hamsters or rabbits. Where the conjugate is used therapeutically, the murine or rabbit antibody can optionally be chimeric or humanized.

Fragments or recombinant variants of antibodies containing a _CH2 domain may be, for example,

· Antibody formats containing only the heavy chain domain (shark antibody/IgNAR(VH- _CH 1- _CH 2- _CH 3- _CH 4- _CH 5) ₂ or camel antibody/hcIgG (VH- _CH 2 -C _H 3) ₂ )

·Single chain antibody (scFV)-Fc(VH-VL-C _H 2-C _H 3) ₂

• Fc fusion peptides, comprising an Fc domain and one or more receptor domains.

Antibodies may also be bispecific (eg, DVD-IgG, crossMab, appended IgG-HC fusion) or bispecific. See Brinkmann and Kontermann; Bispecific antibodies; DrugDiscov Today; 2015; 20(7); pp. 838-47, for review.

In a specific embodiment, the invention relates to a method according to the invention, wherein the antibody is an IgG antibody, in particular an IgG1 antibody.

"IgG" as used herein refers to a polypeptide belonging to the class of antibodies essentially encoded by the recognized immunoglobulin gamma gene. In humans, IgG includes the subclasses or isotypes IgG1, IgG2, IgG3, and IgG4. In mice, IgG includes IgG1, IgG2a, IgG2b, and IgG3. Full-length IgG consists of two identical pairs of two immunoglobulin chains, each pair has a light chain and a heavy chain, each light chain contains the immunoglobulin domains VL and CL, and each heavy chain contains the immunoglobulin structure Domains VH, Cγ1 (also known as CH1), Cγ2 (also known as CH2) and Oγ3 (also known as CH3). In the context of human IgG1, according to the EU index in Kabat, "CH1" refers to positions 118-215, the CH2 domain refers to positions 231-340, and the CH3 domain refers to positions 341-447. IgG1 also includes a hinge domain, which in the case of IgG1 refers to positions 216-230.

The antibody used in the method of the invention or the antibody-payload conjugate of the invention may be or comprise any antibody, preferably any antibody of the IgG type. For example, the antibody may be, but is not limited to, Brentuximab, Trastuzumab, Gemtuzumab, Inotuzumab, Avelumab (Avelumab), Cetuximab (Cetuximab), Rituximab (Rituximab), Daratumumab (Daratumumab), Pertuzumab (Pertuzumab), Vedolizumab (Vedolizumab), Ocrelizumab, Tocilizumab, Ustekinumab, Golimumab, Obinutuzumab, Saxituzumab (Sacituzumab), belantuzumab (Belantamab), polatuzumab (Polatuzumab) and enfortumab (Enfortumab).

Thus, in a specific embodiment, the present invention relates to a method according to the present invention, wherein the antibody is selected from the group consisting of: velbutuximab, trastuzumab, gemtuzumab, iodine Tocilizumab, avelumab, cetuximab, rituximab, daratumumab, pertuzumab, vedolizumab, ocrelizumab, tocilizumab Antibodies, ustekinumab, golimumab, otuzumab, sacetuzumab, belantuzumab, polotuzumab, and ennozumab.

In a specific embodiment, the present invention relates to a method according to the present invention, wherein the antibody is selected from the group consisting of: velbutuximab, gemtuzumab, trastuzumab, intuzumab monoclonal antibody, plotuzumab, ennozumab, sacerituzumab, and belantuzumab.

In a more preferred embodiment, the invention relates to a method according to the invention, wherein the antibody is polotuzumab or trastuzumab or ennozumab.

That is, in specific embodiments, the invention relates to antibody-linker conjugates, wherein the antibody is polotuzumab, and wherein the linker is any of the linkers disclosed herein.

In another embodiment, the invention relates to antibody-linker conjugates, wherein the antibody is trastuzumab, and wherein the linker is any of the linkers disclosed herein.

In another embodiment, the invention relates to antibody-linker conjugates, wherein the antibody is ennosumab, and wherein the linker is any of the linkers disclosed herein.

Antibodies used in the methods according to the invention may be glycosylated antibodies, deglycosylated antibodies or aglycosylated antibodies.

That is, in certain embodiments, the antibody may be an IgG antibody that is preferably glycosylated at residue N297. Thus, in a specific embodiment, the invention relates to a method according to the invention, wherein the IgG antibody is a glycosylated IgG antibody, in particular wherein the IgG antibody is present at residue N297 of the _CH2 domain (EU numbering ) is glycosylated.

As described herein, IgG antibodies glycosylated at residue N297 have several advantages over non-glycosylated antibodies.

However, the antibody may also be a deglycosylated antibody, preferably in which the glycan at residue N297 has been cleaved by the enzyme PNGase F. Further, the antibody may be a non-glycosylated antibody, preferably wherein residue N297 has been replaced by a non-asparagine residue. Methods of deglycosylating antibodies and generating non-glycosylated antibodies are known in the art.

In certain embodiments, the linkers of the invention can be coupled to endogenous Gln residues in the Fc domain of an antibody, or to Gln residues that have been introduced into the antibody through molecular engineering.

Thus, in a specific embodiment, the invention relates to a method according to the invention, wherein the linker-coupled Gln residue is comprised in the Fc domain of the antibody, in particular wherein the linker-coupled Gln residue is IgG Gln residue Q295 of the _CH2 domain of the antibody (EU numbering).

The linker of the present invention can be coupled to any Gln residue in the Fc domain of an antibody, and the antibody can be used as a substrate for microbial transglutaminase. Generally, the term Fc domain as used herein refers to the last two constant region immunoglobulin domains ( _CH2 and _CH3 ) of IgA, IgD and IgG and the last three constant region domains of IgE, IgY and IgM ( _CH 2, _CH 3 and _CH 4). That is, linkers according to the invention can be coupled to the _CH2 , _CH3 and _CH4 (where applicable) domains of antibodies.

In certain embodiments, the endogenous Gln residue may be Gln residue Q295 (EU numbering) of the CH2 domain of an IgG antibody. Thus, in a specific embodiment, the invention relates to a method according to the invention, wherein the Gln residue in the Fc domain of the antibody is Gln residue Q295 (EU numbering) of the CH2 domain of an IgG antibody.

It is important to understand that Q295 is an extremely conserved amino acid residue among IgG-type antibodies. It is conserved among human IgG1, 2, 3, and 4 as well as rabbit and rat antibodies. Therefore, the ability to use Q295 is a considerable advantage for the preparation of therapeutic antibody-payload conjugates or diagnostic conjugates, where the antibodies are often of non-human origin. Therefore, the method according to the invention indeed provides a very versatile and widely applicable tool. Although residue Q295 is extremely conserved among IgG-type antibodies, some IgG-type antibodies (such as mouse and rat IgG2a antibodies) do not have this residue. Accordingly, it will be understood that the antibody used in the method of the invention is preferably an IgG type antibody comprising residue Q295 (EU numbering) of the _CH2 domain.

Further, it has been shown that engineered conjugates using Q295 for payload attachment display good pharmacokinetics and efficacy (Lhospice et al., Site-specific conjugation of monomethyl auristatin E to anti-Cd30 antibodies Improved their pharmacokinetics and therapeutic index in rodent models (Site-Specific Conjugation of MonomethylAuristatin E to Anti-Cd30 Antibodies Improves Their Pharmacokinetics and Therapeutic Index in Rodent Models), Mol Pharm; 2015; 12(6), p. 1863-1871) and capable of carrying even unstable toxins prone to degradation (Dorywalska et al.; Site-dependent degradation of non-cleavable auristatin-based linker-payloads in rodent plasma and its implications for ADC efficacy Impact (Site-DependentDegradation of a Non-Cleavable Auristatin-Based Linker-Payload in RodentPlasma and Its Effect on ADC Efficacy), PLoS ONE; 2015; 10(7):e0132822). It was expected that similar effects would be seen with this site-specific approach, since the same residues would be modified instead of glycosylated antibodies. Glycosylation can further contribute to the overall stability of the ADC, as removal of glycan moieties as described above has been shown to result in reduced antibody stability (Zheng et al.; Effect of glycosylation on monoclonal antibody conformation and stability (The impact of glycosylation on monoclonal antibody conformation and stability), MabsAustin; 2011, 3(6), pp. 568-576).

In the literature discussing coupling of linkers to _CH2 -Gln residues by transglutaminase, the emphasis has been on small, low molecular weight substrates. However, in the prior art literature, in order to achieve such coupling, a deglycosylation step at position N297 or the use of aglycosylated antibody is always described as necessary (WO 2015/015448; WO 2017/025179; WO 2013/092998).

However, very surprisingly, and contrary to all expectations, site-specific coupling of Q295 to glycosylated antibodies is indeed efficient by using the linker structure described above. In particular, coupling of linkers containing toxin molecules was achieved with a coupling efficiency greater than 80%.

Even though Q295 is very close to N297, which is glycosylated in its native state, the methods of the present invention still allow linkers or payloads to be coupled to it using the specified linkers.

As shown, the method according to the invention does not require prior enzymatic deglycosylation of N297, nor the use of non-glycosylated antibodies, nor the substitution of N297 against another amino acid, nor the introduction of the T299A mutation to prevent Glycosylation.

These two points provide significant advantages in manufacturing. In terms of GMP, an enzymatic deglycosylation step is undesirable as it must ensure that both the deglycosylation enzyme (e.g. PNGase F) and the cleaved glycans must be removed from the culture medium.

Furthermore, genetic engineering of the antibody for payload attachment is not required, thus avoiding sequence insertions that could increase immunogenicity and reduce the overall stability of the antibody.

Substitution N297 for another amino acid also has undesirable effects as it may affect the overall stability of the entire Fc domain (Subedi et al., The Structural Role of Antibody N-glycosylation in Receptor Interactions) N-Glycosylation in Receptor Interactions), Structure2015, 23(9), 1573-1583), as well as the efficacy of the entire conjugate, the consequences of which may lead to increased antibody aggregation and reduced solubility (Zheng et al., Effect of glycosylation on monoclonal antibodies The impact of glycosylation on monoclonal antibody conformation and stability, Mabs Austin 2011, 3(6), 568-576), which is particularly important for hydrophobic payloads (such as PBD). Furthermore, the glycan present at N297 has important immunomodulatory effects because it triggers antibody-dependent cellular cytotoxicity (ADCC), etc. These immunomodulatory effects will be lost upon deglycosylation or any of the other methods described above to obtain non-glycosylated antibodies. Further, any sequence modifications to established antibodies may also lead to regulatory issues, which is problematic because generally accepted and clinically validated antibodies are used as the starting point for ADC coupling.

The method according to the invention therefore allows the easy and without disadvantages production of stoichiometrically well-defined ADCs with site-specific payload binding.

In view of the above, it is pointed out that the method of the present invention is preferably used for the coupling of IgG antibodies at residue Q295 (EU numbering) of the _CH 2 domain of the antibody, wherein the antibody is at residue N297 (EU numbering) of the _CH 2 domain No.) is glycosylated. However, it is expressly stated that the methods of the invention also encompass the coupling of deglycosylated or non-glycosylated antibodies at residue Q295 or any other suitable Gln residue of the antibody, where the Gln residue may be endogenous Gln residues or Gln residues introduced through molecular engineering.

Thus, in a specific embodiment, the invention relates to a method according to the invention, wherein the Gln residue coupled to the linker has been introduced into the heavy or light chain of the antibody by molecular engineering.

The term "molecular engineering" as used herein refers to the use of molecular biology methods to manipulate nucleic acid sequences. In the present invention, molecular engineering can be used to introduce Gln residues into the heavy or light chain of an antibody. In general, two different strategies for introducing Gln residues into the heavy or light chain of an antibody are contemplated in the present invention. First, a single residue of the heavy or light chain of the antibody can be replaced by a Gln residue. Second, a Gln-containing peptide tag consisting of two or more amino acid residues can be integrated into the heavy or light chain of an antibody. For this purpose, the peptide tag can be integrated into an internal position of the heavy or light chain, i.e. between two existing amino acid residues of the heavy or light chain or by replacing them, or the peptide tag can be fused (attached) to the heavy chain of the antibody. chain or light chain N-terminus or C-terminus.

For example, the amino residues of the heavy or light chain of the antibody can be substituted with Gln residues, provided that the resulting antibody can be coupled to the linker of the invention by a microbial transglutaminase. In certain embodiments, the antibody is an antibody in which amino acid residue N297 (EU numbering) of the _CH2 domain of the IgG antibody is substituted, particularly wherein the substitution is an N297Q substitution. Antibodies containing the N297Q mutation can be coupled to more than one linker on each heavy chain of the antibody. For example, an antibody containing the N297Q mutation can be coupled to four linkers, one linker coupled to residue Q295 of the first heavy chain of the antibody, one linker coupled to residue Q297Q of the first heavy chain of the antibody, and one linker coupled to residue Q297Q of the first heavy chain of the antibody. A linker is coupled to residue Q295 of the second heavy chain of the antibody, and one linker is coupled to residue N297Q of the second heavy chain of the antibody. Those skilled in the art are aware that replacement of residue N297 of an IgG antibody with a Gln residue results in a non-glycosylated antibody.

Therefore, in a specific embodiment, the invention relates to a method according to the invention, wherein the Gln residue introduced by molecular engineering into the heavy or light chain of the antibody is N297Q of the _CH2 domain of a non-glycosylated IgG antibody (EU number).

In a specific embodiment, the invention relates to a method according to the invention, wherein the Gln residue introduced by molecular engineering into the heavy or light chain of the antibody is comprised in a peptide which has been (a) integrated into the heavy or light chain of the antibody. chain or light chain or (b) fused to the N-terminus or C-terminus of the heavy or light chain of the antibody.

Instead of replacing a single amino acid residue of the antibody, a peptide tag containing a transglutaminase-accessible Gln residue can be introduced into the heavy or light chain of the antibody. Such peptide tags can be fused to the N-terminus or C-terminus of the heavy or light chain of the antibody. Alternatively, the peptide tag can be inserted into the heavy or light chain of the antibody at a suitable location. Preferably, a peptide tag comprising a transglutaminase accessible Gln residue is fused to the C-terminus of the heavy chain of the antibody. Even more preferably, a peptide tag comprising a transglutaminase accessible Gln residue is fused to the C-terminus of the heavy chain of the IgG antibody. Several peptide tags that can be fused to the C-terminus of the heavy chain of an antibody and used as substrates for microbial transglutaminases are described in WO 2012/059882 and WO 2016/144608.

Therefore, in a specific embodiment, the invention relates to a method according to the invention, wherein a peptide comprising a Gln residue has been fused to the C-terminus of the heavy chain of the antibody.

Exemplary peptide tags that can be introduced into the heavy or light chain of an antibody, particularly fused to the C-terminus of the antibody heavy chain, are LLQGG (SEQ ID NO: 16), LLQG (SEQ ID NO: 17), LSLSQG (SEQ ID NO:18), GGGLLQGG (SEQ ID NO:19), GLLQG (SEQ ID NO:20), LLQ (SEQ ID NO:21), GSPLAQSHGG (SEQ ID NO:22), GLLQGGG (SEQ ID NO:23), GLLQGG (SEQ ID NO:24), GLLQ (SEQ ID NO:25), LLQLLQGA (SEQ ID NO:26), LLQGA (SEQ ID NO:27), LLQYQGA (SEQ ID NO:28), LLQGSG (SEQ ID NO: 29), LLQYQG (SEQ ID NO:30), LLQLLQG (SEQ ID NO:31), SLLQG (SEQ ID NO:32), LLQLQ (SEQ ID NO:33), LLQLLQ (SEQ ID NO:34), LLQGR (SEQ ID NO:35), EEQYASTY(SEQ ID NO:36), EEQYQSTY(SEQ ID NO:37), EEQYNSTY(SEQ ID NO:38), EEQYQS(SEQ ID NO:39), EEQYQST(SEQ ID NO:40), EQYQSTY(SEQ ID NO:41), QYQS(SEQ ID NO:42), QYQSTY(SEQ ID NO:43), YRYRQ(SEQ ID NO:44), DYALQ(SEQ ID NO:45), FGLQRPY(SEQ ID NO: 46), EQKLISEEDL (SEQ ID NO:47), LQR (SEQ ID NO:48) and YQR (SEQ ID NO:49).

Those skilled in the art are aware of the substitution of amino acid residues of antibodies by methods of molecular cloning, for example, as described in Sambrook, Joseph. (2001). Molecular cloning: a laboratory manual, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press. methods of introducing peptide tags into antibodies.

Generally, those skilled in the art are aware of methods for determining where on an antibody a linker is coupled. For example, the conjugation site can be determined by proteolytic digestion of the antibody-payload conjugate and LC-MS analysis of the resulting fragments. For example, samples can be deglycosylated with GlyciNATOR (Genovis) according to instructions and subsequently digested separately with trypsin gold (mass spectrometry grade, Promega). Therefore, 1 μg of protein can be incubated with 50 ng of trypsin overnight at 37°C. LC-MS analysis can be performed using a nanoAcquity HPLC system coupled to a Synapt-G2 mass spectrometer (Waters). To do this, 100 ng of peptide solution can be loaded onto an Acquity UPLC Symmetry C18 capture column (Waters, part no. 186006527) and mixed with a flow rate of 5 μL/min in 1% Buffer A (water, 0.1% formic acid) and 99% Buffer B ( Acetonitrile, 0.1% formic acid) for 3 min. The peptide can then be eluted with a linear gradient from 3% to 65% buffer B over 25 min. Data can be acquired in resolution mode with positive polarity and in the mass range from 50 to 2000 m/z. Other instrument settings can be as follows: capillary voltage 3.2 kV, sampling cone 40 V, extraction cone 4.0 V, source temperature 130°C, cone gas 35 L/h, nanoflow gas 0.1 bar, and purge gas 150 L/h. The mass spectrometer can be calibrated with [Glu1]-fibrin peptide.

Further, those skilled in the art are aware of methods for determining the drug to antibody (DAR) ratio or payload to antibody ratio of an antibody-payload construct. For example, DAR can be determined by hydrophobic interaction chromatography (HIC) or LC-MS.

For hydrophobic interaction chromatography (HIC), samples can be adjusted to 0.5M ammonium sulfate and used in solutions ranging from A (1.5M ammonium sulfate, 25mm Tris-HCl, pH 7.5) to B (20% isopropanol, 25mm-Tris -HCl, pH 7.5), was evaluated through a MAB PAK HIC butyl column (5 μM, 4.6x 100 mm, Thermo Scientific) over 20 min at 1 mL/min and 30°C. Typically, 40 μg of sample can be used and the signal can be recorded at 280 nm. The relative HIC retention time (HIC-RRT) can be calculated by dividing the absolute retention time of the ADC DAR2 species by the retention time of the corresponding unconjugated mAb.

For LC-MS DAR determination, the ADC can be diluted with NH ₄ HCO ₃ to a final concentration of 0.025 mg/mL. Subsequently, 40 μL of the solution can be reduced with 1 μL of TCEP (500mM) for 5 min at room temperature, then alkylated by adding 10 μL of chloroacetamide (200mM), and then incubated in the dark at 37°C overnight. For reversed-phase chromatography, the Dionex U3000 system can be used in combination with Chromeleon software. The system can be equipped with an RP-1000 column heated to 70°C ( 5 μm, 1.0 × 100 mm, Sepax) and a UV detector set to a wavelength of 214 nm. Solvent A may consist of water containing 0.1% formic acid, while solvent B may include 85% acetonitrile containing 0.1% formic acid. Reduced and alkylated samples can be loaded onto the column and separated by a gradient from 30-55% solvent B in 14 minutes. The liquid chromatography system can be connected to the Synapt-G2 mass spectrometer for identification of DAR species. The capillary voltage of the mass spectrometer can be set to 3kV, the sampling cone can be set to 30V, and the extraction cone can add up to a value of 5V. The source temperature can be set to 150°C, the desolvation temperature to 500°C, the cone gas to 20l/h, the desolvation gas to 600l/h, and acquisition and scanning can be performed in positive mode over a mass range of 600-5000Da The time is 1s. The instrument can be calibrated with sodium iodide. Deconvolution of the spectra can be performed using MassLynx's MaxEnt1 algorithm until convergence. After assigning DAR species to chromatographic peaks, DAR can be calculated based on the integrated peak area of reversed-phase chromatography.

In a specific embodiment, the invention relates to a method according to the invention, wherein the linker is coupled to the γ-carboxamide group of a Gln residue contained in the antibody.

That is to say, the linker according to the present invention is preferably coupled to the amide group in the side chain of the Gln residue contained in the antibody, preferably any one of the Gln residues disclosed herein, more preferably Gln residue Q295 (EU numbering ).

In a particular embodiment, the invention relates to a method according to the invention, wherein the linker is adapted to be present in an amount of at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95% coupling efficiency for coupling to glycosylated antibodies.

That is, in certain embodiments, the linker may be capable of operating with at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% Linkers for efficient coupling to glycosylated antibodies. In preferred embodiments, the linker may be a linker capable of coupling to the glycosylated antibody with at least 70% efficiency. In another preferred embodiment, the linker may be a linker capable of coupling to the glycosylated antibody with at least 75% efficiency. In another preferred embodiment, the linker may be a linker capable of coupling to the glycosylated antibody with at least 80% efficiency. In another preferred embodiment, the linker may be a linker capable of coupling to the glycosylated antibody with at least 85% efficiency. In another preferred embodiment, the linker may be a linker capable of coupling to the glycosylated antibody with at least 90% efficiency. In another preferred embodiment, the linker may be a linker capable of coupling to the glycosylated antibody with at least 95% efficiency. Preferably, the glycosylated antibody is a glycosylated IgG antibody, more preferably an IgG antibody glycosylated at residue N297 (EU numbering).

Those skilled in the art are aware of methods to determine the efficiency of coupling an antibody to a specific linker. For example, coupling efficiency can be determined as described herein. That is, antibodies, particularly IgG1 antibodies, can be prepared at 37°C or as described in Example 1 in a suitable buffer at a concentration of 1-5 mg/mL with 5-20 eq molar equivalents of linker per mg of antibody and Incubate with 3-6U of microbial transglutaminase for 20-48 hours. After the incubation period, coupling efficiency can be determined by LC-MS analysis under reducing conditions. The microbial transglutaminase may be MTG from Streptomyces moehara available from Zedira (Germany). Suitable buffers may be Tris, MOPS, HEPES, PBS or BisTris buffer. However, it should be understood that the choice of buffer system may vary and is highly dependent on the chemistry of the linker. However, one skilled in the art will be able to identify optimal buffering conditions based on this disclosure. Alternatively, the coupling efficiency can be improved as shown by Spycher et al. (Dual, Site-Specific Modification of Antibodies by Using Solid-Phase Immobilized MicrobialTransglutaminase) , ChemBioChem 2019 18(19):1923-1927) The assay was performed as described in Benjamin et al. (Thiolation of Q295: Site-specific conjugation of hydrophobic payloads that do not require genetic engineering) Specific Conjugation of Hydrophobic Payloads without the Need for Genetic Engineering), Mol.Pharmaceutics 2019, 16:2795-2807) for analysis.

In certain embodiments, antibodies can be coupled as described in Example 1. That is, 5 mg/ml of native glycosylated monoclonal antibody can be incubated for 24 hours at 37°C in a rotary thermomixer in 50 mM Tris pH 7.6 containing a concentration of 5 U/mg of antibody. of microbial transglutaminase (MTG, Zedira) and 5 molar equivalents of the indicated linker-payload.

In a specific embodiment, the invention relates to a method according to the invention, wherein the microbial transglutaminase is derived from the genus Streptomyces, in particular Streptomyces moehara.

That is, the microbial transglutaminase used in the method of the present invention may be derived from the genus Streptomyces (in particular, derived from Streptomyces moehara), preferably having 80% sequence identity with the native enzyme. Therefore, MTG can be a native enzyme, or it can be an engineered variant of a natural enzyme.

Such microbial transglutaminase is commercially available from Zedira (Germany). It is produced recombinantly in E. coli. Streptomyces moehara transglutaminase has the amino acid sequence disclosed in SEQ ID NO:12. S. moehara MTG variants with other amino acid sequences have been reported and are also included in the present invention (SEQ ID NO: 13 and 14).

In another embodiment, a microbial transglutaminase from Streptomyces ladakanum (formerly Streptoverticillium ladakanum) may be used. Streptomyces dakari transglutaminase (US Patent No. US 6660510 B2) has the amino acid sequence disclosed in SEQ ID NO: 15.

Both of the above transglutaminase enzymes can undergo sequence modification. In several embodiments, a transglutaminase having 80%, 85%, 90%, or 95% or more sequence identity to any one of SEQ ID NOs: 12-15 may be used.

Another suitable microbial transglutaminase is commercially available from Ajinomoto as ACTIVA TG. Compared to Zedira's transglutaminase, ACTIVA TG lacks the 4 N-terminal amino acids but has similar activity.

Other microbial transformations useful in the present invention are disclosed in Kieliszek and Misiewicz (Folia Microbiol (Praha). 2014;59(3):241–250), WO2015/191883A1, WO2008/102007A1 and US 2010/0143970 Aminamidase, the contents of which are fully incorporated herein by reference.

In certain embodiments, mutant variants of microbial transglutaminase enzymes can be used to couple linkers to antibodies. That is, the microbial transglutaminase used in the method of the invention may be a variant of the Streptomyces moehara transglutaminase as listed in SEQ ID NO: 12 or 13. In certain embodiments, a recombinant Streptomyces moehara transglutaminase as set forth in SEQ ID NO: 12 can comprise mutation G254D. In certain embodiments, a recombinant Streptomyces moehara transglutaminase as set forth in SEQ ID NO: 12 can comprise mutations G254D and E304D. In certain embodiments, a recombinant Streptomyces moehara transglutaminase as set forth in SEQ ID NO: 12 can comprise mutations D8E and G254D. In certain embodiments, a recombinant Streptomyces moehara transglutaminase as set forth in SEQ ID NO: 12 can comprise mutations E124A and G254D. In certain embodiments, a recombinant Streptomyces moehara transglutaminase as set forth in SEQ ID NO: 12 can comprise mutations A216D and G254D. In certain embodiments, a recombinant Streptomyces moehara transglutaminase as set forth in SEQ ID NO: 12 can comprise mutations G254D and K331T.

Microbial transglutaminase can be added to the coupling reaction at any concentration that allows efficient binding of the antibody to the linker. In certain embodiments, the concentration of microbial transglutaminase in a coupling reaction can depend on the amount of antibody used in the same reaction. For example, microbial transglutaminase can be administered at less than 100 U/mg antibody, 90 U/mg antibody, 80 U/mg antibody, 70 U/mg antibody, 60 U/mg antibody, 50 U/mg antibody, 40 U/mg antibody, 30 U/mg Concentrations of antibody, 20U/mg antibody, 10U/mg antibody, or 6U/mg antibody were added to the coupling reaction. In certain embodiments, microbial transglutaminase can be added to the coupling reaction at a concentration of 1, 3, 5, or 6 U/mg of antibody.

That is, in certain embodiments, the microbial transglutaminase can be administered at 1-20 U/mg antibody, preferably 1-10 U/mg antibody, more preferably 1-7.5 U/mg antibody, even more preferably 2- A concentration of 6 U/mg antibody, even more preferably 2-4 U/mg antibody or most preferably 3 U/mg antibody is added to the coupling reaction.

The method according to the invention involves the use of microbial transglutaminase. It should be noted, however, that equivalent reactions can be performed by enzymes of non-microbial origin containing transglutaminase activity. Therefore, the antibody-linker conjugates of the invention can also be produced using enzymes of non-microbial origin that contain transglutaminase activity.

Antibodies can be added to the coupling reaction at any concentration. However, it is preferred to add the antibody to the coupling reaction at a concentration of 0.1-20 mg/ml. That is, in a specific embodiment, the present invention relates to a method according to the present invention, wherein the antibody is present at 0.1-20 mg/mL, preferably 0.25-15 mg/mL, more preferably 0.5-12.5 mg/mL, even more preferably 1 - A concentration of 10 mg/mL, even more preferably 2-7.5 mg/mL, most preferably about 5 mg/mL is added to the coupling reaction.

Alternatively, the antibody may be added to the coupling reaction at a concentration of 1-20 mg/ml, preferably 2.5-20 mg/ml, more preferably 5-20 mg/ml, most preferably 5-17 mg/ml.

To obtain efficient coupling, the linker is preferably added to the antibody in molar excess. That is, in certain embodiments, the antibody is mixed with at least 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 molar equivalents of linker.

That is, in a specific embodiment, the invention relates to a method according to the invention, wherein the antibody is combined with 2-100 molar equivalents of a linker, preferably 2-80 molar equivalents of a linker, more preferably 2-70 molar equivalents of a linker , even more preferably 2-60 molar equivalents of linkers, even more preferably 2-50 molar equivalents of linkers, even more preferably 2-40 molar equivalents of linkers, even more preferably 2-30 molar equivalents of linkers, even more preferably 2- 25 molar equivalents of linker, even more preferably 2-20 molar equivalents of linker, even more preferably 2-15 molar equivalents of linker, most preferably 2-10 molar equivalents of linker are contacted.

Alternatively, the antibody may be combined with 2.5-100 molar equivalents of linker, preferably 2.5-80 molar equivalents of linker, more preferably 2.5-70 molar equivalents of linker, even more preferably 2.5-60 molar equivalents of linker, even more preferably 2.5- 50 molar equivalents of linker, even more preferably 2.5-40 molar equivalents of linker, even more preferably 2.5-30 molar equivalents of linker, even more preferably 2.5-20 molar equivalents of linker, even more preferably 2.5-15 molar equivalents of linker, Even more preferably 2.5 to 10 molar equivalents of the linker are contacted, most preferably 2.5 to 8 molar equivalents of the linker are contacted.

Alternatively, the antibody may be combined with 5-100 molar equivalents of linker, preferably 5-80 molar equivalents of linker, more preferably 5-70 molar equivalents of linker, even more preferably 5-60 molar equivalents of linker, even more preferably 5- 50 molar equivalents of linker, even more preferably 5-40 molar equivalents of linker, even more preferably 5-30 molar equivalents of linker, even more preferably 5-20 molar equivalents of linker, even more preferably 5-15 molar equivalents of linker, Most preferred are 5-10 molar equivalent linker contacts.

The method according to the invention is preferably carried out in a pH range of 6 to 9. Therefore, in a preferred embodiment, the invention relates to a method according to the invention, wherein the coupling of the linker to the antibody is in the pH range of 6 to 8.5, more preferably in the pH range of 6.5 to 8, even more preferably in Achieved within a pH range of 7 to 8. In a most preferred embodiment, the invention relates to a method according to the invention, wherein coupling of the linker to the antibody is achieved at pH 7.6.

The method of the present invention can be performed in any buffer suitable for coupling the payload to the linker. Buffers suitable for use in the methods of the invention include, but are not limited to, Tris, MOPS, HEPES, PBS or BisTris buffers. The concentration of the buffer depends inter alia on the concentration of the antibody and/or linker and may vary in the range of 10-1000mM, 10-500mM, 10-400mM, 10-250mM, 10-150mM or 10-100mM. Further, the buffer may include any salt concentration suitable for carrying out the methods of the invention. For example, the buffer used in the method of the present invention may have ≤150mM, ≤140mM, ≤130mM, ≤120mM, ≤110mM, ≤100mM, ≤90mM, ≤80mM, ≤70mM, ≤60mM, ≤50mM, ≤40mM, ≤30mM , a salt concentration of ≤20mM or ≤10mM, or no salt. In a specific embodiment, the method of the invention is carried out in 50mM Tris (pH 7.6), preferably without salt.

It is important to note that optimal reaction conditions (e.g., pH, buffer, salt concentration) may vary from payload to payload and depend to some extent on the physicochemical properties of the linker and/or payload. However, one skilled in the art will not need undue experimentation to determine reaction conditions suitable for carrying out the method of the present invention.

It is to be understood that the present application includes any combination of the above disclosed linkers, antibody MTG and/or buffer concentrations.

In a preferred embodiment, the present invention relates to a method for producing an antibody-linker conjugate by microbial transglutaminase (MTG), the method comprising adding (as shown in the N→C direction) (Sp ₁ a step of coupling a linker of the )-RK-(Sp ₂ )-B-(Sp ₃ ) or (Sp ₁ )-B-(Sp ₂ )-RK-(Sp ₃ ) structure to a Gln residue contained in the antibody, in,

·(Sp ₁ ) is a chemical spacer or does not exist;

·(Sp ₂ ) is a chemical spacer or does not exist;

·(Sp ₃ ) is a chemical spacer or does not exist;

·R is arginine or arginine derivative or arginine mimetic;

·K is lysine or lysine derivative or lysine mimetic;

·B is the connection part or payload;

wherein the linker is coupled to a Gln residue comprised in the antibody via a primary amine comprised in the side chain of a lysine residue, lysine derivative or lysine mimetic; and

wherein the antibody is contacted with 2-80 molar equivalents of the linker; and/or

wherein the microbial transglutaminase is added to the coupling reaction at a concentration of 1-20 U/mg antibody, and optionally, wherein the antibody is added to the coupling reaction at a concentration of 0.1-20 mg/mL.

In a more preferred embodiment, the present invention relates to a method for producing an antibody-linker conjugate by microbial transglutaminase (MTG), the method comprising adding (as shown in the N→C direction) (Sp ₁ ) The step of coupling the linker of the structure of -RK-(Sp ₂ )-B-(Sp ₃ ) or (Sp ₁ )-B-(Sp ₂ )-RK-(Sp ₃ ) to the Gln residue contained in the antibody ,in,

·(Sp ₁ ) is a chemical spacer or does not exist;

·(Sp ₂ ) is a chemical spacer or does not exist;

·(Sp ₃ ) is a chemical spacer or does not exist;

·R is arginine or arginine derivative or arginine mimetic;

·K is lysine or lysine derivative or lysine mimetic;

·B is the connection part or payload;

wherein the linker is coupled to a Gln residue comprised in the antibody via a primary amine comprised in the side chain of a lysine residue, lysine derivative or lysine mimetic; and

wherein the antibody is contacted with 2-50 molar equivalents of the linker; and/or

wherein the microbial transglutaminase is added to the coupling reaction at a concentration of 1-10 U/mg of antibody, and optionally, wherein the antibody is added to the coupling reaction at a concentration of 1-20 mg/mL.

In an even more preferred embodiment, the present invention relates to a method for the production of antibody-linker conjugates by microbial transglutaminase (MTG), the method comprising adding (as shown in the N→C direction) ( The linker of Sp ₁ )-RK-(Sp ₂ )-B-(Sp ₃ ) or (Sp ₁ )-B-(Sp ₂ )-RK-(Sp ₃ ) structure is coupled to the Gln residue contained in the antibody. steps, where,

·(Sp ₁ ) is a chemical spacer or does not exist;

·(Sp ₂ ) is a chemical spacer or does not exist;

·(Sp ₃ ) is a chemical spacer or does not exist;

·R is arginine or arginine derivative or arginine mimetic;

·K is lysine or lysine derivative or lysine mimetic;

·B is the connection part or payload;

wherein the linker is coupled to a Gln residue comprised in the antibody via a primary amine comprised in the side chain of a lysine residue, lysine derivative or lysine mimetic; and

wherein the antibody is contacted with 2-30 molar equivalents of the linker; and/or

wherein microbial transglutaminase is added to the coupling reaction at a concentration of 2-10 U/mg antibody, and optionally, wherein the antibody is added to the coupling reaction at a concentration of 5-20 mg/mL.

In an even more preferred embodiment, the present invention relates to a method for the production of antibody-linker conjugates by microbial transglutaminase (MTG), the method comprising adding (as shown in the N→C direction) ( The linker of Sp ₁ )-RK-(Sp ₂ )-B-(Sp ₃ ) or (Sp ₁ )-B-(Sp ₂ )-RK-(Sp ₃ ) structure is coupled to the Gln residue contained in the antibody. steps, where,

·(Sp ₁ ) is a chemical spacer or does not exist;

·(Sp ₂ ) is a chemical spacer or does not exist;

·(Sp ₃ ) is a chemical spacer or does not exist;

·R is arginine or arginine derivative or arginine mimetic;

·K is lysine or lysine derivative or lysine mimetic;

·B is the connection part or payload;

wherein the linker is coupled to a Gln residue comprised in the antibody via a primary amine comprised in the side chain of a lysine residue, lysine derivative or lysine mimetic; and

wherein the antibody is contacted with about 2-20 molar equivalents of the linker; and/or wherein the microbial transglutaminase is added to the coupling reaction at a concentration of 2-10 U/mg antibody, and optionally, wherein the antibody is Add to the coupling reaction at a concentration of 5-20 mg/mL.

In an even more preferred embodiment, the present invention relates to a method for the production of antibody-linker conjugates by microbial transglutaminase (MTG), the method comprising adding (as shown in the N→C direction) ( The linker of Sp ₁ )-RK-(Sp ₂ )-B-(Sp ₃ ) or (Sp ₁ )-B-(Sp ₂ )-RK-(Sp ₃ ) structure is coupled to the Gln residue contained in the antibody. steps, where,

·(Sp ₁ ) is a chemical spacer or does not exist;

·(Sp ₂ ) is a chemical spacer or does not exist;

·(Sp ₃ ) is a chemical spacer or does not exist;

·R is arginine or arginine derivative or arginine mimetic;

·K is lysine or lysine derivative or lysine mimetic;

·B is the connection part or payload;

wherein the linker is coupled to a Gln residue comprised in the antibody via a primary amine comprised in the side chain of a lysine residue, lysine derivative or lysine mimetic; and

wherein the antibody is contacted with about 2.5-15 molar equivalents of the linker; and/or wherein the microbial transglutaminase is added to the coupling reaction at a concentration of 2-10 U/mg antibody, and optionally, wherein the antibody is Add to the coupling reaction at a concentration of 5-20 mg/mL.

In a most preferred embodiment, the present invention relates to a method for producing an antibody-linker conjugate by microbial transglutaminase (MTG), the method comprising adding (as shown in the N→C direction) (Sp ₁ ) The step of coupling the linker of the structure of -RK-(Sp ₂ )-B-(Sp ₃ ) or (Sp ₁ )-B-(Sp ₂ )-RK-(Sp ₃ ) to the Gln residue contained in the antibody ,in,

·(Sp ₁ ) is a chemical spacer or does not exist;

·(Sp ₂ ) is a chemical spacer or does not exist;

·(Sp ₃ ) is a chemical spacer or does not exist;

·R is arginine or arginine derivative or arginine mimetic;

·K is lysine or lysine derivative or lysine mimetic;

·B is the connection part or payload;

wherein the linker is coupled to a Gln residue comprised in the antibody via a primary amine comprised in the side chain of a lysine residue, lysine derivative or lysine mimetic; and

wherein the antibody is contacted with about 2.5-10 molar equivalents of the linker; and/or wherein the microbial transglutaminase is added to the coupling reaction at a concentration of 2-10 U/mg antibody, and optionally, wherein the antibody is Add to the coupling reaction at a concentration of 5-20 mg/mL.

In a specific embodiment, the invention relates to antibody-linker conjugates prepared using the methods of the invention.

That is, the present invention relates to an antibody-linker conjugate produced by any of the above steps.

In a specific embodiment, the invention relates to an antibody-linker conjugate comprising:

a) Antibodies; and

b) Joint, which includes the following structures:

(Sp ₁ )-RK-(Sp ₂ )-B-(Sp ₃ ) or

(Sp ₁ )-B-(Sp ₂ )-RK-(Sp ₃ ); where

·(Sp ₁ ) is a chemical spacer or does not exist;

·(Sp ₂ ) is a chemical spacer or does not exist;

·(Sp ₃ ) is a chemical spacer or does not exist;

·R is arginine or arginine derivative or arginine mimetic;

·K is lysine or lysine derivative or lysine mimetic;

·B is the connection part or payload;

Wherein, the linker is coupled to the antibody via an isopeptide bond between the γ-carboxamide group of the glutamine residue included in the antibody and the lysine residue included in the RK motif included in the linker, Formed between primary amines contained in the side chains of lysine derivatives or lysine mimetics.

That is, the invention further relates to antibody-linker conjugates that have been produced using the methods of the invention. In particular, the invention relates to antibodies coupled at a glutamine residue contained in the heavy or light chain of the antibody to any of the linkers disclosed herein for use in the methods of the invention. That is, all linkers that have been disclosed above for use in the methods of the invention can be included in the antibody-linker constructs of the invention. Preferably, the linker of the invention is coupled to a glutamine residue in the antibody via an amide bond between the amide side chain of the glutamine residue comprised in the antibody and a residue comprised in the RK motif of the linker Formed between primary amines contained in K. In certain embodiments, the primary amine comprised in residue K is an amine group comprised in the side chain of a lysine residue, lysine mimetic, or lysine derivative, as disclosed herein. In certain embodiments, K is a lysine residue and the primary amine via which the linker is coupled to the antibody is an epsilon-amino group contained in the lysine residue.

The chemical spacer included in the antibody-linker constructs disclosed herein can be any of the RK-containing linkers disclosed herein. That is, the joint may be a joint that includes a single connecting portion or payload B, or it may be a joint that includes two or more connecting portions and/or payloads B ₁ , B _{2 ,} etc.

In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the chemical spacers (Sp ₁ ), (Sp ₂ ) and (Sp ₃ ) each independently comprise from 0 to 12 amino acid residues. base.

In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the linker comprises no more than 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 , 5, 4 amino acid residues.

In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the net charge of the linker is neutral or positive.

In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the linker does not comprise negatively charged amino acid residues.

In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the linker comprises an amino acid sequence selected from the group consisting of: RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO:3) and RKR (SEQ ID NO:4).

In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the linker comprises an amino acid sequence selected from the group consisting of: RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2) and ARK (SEQ ID NO:3).

In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the linker comprises the amino acid sequence RKAA (SEQ ID NO: 1).

In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the linker comprises the amino acid sequence RK-Val-Cit (SEQ ID NO: 54).

In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein B is a linker moiety.

In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein linking moiety B comprises

·Bioorthogonal labeling groups, or

· Non-bioorthogonal entities for cross-linking.

In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the bioorthogonal labeling group or the non-bioorthogonal entity for cross-linking consists of at least one molecule or moiety or contains at least A molecule or moiety selected from the group consisting of:

·–NN≡N or –N ₃ ;

·Lys(N ₃ );

·Tetrazine;

·Alkynes;

·Strained cyclooctyne;

·BCN;

·Strained olefin;

·Photoreactive groups;

·aldehyde;

·Acyl trifluoroborate;

·Protein degradation agent ('PROTAC');

·Cyclopentadiene/spirocyclopentadiene;

·Thio-selective electrophile;

·-SH; and

·Cysteine.

That is, the antibody-linker conjugate according to the present invention may be an antibody coupled to a linker comprising one or more linking moieties. Such antibody-linker conjugates can later be customized with one or more payloads, particularly with payloads capable of being adapted for coupling to one or more linking moieties.

In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein one or more payloads have been coupled to linker B.

In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein one or more payloads have been coupled to linker B via a click reaction.

That is, the antibody-linker conjugate according to the invention may be an antibody-payload conjugate produced in the two-step process disclosed herein.

In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein B is the payload.

In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the payload comprises at least one of the following:

·toxin;

·Cytokines;

·Growth factors;

·Radionuclides;

·hormone;

·Antiviral agents;

·Antibacterial agents;

·Fluorescent dyes;

·Immune modulators/immunostimulants;

·Half-life increased part;

·Solubility increased part;

·Polymer-toxin conjugates;

·Nucleic acid;

·Biotin or streptavidin moiety;

·Vitamins;

·Protein degradation agent (‘PROTAC’);

·Target binding moiety; and/or

·Anti-inflammatory agent.

In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the toxin is selected from at least one of the group consisting of:

·pyrrolobenzodiazepines (e.g., PBD);

·Auristatin (such as MMAE, MMAF);

Maytansinoids (e.g., maytansinoids, DM1, DM4, DM21);

·Becarcinomycin;

·Nicotinamide phosphoribosyltransferase (NAMPT) inhibitor;

·Microtubulysin;

· Enedyne (e.g., calicheamicin);

·Anthracycline derivatives (PNU) (e.g., doxorubicin);

·Pyrrolyl kinesin spindle protein (KSP) inhibitor;

·Nodulin;

·Drug efflux pump inhibitors;

·Sandromycin;

·Amanitanic acid (e.g., alpha-amanitinine); and

· Camptothecins (e.g., ixotecan, drotecan).

In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the chemical spacer ( _Sp2 ) comprises a self-cleaving moiety.

In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the self-cleaving moiety is directly attached to the payload B.

In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the self-cleaving moiety comprises a p-aminobenzylcarbamoyl (PABC) moiety.

That is, the antibody-linker conjugate according to the invention may be an antibody-payload conjugate produced in the one-step method disclosed herein.

The antibody comprised in the antibody-linker conjugate according to the invention may be any one of the antibodies disclosed herein for use in the method according to the invention, in particular any one of the IgG type antibodies. That is, the antibodies comprised in the antibody-linker conjugates according to the invention may comprise the same glycosylation pattern, mutations and/or modifications as the antibodies disclosed herein for use in the methods according to the invention.

In a specific embodiment, the invention relates to an antibody-payload conjugate according to the invention, wherein the linker is Figure 1, Figure 2, Figure 3, Figure 8, Figure 9, Figure 14, Figure 15, Figure 17, Figure 18, Figure 19, Figure 20, Figure 21, Figure 22, Figure 23, Figure 24, Figure 25, Figure 26, Figure 27, Figure 28, Figure 29, Figure 30, Figure 31, Figure 32, Figure 33 or Figure 34 Any of the connectors shown in .

In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the antibody is an IgG antibody, in particular an IgG1 antibody.

In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the linker-coupled Gln residue is comprised in the Fc domain of the antibody, in particular wherein the linker-coupled Gln residue is Gln residue Q295 (EU numbering) of the _CH2 domain of an IgG antibody.

In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the Gln residue coupled to the linker has been introduced into the heavy or light chain of the antibody by molecular engineering.

In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the Gln residue introduced by molecular engineering into the heavy or light chain of the antibody is the _CH 2 domain of a non-glycosylated IgG antibody N297Q (EU number).

In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the Gln residue of the heavy or light chain of the antibody introduced by molecular engineering is comprised in a peptide which has been (a) integrated to the heavy or light chain of the antibody or (b) fused to the N-terminus or C-terminus of the heavy or light chain of the antibody.

In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein a peptide comprising a Gln residue has been fused to the C-terminus of the heavy chain of the antibody.

In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the IgG antibody is a glycosylated IgG antibody, in particular wherein the IgG antibody is present at residue N297 of the _CH 2 domain (EU No.) glycosylation.

In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the antibody is selected from the group consisting of: velbutuximab, trastuzumab, gemtuzumab, Ointuzumab, avelumab, cetuximab, rituximab, daratumumab, pertuzumab, vedolizumab, ocrelizumab, tom Tizumab, ustekinumab, golimumab, otuzumab, sacerituzumab, belantuzumab, polotuzumab, and ennozumab.

In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the antibody is selected from the group consisting of: velbutuximab, gemtuzumab, trastuzumab, Ointuzumab, polotuzumab, ennozumab, sacerituzumab, and belantuzumab.

In a more preferred embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the antibody is polotuzumab or trastuzumab or ennozumab.

In a specific embodiment, the invention relates to an antibody-drug conjugate. That is, the antibody can be coupled to a linker according to the invention, wherein the linker contains one or more toxins.

Thus, in a specific embodiment, the invention relates to an antibody-drug conjugate comprising:

a) IgG antibodies; and

b) A linker comprising drug moiety B, wherein drug moiety B is covalently linked to an amino acid sequence selected from the group consisting of: RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 2) NO:3) or RKR (SEQ ID NO:4);

Wherein, the linker is coupled to the IgG antibody via an isopeptide bond between the γ-carboxamide group of glutamine residue Q295 (EU numbering) of the _CH2 domain of the antibody and the lysine contained in the linker. Formed between primary amines contained in the side chains of acid residues.

In certain embodiments, the invention relates to an antibody-drug conjugate comprising:

a) IgG antibodies; and

b) a linker comprising drug moiety B, wherein drug moiety B is covalently linked to an amino acid sequence comprising or consisting of the sequence RK-Val-Cit (SEQ ID NO: 54);

Wherein, the linker is coupled to the IgG antibody via an isopeptide bond between the γ-carboxamide group of glutamine residue Q295 (EU numbering) of the _CH2 domain of the antibody and the lysine contained in the linker. Formed between primary amines contained in the side chains of acid residues.

That is, in certain embodiments, the linker may comprise the sequence RKAA (SEQ ID NO:1), RKA (SEQ ID NO:2), ARK (SEQ ID NO:3), RKR (SEQ ID NO:4), or Any of RK-Val-Cit (SEQ ID NO:54), wherein the linker is coupled to a glutamine residue in the antibody via a primary amine contained in residue K. It should be understood that drug moiety B need not be directly linked to structure RKAA (SEQ ID NO:1), RKA (SEQ ID NO:2), ARK (SEQ ID NO:3), RKR (SEQ ID NO:4) or RK-Val -Cit(SEQ ID NO:54). Alternatively, drug moiety B may be indirectly linked to structure RKAA (SEQ ID NO:1), RKA (SEQ ID NO:2), ARK (SEQ ID NO:3), RKR (SEQ ID NO:4) or RK-Val -Cit(SEQ ID NO:54). For example, the linker may include a linker between drug moiety B and the structure RKAA (SEQ ID NO:1), RKA (SEQ ID NO:2), ARK (SEQ ID NO:3), RKR (SEQ ID NO:4), or RK-Val -Cit (SEQ ID NO:54) other chemical structures between. Such chemical structure may be any structure disclosed herein for chemical spacers (Sp ₁ ), (Sp ₂ ) or (Sp ₃ ). In certain embodiments, the linker may comprise a linker between drug moiety B and the structure RKAA (SEQ ID NO:1), RKA (SEQ ID NO:2), ARK (SEQ ID NO:3), RKR (SEQ ID NO:4 ) or one or more amino acid residues between RK-Val-Cit (SEQ ID NO:54). In certain embodiments, the linker may comprise a linker between drug moiety B and the structure RKAA (SEQ ID NO:1), RKA (SEQ ID NO:2), ARK (SEQ ID NO:3), RKR (SEQ ID NO:4 ) or one or more PEG moieties between RK-Val-Cit (SEQ ID NO:54). In certain embodiments, the linker may comprise a linker between drug moiety B and the structure RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4) or the cleavable and/or self-cleavable moiety between RK-Val-Cit (SEQ ID NO:54).

That is, in a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the drug moiety B is linked via a self-cleaving moiety to the N-terminus or C-terminus of the amino acid sequence contained in the linker .

In a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the self-cleaving moiety comprises a p-aminobenzylcarbamoyl (PABC) moiety.

That is, the self-cleaving moiety comprised in the linker according to the present invention may be any of the self-cleaving moieties disclosed herein. In certain embodiments, the self-cleaving moiety may be a PABC or methylamino group as disclosed herein.

In a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the IgG antibody is a glycosylated IgG antibody, in particular wherein the IgG antibody is present at residue N297 of the _CH2 domain (EU No.) glycosylation.

In a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the IgG antibody is an IgG1 antibody.

That is, the antibody is preferably an IgG antibody, especially an IgG1 antibody, especially wherein the IgG or IgG1 antibody is glycosylated at residue N297 (EU numbering).

Antibody-drug conjugates may contain one or more toxins disclosed herein. Thus, in a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the drug is a toxin selected from the group consisting of:

·pyrrolobenzodiazepines (e.g., PBD);

·Auristatin (such as MMAE, MMAF);

Maytansinoids (e.g., maytansinoids, DM1, DM4, DM21);

·Becarcinomycin;

·Nicotinamide phosphoribosyltransferase (NAMPT) inhibitor;

·Microtubulysin;

· Enedyne (e.g., calicheamicin);

·Anthracycline derivatives (PNU) (e.g., doxorubicin);

·Pyrrolyl kinesin spindle protein (KSP) inhibitor;

·Nodulin;

·Drug efflux pump inhibitors;

·Sandromycin;

·Amanitanic acid (e.g., alpha-amanitinine); and

· Camptothecins (e.g., ixotecan, drotecan).

It will be appreciated that the toxin can be coupled directly to the linker by chemical synthesis. However, in other embodiments, the toxin can also be linked to the linker moiety contained in the linker in a two-step process.

In a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure RKAA-B or RKAA-(linker molecule)-B.

That is, payload B can be coupled directly to the C-terminus of an alanine residue, or can be coupled to the C-terminus of an alanine residue via a linker molecule. It should be understood that the choice of linker molecule depends largely on the functional groups available in the payload B. Disclosed herein are linker molecules suitable for coupling payloads with different functional groups to peptides. Linker molecules may be cleavable or non-cleavable linker molecules. In particular, the linker molecule may comprise a self-cleaving moiety, particularly any one of the self-cleaving moieties disclosed herein. Thus, in a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure RKAA-(self-cleaving moiety)-B.

Alternatively, the payload can be coupled to the N-terminus of the arginine residue directly or via a linker molecule (eg, via any of the linker molecules disclosed herein). That is, in a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure B-RKAA or B-(linker molecule)-RKAA. In a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of structure B-(self-cleaving moiety)-RKAA. In certain embodiments, the self-cleaving moiety comprised in Structure B-(self-cleaving moiety)-RKAA can be a self-cleaving moiety comprising an ortho-hydroxyl protected aryl sulfate (OHPAS) moiety disclosed herein.

In a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker has the structure RKAA-PABC-B, especially wherein B is auristatin or maytansinoid, especially wherein , auristatin is a MMAE, and where the maytansinoids are DM1 or maytansinoids.

In certain embodiments, the linker can have the structure RKAA-PABC-B. That is, the linker may comprise a linear peptide RKAA in which the carboxyl group of the C-terminal alanine residue is coupled via an amide bond to the amino group contained in PABC. Toxin B can attach to PABC through carbamate formation. It should be understood that not all toxins contain functional groups that allow carbamate formation with PABC. Therefore, the toxin can be linked to PABC via a linker.

In certain embodiments, the toxin may be a toxin containing a primary or secondary amine. In certain embodiments, the toxin may be MMAE or maytansine.

In certain embodiments, the linker may have a protected N-terminus. In certain embodiments, the N-terminus can be acetylated. In certain embodiments, the linker is the linker shown in Figure 1 or Figure 8.

In certain embodiments, the linker can have the structure RKAA-PABC-MMAE. In certain embodiments, the linker can have the structure RKAA-(PEG) _n -PABC-MMAE, where n is an integer between 2 and 20. In certain embodiments, the linker can have the structure RKAA-(PEG) ₂ -PABC-MMAE. In certain embodiments, the linker can have the structure RKAA-MMAE. In certain embodiments, the linker can have the structure RKAA-Val-Cit-PABC-MMAE. In certain embodiments, linkers may include additional linkers between the PABC moiety and the MMAE. In certain embodiments, the additional linker may be a p-nitrophenol (PNP) group.

It must be noted that linkers can include other self-cleaving moieties besides PABC. That is, the linker may have the structure RKAA-(self-cleaving moiety)-toxin. Those skilled in the art are aware of other self-cleaving moieties that may be used in the present invention. Further, those skilled in the art are aware of toxins that may be coupled to self-cleaving moieties, optionally via additional linkers.

In certain embodiments, the toxin can be a toxin that contains a hydroxyl group, and the linker can contain a self-cleaving methylamine group. That is, the linker can have the structure RKAA-(NH)-( _CH3 )-O-toxin. In certain embodiments, the hydroxyl-containing toxin may be a camptothecin (such as ixotecan or an ixotecan derivative, particularly ixotecan derivative Dxd) or an anthracycline (such as, PNU- 159682).

In certain embodiments, the toxin can be a toxin containing a thiol group and the linker can contain a self-cleaving methylamine group. That is, the linker can have the structure RKAA-(NH)-( _CH3 )-S-toxin. In certain embodiments, the thiol-containing toxin may be a maytansinoid (such as DM1) or a thiol-containing derivative thereof.

In a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure RKA-B or RKA-(linker molecule)-B.

That is, payload B can be coupled directly to the C-terminus of an alanine residue, or can be coupled to the C-terminus of an alanine residue via a linker molecule. It should be understood that the choice of linker molecule depends largely on the functional groups available in the payload B. Disclosed herein are linker molecules suitable for coupling payloads with different functional groups to peptides. Linker molecules may be cleavable or non-cleavable linker molecules. In particular, the linker molecule may comprise a self-cleaving moiety, particularly any one of the self-cleaving moieties disclosed herein. Thus, in a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure RKA-(self-cleaving moiety)-B.

Alternatively, the payload can be coupled to the N-terminus of the arginine residue directly or via a linker molecule (eg, via any of the linker molecules disclosed herein). That is, in a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure B-RKA or B-(linker molecule)-RKA. In a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of structure B-(self-cleaving moiety)-RKA. In certain embodiments, the self-cleaving moiety comprised in Structure B-(self-cleaving moiety)-RKA can be a self-cleaving moiety comprising an ortho-hydroxyl protected aryl sulfate (OHPAS) moiety disclosed herein.

In a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker has the structure RKA-PABC-B, especially wherein B is auristatin or maytansinoid, especially wherein , auristatin is a MMAE, and where the maytansinoids are DM1 or maytansinoids.

In certain embodiments, the linker can have the structure RKA-PABC-B. That is, the linker may comprise a linear peptide RKA, in which the carboxyl group of the C-terminal alanine residue is coupled via an amide bond to the amino group contained in PABC. Toxin B can attach to PABC through carbamate formation. It should be understood that not all toxins contain functional groups that allow carbamate formation with PABC. Therefore, the toxin can be linked to PABC via a linker.

In certain embodiments, the toxin may be a toxin containing a primary or secondary amine. In certain embodiments, the toxin may be MMAE or maytansine.

In certain embodiments, the linker may have a protected N-terminus. In certain embodiments, the N-terminus can be acetylated. In certain embodiments, the linker is the linker shown in Figure 2.

In certain embodiments, the linker can have the structure RKA-PABC-MMAE. In certain embodiments, the linker can have the structure RKA-(PEG) _n -PABC-MMAE, where n is an integer between 2 and 20. In certain embodiments, the linker can have the structure RKA-(PEG) ₂ -PABC-MMAE. In certain embodiments, the linker can have the structure RKA-MMAE. In certain embodiments, the linker can have the structure RKA-Val-Cit-PABC-MMAE. In certain embodiments, linkers may include additional linkers between the PABC moiety and the MMAE. In certain embodiments, the additional linker may be a p-nitrophenol (PNP) group.

It must be noted that linkers can include other self-cleaving moieties besides PABC. That is, the linker may have the structure RKA-(self-cleaving moiety)-toxin. Those skilled in the art are aware of other self-cleaving moieties that may be used in the present invention. Further, those skilled in the art are aware of toxins that may be coupled to self-cleaving moieties, optionally via additional linkers.

In certain embodiments, the toxin can be a toxin that contains a hydroxyl group, and the linker can contain a self-cleaving methylamine group. That is, the linker can have the structure RKA-(NH)-( _CH3 )-O-toxin. In certain embodiments, the hydroxyl-containing toxin may be a camptothecin (such as ixotecan or an ixotecan derivative, particularly ixotecan derivative Dxd) or an anthracycline (such as, PNU- 159682).

In certain embodiments, the toxin can be a toxin containing a thiol group and the linker can contain a self-cleaving methylamine group. That is, the linker can have the structure RKA-(NH)-( _CH3 )-S-toxin. In certain embodiments, the thiol-containing toxin may be a maytansinoid (such as DM1) or a thiol-containing derivative.

In a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure ARK-B or ARK-(linker molecule)-B.

That is, payload B can be coupled directly to the C-terminus of a lysine residue, or can be coupled to the C-terminus of a lysine residue via a linker molecule. It should be understood that the choice of linker molecule depends largely on the functional groups available in the payload B. Disclosed herein are linker molecules suitable for coupling payloads with different functional groups to peptides. Linker molecules may be cleavable or non-cleavable linker molecules. In particular, the linker molecule may comprise a self-cleaving moiety, particularly any one of the self-cleaving moieties disclosed herein. Thus, in a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure ARK-(auto-cleaving moiety)-B.

Alternatively, the payload can be coupled to the N-terminus of the alanine residue directly or via a linker molecule (eg, via any of the linker molecules disclosed herein). That is, in a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure B-ARK or B-(linker molecule)-ARK. In a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of structure B-(self-cleaving moiety)-ARK. In certain embodiments, the self-cleaving moiety comprised in structure B-(auto-cleaving moiety)-ARK can be a self-cleaving moiety comprising an ortho-hydroxyl protected aryl sulfate (OHPAS) moiety disclosed herein.

In a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker has the structure ARK-PABC-B, especially wherein B is auristatin or maytansinoid, especially wherein , auristatin is a MMAE, and where the maytansinoids are DM1 or maytansinoids.

In certain embodiments, the linker can have the structure ARK-PABC-B. That is, the linker may comprise a linear peptide ARK, in which the carboxyl group of the C-terminal lysine residue is coupled via an amide bond to the amino group contained in PABC. Toxin B can attach to PABC through carbamate formation. It should be understood that not all toxins contain functional groups that allow carbamate formation with PABC. Therefore, the toxin can be linked to PABC via a linker.

In certain embodiments, the toxin may be a toxin containing a primary or secondary amine. In certain embodiments, the toxin may be MMAE or maytansine.

In certain embodiments, the linker may have a protected N-terminus. In certain embodiments, the N-terminus can be acetylated. In certain embodiments, the linker is the linker shown in Figure 3.

In certain embodiments, the linker may have the structure ARK-PABC-MMAE. In certain embodiments, the linker can have the structure ARK-(PEG) _n -PABC-MMAE, where n is an integer between 2 and 20. In certain embodiments, the linker can have the structure ARK-(PEG) ₂ -PABC-MMAE (see Figure 14). In certain embodiments, the linker may have the structure ARK-MMAE. In certain embodiments, the linker can have the structure ARK-Val-Cit-PABC-MMAE. In certain embodiments, linkers may include additional linkers between the PABC moiety and the MMAE. In certain embodiments, the additional linker may be a p-nitrophenol (PNP) group.

It must be noted that linkers can include other self-cleaving moieties besides PABC. That is, the linker may have the structure ARK-(self-cleaving moiety)-toxin. Those skilled in the art are aware of other self-cleaving moieties that may be used in the present invention. Further, those skilled in the art are aware of toxins that may be coupled to self-cleaving moieties, optionally via additional linkers.

In certain embodiments, the toxin can be a toxin that contains a hydroxyl group, and the linker can contain a self-cleaving methylamine group. That is, the linker can have the structure ARK-(NH)-( _CH3 )-O-toxin. In certain embodiments, the hydroxyl-containing toxin may be a camptothecin (such as ixotecan or an ixotecan derivative, particularly ixotecan derivative Dxd) or an anthracycline (such as, PNU- 159682).

In certain embodiments, the toxin can be a toxin containing a thiol group and the linker can contain a self-cleaving methylamine group. That is, the linker can have the structure ARK-(NH)-( _CH3 )-O-toxin (similar to Figure 15). In certain embodiments, the thiol-containing toxin may be a maytansinoid (such as DM1) or a thiol-containing derivative thereof.

In certain embodiments, the linker is the linker shown in Figure 14 or Figure 15.

In a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure RKR-B or RKR-(linker molecule)-B.

That is, payload B can be coupled directly to the C-terminus of the arginine residue, or can be coupled to the C-terminus of the arginine residue via a linker molecule. It should be understood that the choice of linker molecule depends largely on the functional groups available in the payload B. Disclosed herein are linker molecules suitable for coupling payloads with different functional groups to peptides. Linker molecules may be cleavable or non-cleavable linker molecules. In particular, the linker molecule may comprise a self-cleaving moiety, particularly any one of the self-cleaving moieties disclosed herein. Thus, in a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure RKR-(self-cleaving moiety)-B.

Alternatively, the payload can be coupled to the N-terminus of the arginine residue directly or via a linker molecule (eg, via any of the linker molecules disclosed herein). That is, in a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure B-RKR or B-(linker molecule)-RKR. In certain embodiments, the linker can be a dicarboxylic acid linker (see Figure 9). In a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of structure B-(self-cleaving moiety)-RKR. In certain embodiments, the self-cleaving moiety comprised in Structure B-(self-cleaving moiety)-RKR can be a self-cleaving moiety comprising an ortho-hydroxyl protected aryl sulfate (OHPAS) moiety disclosed herein.

In a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker has the structure RKR-PABC-B, especially wherein B is auristatin or maytansinoid, especially wherein , auristatin is a MMAE, and where the maytansinoids are DM1 or maytansinoids.

In certain embodiments, the linker can have the structure RKR-PABC-B. That is, the linker may comprise a linear peptide RKR in which the carboxyl group of the C-terminal arginine residue is coupled via an amide bond to the amino group contained in the PABC. Toxin B can attach to PABC through carbamate formation. It should be understood that not all toxins contain functional groups that allow carbamate formation with PABC. Therefore, the toxin can be linked to PABC via a linker.

In certain embodiments, the toxin may be a toxin containing a primary or secondary amine. In certain embodiments, the toxin may be MMAE or maytansine.

In certain embodiments, the linker may have a protected N-terminus. In certain embodiments, the N-terminus can be acetylated.

In certain embodiments, the linker can have the structure RKR-PABC-MMAE. In certain embodiments, the linker can have the structure RKR-(PEG) _n -PABC-MMAE, where n is an integer between 2 and 20. In certain embodiments, the linker can have the structure RKR-(PEG) ₂ -PABC-MMAE. In certain embodiments, the linker can have the structure RKR-MMAE. In certain embodiments, the linker can have the structure RKR-Val-Cit-PABC-MMAE. In certain embodiments, linkers may include additional linkers between the PABC moiety and the MMAE. In certain embodiments, the additional linker may be a p-nitrophenol (PNP) group.

It must be noted that linkers can include other self-cleaving moieties besides PABC. That is, the linker may have the structure RKR-(self-cleaving moiety)-toxin. Those skilled in the art are aware of other self-cleaving moieties that may be used in the present invention. Further, those skilled in the art are aware of toxins that may be coupled to self-cleaving moieties, optionally via additional linkers.

In certain embodiments, the toxin can be a toxin that contains a hydroxyl group, and the linker can contain a self-cleaving methylamine group. That is, the linker can have the structure RKR-(NH)-( _CH3 )-O-toxin. In certain embodiments, the hydroxyl-containing toxin may be a camptothecin (such as ixotecan or an ixotecan derivative, particularly ixotecan derivative Dxd) or an anthracycline (such as, PNU- 159682).

In certain embodiments, the toxin can be a toxin containing a thiol group and the linker can contain a self-cleaving methylamine group. That is, the linker can have the structure RKR-(NH)-( _CH3 )-O-toxin (similar to Figure 15). In certain embodiments, the thiol-containing toxin may be a maytansinoid (such as DM1) or a thiol-containing derivative thereof.

In a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure RK-Val-Cit-B or RK-Val-Cit-(linker molecule)-B .

That is, payload B can be coupled directly to the C-terminus of the citrulline residue, or can be coupled to the C-terminus of the citrulline residue via a linker molecule. It should be understood that the choice of linker molecule depends largely on the functional groups available in the payload B. Disclosed herein are linker molecules suitable for coupling payloads with different functional groups to peptides. Linker molecules may be cleavable or non-cleavable linker molecules. In particular, the linker molecule may comprise a self-cleaving moiety, particularly any one of the self-cleaving moieties disclosed herein. Thus, in a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker comprises or consists of the structure RK-Val-Cit-(self-cleaving moiety)-B.

In a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the linker has the structure RK-Val-Cit-PABC-B, in particular wherein B is auristatin or maytansinoid , especially wherein auristatin is MMAE, and wherein the maytansinoid is DM1 or maytansine.

In certain embodiments, the linker can have the structure RK-Val-Cit-PABC-B. That is, the linker may comprise the linear peptide RK-Val-Cit, in which the carboxyl group of the C-terminal citrulline residue is coupled to the amino group contained in the PABC via an amide bond. Toxin B can attach to PABC through carbamate formation. It should be understood that not all toxins contain functional groups that allow carbamate formation with PABC. Therefore, the toxin can be linked to PABC via a linker.

In certain embodiments, the toxin may be a toxin containing a primary or secondary amine. In certain embodiments, the toxin may be MMAE or maytansine.

In certain embodiments, the linker may have a protected N-terminus. In certain embodiments, the N-terminus can be acetylated.

In certain embodiments, the linker can have the structure RK-Val-Cit-PABC-MMAE. In certain embodiments, the linker can have the structure RK-(PEG) _n -Val-Cit-PABC-MMAE, where n is an integer between 2 and 20. In certain embodiments, the linker can have the structure RK-(PEG-Val-Cit-PABC-MMAE. In certain embodiments, the linker can have the structure RK-Val-Cit-MMAE. In certain embodiments, Linkers can include additional linkers between the PABC moiety and MMAE. In certain embodiments, the additional linkers can be p-nitrophenol (PNP) groups.

It must be noted that linkers can include other self-cleaving moieties besides PABC. That is, the linker may have the structure RK-Val-Cit-(self-cleaving moiety)-toxin. Those skilled in the art are aware of other self-cleaving moieties that may be used in the present invention. Further, those skilled in the art are aware of toxins that may be coupled to self-cleaving moieties, optionally via additional linkers.

In certain embodiments, the toxin can be a toxin that contains a hydroxyl group, and the linker can contain a self-cleaving methylamine group. That is, the linker can have the structure RK-Val-Cit-(NH)-( _CH3 )-O-toxin. In certain embodiments, the hydroxyl-containing toxin may be a camptothecin (such as ixotecan or an ixotecan derivative, particularly ixotecan derivative Dxd) or an anthracycline (such as, PNU- 159682).

In certain embodiments, the toxin can be a toxin containing a thiol group and the linker can contain a self-cleaving methylamine group. That is, the linker may have the structure RK-Val-Cit-(NH)-( _CH3 )-S-toxin. In certain embodiments, the thiol-containing toxin may be a maytansinoid (such as DM1) or a thiol-containing derivative thereof.

In a specific embodiment, the present invention relates to an antibody-linker conjugate or an antibody-drug conjugate comprising the antibody polotuzumab or alternatively an anti-CD79b antibody.

That is, in a specific embodiment, the invention relates to an antibody-linker conjugate comprising:

a) Polotuzumab or anti-CD79b antibody; and

b) Joint, which includes the following structures:

(Sp ₁ )-RK-(Sp ₂ )-B-(Sp ₃ ) or

(Sp ₁ )-B-(Sp ₂ )-RK-(Sp ₃ ); where

·(Sp ₁ ) is a chemical spacer or does not exist;

·(Sp ₂ ) is a chemical spacer or does not exist;

·(Sp ₃ ) is a chemical spacer or does not exist;

·R is arginine or arginine derivative or arginine mimetic;

·K is lysine or lysine derivative or lysine mimetic;

·B is the connection part or payload;

Wherein, the linker is coupled to polotuzumab or anti-CD79b antibody via an isopeptide bond in the γ-carboxylic acid of a glutamine residue contained in polotuzumab or anti-CD79b antibody. The amide group is formed between a primary amine contained in the side chain of a lysine residue, a lysine derivative or a lysine mimetic contained in the RK motif contained in the linker.

In a specific embodiment, the invention relates to an antibody-payload conjugate comprising polotuzumab or an anti-CD79b antibody, wherein the linker is Figure 1, Figure 2, Figure 3, Figure 8, Figure 9, Figure 14, Figure 15, Figure 17, Figure 18, Figure 19, Figure 20, Figure 21, Figure 22, Figure 23, Figure 24, Figure 25, Figure 26, Figure 27, Figure 28, Figure 29, Figure 30, Figure 31 , any of the connectors shown in Figure 32, Figure 33 or Figure 34.

Polatuzumab is commercially available as the antibody-drug conjugate Polatuzumabvedotin and sold under the name Polivy. Polotuzumab vedotin includes the anti-CD79b antibody polotuzumab vedotin and the linker maleimidocaproyl-L-valine-L-citrulline-PAC-MMAE (mc -vc-PABC-MMAE), which is commonly known as vedotin. Polotuzumab Vedotin's mc-vc-PABC-MMAE linker couples to free cysteine residues contained in the antibody. The antibody polotuzumab has been disclosed in WO 2009/012268, the entire content of which is incorporated herein by reference in its entirety. Further, engineered cysteine variants of polotuzumab have been disclosed in WO 2009/099728, which is also incorporated herein by reference in its entirety.

The inventors have shown that polotuzumab conjugates comprising a linker according to the invention have a longer half-life in plasma compared to the commercially available conjugate polotuzumab vedotin. Therefore, antibody-linker conjugates conjugated by microbial transglutaminase to the linkers of the invention are surprisingly more efficient than those produced by other techniques, such as by coupling a maleimide-containing linker to the cysteine of the antibody. residue coupling) produces more stable antibodies. Therefore, antibody-linker conjugates according to the invention are more likely to reach their target cells or tissues without prematurely losing their payload.

In certain embodiments, the antibody is polotuzumab, comprising a heavy chain set forth in SEQ ID NO:5 and a light chain set forth in SEQ ID NO:6. However, the present invention also includes variants of polotuzumab, wherein the heavy and/or light chain corresponds to SEQ ID NO: 5 and/or SEQ ID NO: 6, respectively, by at least 80%, at least 85%, at least 90%, at least 95% sequence identity. In particular, the antibody may comprise any sequence variant disclosed in WO 2009/012268 or WO 2009/099728.

It is preferred herein that the polotuzumab or anti-CD79b antibody is present in the antibody-linker conjugate in a glycosylated form. That is, polotuzumab or anti-CD79b antibodies are preferably glycosylated at residue N297 (EU numbering). However, polotuzumab or anti-CD79b antibodies can also be deglycosylated as described herein.

Polotuzumab or anti-CD79b antibodies may be coupled to any of the linkers disclosed herein, particularly for use in methods according to the invention. Preferably, the linker is coupled to glutamine residue Q295 (EU numbering) of the antibody in an MTG-catalyzed manner. However, the linker may also be coupled to an engineered glutamine residue (such as N297Q (EU numbering)) and/or to any of the glutamine-containing tags disclosed herein.

The linker coupled to polotuzumab or anti-CD79b antibody may include a single linker moiety or payload B, or may include multiple linker moieties and/or payloads B ₁ , B ₂ , etc.

That is, in certain embodiments, the linker included in the polotuzumab-linker conjugate can include one or more linking moieties B. Such polotuzumab-linker conjugates can then be functionalized with a suitable payload in the two-step process disclosed herein.

In certain embodiments, the linker included in the polotuzumab-payload conjugate can comprise one or more payload Bs. Such polotuzumab-payload conjugates can be obtained in a two-step process, where the linker containing the linking moiety has been conjugated to polotuzumab in the first step, and the payload has been Connect with the connection part in the second step. Alternatively, polotuzumab-payload conjugates can be obtained in a one-step process in which the payload-containing linker is coupled directly to polotuzumab.

In certain embodiments, the invention relates to an antibody-drug conjugate comprising polotuzumab or an anti-CD79B antibody. That is, the linker included in the antibody-drug conjugate can comprise one or more toxins described herein.

In certain embodiments, an antibody-drug conjugate comprising plotuzumab or an anti-CD79b antibody can comprise the amino acid sequence RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK ( SEQ ID NO:3), RKR (SEQ ID NO:4) or RK-Val-Cit (SEQ ID NO:54).

That is, in a specific embodiment, the invention relates to an antibody-drug conjugate comprising:

a) Polotuzumab or anti-CD79b antibody; and

b) A linker comprising drug moiety B, wherein drug moiety B is covalently linked to an amino acid sequence selected from the group consisting of: RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 2) NO:3), RKR (SEQ ID NO:4) or RK-Val-Cit (SEQ ID NO:54);

Wherein, the linker is coupled to polotuzumab or anti-CD79b antibody via an isopeptide bond at the γ-carboxamide of glutamine residue Q295 (EU numbering) of the _CH2 domain of the antibody The group is formed between a primary amine contained in the side chain of a lysine residue contained in the linker.

Preferably, the antibody is polotuzumab comprising the heavy chain shown in SEQ ID NO:5 and the light chain shown in SEQ ID NO:6. Therefore, in a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the IgG antibody is polotuzumab or comprises a heavy chain as shown in SEQ ID NO:5 and Antibodies to the light chain as shown in SEQ ID NO:6.

In certain embodiments, linkers coupled to polotuzumab or anti-CD79b antibodies can comprise the structures RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3 ), RKR (SEQ ID NO:4) or RK-Val-Cit (SEQ ID NO:54). In certain embodiments, the structure RKAA (SEQ ID NO:1), RKA (SEQ ID NO:2), ARK (SEQ ID NO:3), RKR (SEQ ID NO:4) or RK-Val-Cit is included. The linker of (SEQ ID NO:54) can be coupled to residue Q295 of polotuzumab or anti-CD79b antibodies via a primary amine contained in residue K.

In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RKAA-B disclosed herein, wherein B is preferably a toxin. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RKAA-(self-cleaving moiety)-B disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the C-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a PABC or a methylamine-containing group.

In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RKAA-PABC-B. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RKAA-PABC-MMAE (see Figure 1). In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RKAA-PABC-maytansine (see Figure 8). In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RKAA-MMAE. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RKAA-PABC-PNP-MMAE. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RKAA-(NH)-(CH ₃ )-OB, where O is the oxygen atom of the ether bond and B is a hydroxyl-containing of toxins. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RKAA-(NH)-( _CH3 )-O-camptothecin. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RKAA-(NH)-( _CH3 )-SB, where S is a sulfur atom of a thioether bond and B is a linker containing Thiol toxins. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RKAA-(NH)-( _CH3 )-S-DM1.

In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker B-RKAA disclosed herein, wherein B is preferably a toxin. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker B-(self-cleaving moiety)-RKAA disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the N-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a self-cleaving moiety containing an ortho-hydroxyl protected aryl sulfate (OHPAS) moiety. In certain embodiments, similar to Figure 9, amine-containing payload B can be coupled to the N-terminal arginine moiety via a dicarboxylic acid linker.

In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RKA-B disclosed herein, wherein B is preferably a toxin. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RKA-(self-cleaving moiety)-B disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the C-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a PABC or a methylamine-containing group.

In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RKA-PABC-B. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RKA-PABC-MMAE (see Figure 2). In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RKA-PABC-maytansine. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RKA-MMAE. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RKA-PABC-PNP-MMAE. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RKA-(NH)-(CH ₃ )-OB, where O is the oxygen atom of the ether bond and B is a hydroxyl-containing of toxins. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RKA-(NH)-( _CH3 )-O-camptothecin. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RKA-(NH)-( _CH3 )-SB, where S is a sulfur atom of a thioether bond and B is a linker containing Thiol toxins. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RKA-(NH)-( _CH3 )-S-DM1.

In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker B-RKA disclosed herein, wherein B is preferably a toxin. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker B-(self-cleaving moiety)-RKA disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the N-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a self-cleaving moiety that includes an ortho-hydroxyl protected aryl sulfate (OHPAS) moiety. In certain embodiments, similar to Figure 9, amine-containing payload B can be coupled to the N-terminal arginine moiety via a dicarboxylic acid linker.

In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker ARK-B disclosed herein, wherein B is preferably a toxin. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker ARK-(auto-cleaving moiety)-B disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the C-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a PABC or a methylamine-containing group.

In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker ARK-PABC-B. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker ARK-PABC-MMAE (see Figure 3). In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker ARK-PABC-maytansine. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker ARK-MMAE. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker ARK-PABC-PNP-MMAE. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker ARK-(NH)-(CH ₃ )-OB, where O is the oxygen atom of the ether bond, and B is a hydroxyl-containing of toxins. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker ARK-(NH)-( _CH3 )-O-camptothecin. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker ARK-(NH)-(CH ₃ )-SB, where S is a sulfur atom of a thioether bond and B is a sulfur atom containing a thioether bond. Thiol toxins. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker ARK-(NH)-( _CH3 )-S-DM1.

In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker B-ARK disclosed herein, wherein B is preferably a toxin. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker B-(self-cleaving moiety)-ARK disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the N-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a self-cleaving moiety that includes an ortho-hydroxyl protected aryl sulfate (OHPAS) moiety.

In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RKR-B disclosed herein, wherein B is preferably a toxin. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RKR-(self-cleaving moiety)-B disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the C-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a PABC or a methylamine-containing group.

In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RKR-PABC-B. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RKR-PABC-MMAE. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RKR-PABC-maytansine. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RKR-MMAE. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RKR-PABC-PNP-MMAE. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RKR-(NH)-(CH ₃ )-OB, where O is the oxygen atom of the ether bond and B is a hydroxyl-containing of toxins. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RKR-(NH)-( _CH3 )-O-camptothecin. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RKR-(NH)-( _CH3 )-SB, where S is a sulfur atom of a thioether bond and B is a linker containing Thiol toxins. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RKR-(NH)-( _CH3 )-S-DM1.

In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker B-RKR disclosed herein, wherein B is preferably a toxin. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker B-(self-cleaving moiety)-RKR disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the N-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a self-cleaving moiety that includes an ortho-hydroxyl protected aryl sulfate (OHPAS) moiety. In certain embodiments, as illustrated in Figure 9, amine-containing payload B can be coupled to an N-terminal arginine moiety via a dicarboxylic acid linker.

In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RK-Val-Cit-B disclosed herein, wherein B is preferably a toxin. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RK-Val-Cit-(self-cleaving moiety)-B disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the C-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a PABC or a methylamine-containing group.

In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RK-Val-Cit-PABC-B. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RK-Val-Cit-PABC-MMAE. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RK-Val-Cit-PABC-maytansine. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RK-Val-Cit-MMAE. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RK-Val-Cit-PABC-PNP-MMAE. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RK-Val-Cit-(NH)-(CH ₃ )-OB, wherein O is the oxygen atom of the ether bond, and B is a hydroxyl-containing toxin. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RK-Val-Cit-(NH)-( _CH3 )-O-camptothecin. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RK-Val-Cit-(NH)-(CH ₃ )-SB, where S is the sulfur atom of the thioether bond, And B is a thiol-containing toxin. In certain embodiments, polotuzumab or anti-CD79b antibodies can be coupled to the linker RK-Val-Cit-(NH)-( _CH3 )-S-DM1.

In a specific embodiment, the present invention relates to an antibody-linker conjugate or an antibody-drug conjugate comprising the antibody trastuzumab or alternatively an anti-HER2 antibody.

That is, in a specific embodiment, the invention relates to an antibody-linker conjugate comprising:

a) Polotuzumab or anti-HER2/neu antibody; and

b) Joint, which includes the following structures:

(Sp ₁ )-RK-(Sp ₂ )-B-(Sp ₃ ) or

(Sp ₁ )-B-(Sp ₂ )-RK-(Sp ₃ ); where

·(Sp ₁ ) is a chemical spacer or does not exist;

·(Sp ₂ ) is a chemical spacer or does not exist;

·(Sp ₃ ) is a chemical spacer or does not exist;

·R is arginine or arginine derivative or arginine mimetic;

·K is lysine or lysine derivative or lysine mimetic;

·B is the connection part or payload;

Wherein, the linker is coupled to trastuzumab or anti-HER2/neu antibody via an isopeptide bond, and the isopeptide bond is in the γ of the glutamine residue included in trastuzumab or anti-HER2/neu antibody. - a carboxamide group is formed between a primary amine contained in the side chain of a lysine residue, a lysine derivative or a lysine mimetic contained in the RK motif contained in the linker.

In a specific embodiment, the invention relates to an antibody-payload conjugate comprising trastuzumab or an anti-HER2/neu antibody, wherein the linker is Figure 1, Figure 2, Figure 3, Figure 8, Figure 9 , Figure 14, Figure 15, Figure 17, Figure 18, Figure 19, Figure 20, Figure 21, Figure 22, Figure 23, Figure 24, Figure 25, Figure 26, Figure 27, Figure 28, Figure 29, Figure 30, Figure 31. Any of the connectors shown in Figure 32, Figure 33 or Figure 34.

Trastuzumab is commercially available as the antibody-drug conjugate Trastuzumab emtansine and sold under the name Kadcyla. Trastuzumab contains the anti-HER2/neu antibody trastuzumab and toxin DM1, which is synthesized via N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane A -1-carboxylate (SMCC) linker was conjugated to trastuzumab. The linker-DM1 construct can be coupled to up to eight different lysine residues contained in the antibody, resulting in antibodies with different drug-to-antibody ratios. Preferably, trastuzumab comprises a heavy chain as set forth in SEQ ID NO:7 and a light chain as set forth in SEQ ID NO:8. However, the present invention also includes variants of trastuzumab in which the heavy chain and/or the light chain are at least 80%, at least 85%, and at least 90% identical to SEQ ID NO:7 and/or SEQ ID NO:8, respectively. %, at least 95% sequence identity. In certain embodiments, the antibody may be an anti-HER2/neu antibody, such as disclosed in, but not limited to, WO 1998/006692, WO 1999/905536, WO 2003/087131, all of which are incorporated herein by reference.

It is preferred herein that the trastuzumab or anti-HER2/neu antibody is present in the antibody-linker conjugate in a glycosylated form. That is, trastuzumab or anti-HER2/neu antibodies are preferably glycosylated at residue N297 (EU numbering). However, trastuzumab or anti-HER2/neu antibodies can also be deglycosylated as described herein.

Trastuzumab or anti-HER2/neu antibodies may be coupled to any of the linkers disclosed herein, particularly for use in methods according to the invention. Preferably, the linker is coupled to glutamine residue Q295 (EU numbering) of the antibody in an MTG-catalyzed manner. However, the linker may also be coupled to an engineered glutamine residue (such as N297Q (EU numbering)) and/or to any of the glutamine-containing tags disclosed herein.

Linkers coupled to trastuzumab or anti-HER2/neu antibodies may include a single linker or payload B, or may include multiple linkers and/or payloads B ₁ , B ₂ , etc.

That is, in certain embodiments, the linker included in the trastuzumab-linker conjugate can include one or more linking moieties B. Such trastuzumab-linker conjugates can then be functionalized with a suitable payload in the two-step process disclosed herein.

In certain embodiments, the linker included in the trastuzumab-payload conjugate can contain one or more Payload Bs. Such trastuzumab-payload conjugates can be obtained in a two-step process, where the linker containing the linking moiety has been conjugated to trastuzumab in the first step, and the payload has been coupled to the trastuzumab in the second step. Connect with the connection part in the step. Alternatively, trastuzumab-payload conjugates can be obtained in a one-step process in which the payload-containing linker is coupled directly to trastuzumab.

In certain embodiments, the invention relates to an antibody-drug conjugate comprising trastuzumab or an anti-HER2/neu antibody. That is, the linker included in the antibody-drug conjugate can comprise one or more toxins described herein.

In certain embodiments, an antibody-drug conjugate comprising trastuzumab or an anti-HER2/neu antibody can comprise the amino acid sequence RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO:3), RKR (SEQ ID NO:4) or RK-Val-Cit (SEQ ID NO:54).

That is, in a specific embodiment, the invention relates to an antibody-drug conjugate comprising:

a) trastuzumab or anti-HER2/neu antibody; and

b) A linker comprising drug moiety B, wherein drug moiety B is covalently linked to an amino acid sequence selected from the group consisting of: RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 2) NO:3), RKR (SEQ ID NO:4) or RK-Val-Cit (SEQ ID NO:54);

Among them, the linker is coupled to trastuzumab or anti-HER2/neu antibody via an isopeptide bond at the γ-carboxylic acid of glutamine residue Q295 (EU numbering) of the _CH2 domain of the antibody. An amide group is formed between a primary amine contained in the side chain of a lysine residue contained in the linker.

Preferably, the antibody is trastuzumab comprising the heavy chain shown in SEQ ID NO:7 and the light chain shown in SEQ ID NO:8. Therefore, in a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the IgG antibody is trastuzumab or comprises a heavy chain as set forth in SEQ ID NO:7 and as Antibodies to the light chain shown in SEQ ID NO:8.

In certain embodiments, linkers coupled to trastuzumab or anti-HER2/neu antibodies can comprise the structures RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO :3), RKR (SEQ ID NO:4) or RK-Val-Cit (SEQ ID NO:54). In certain embodiments, the structure RKAA (SEQ ID NO:1), RKA (SEQ ID NO:2), ARK (SEQ ID NO:3), RKR (SEQ ID NO:4) or RK-Val-Cit is included. The linker of (SEQ ID NO:54) can be coupled to residue Q295 of trastuzumab or anti-HER2/neu antibodies via a primary amine contained in residue K.

In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RKAA-B disclosed herein, wherein B is preferably a toxin. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RKAA-(self-cleaving moiety)-B disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the C-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a PABC or a methylamine-containing group.

In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RKAA-PABC-B. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RKAA-PABC-MMAE (see Figure 1). In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RKAA-PABC-maytansine (see Figure 8). In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RKAA-MMAE. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RKAA-PABC-PNP-MMAE. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RKAA-(NH)-(CH ₃ )-OB, where O is the oxygen atom of the ether bond, and B is Hydroxyl-containing toxins. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RKAA-(NH)-( _CH3 )-O-camptothecin. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RKAA-(NH)-(CH ₃ )-SB, where S is the sulfur atom of the thioether bond, and B It is a thiol-containing toxin. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RKAA-(NH)-( _CH3 )-S-DM1.

In certain embodiments, tocilizumab or anti-HER2/neu antibodies can be coupled to the linker B-RKAA disclosed herein, wherein B is preferably a toxin. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker B-(self-cleaving moiety)-RKAA disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the N-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a self-cleaving moiety that includes an ortho-hydroxyl protected aryl sulfate (OHPAS) moiety. In certain embodiments, similar to Figure 9, amine-containing payload B can be coupled to the N-terminal arginine moiety via a dicarboxylic acid linker.

In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RKA-B disclosed herein, wherein B is preferably a toxin. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RKA-(self-cleaving moiety)-B disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the C-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a PABC or a methylamine-containing group.

In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RKA-PABC-B. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RKA-PABC-MMAE (see Figure 2). In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RKA-PABC-maytansine. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RKA-MMAE. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RKA-PABC-PNP-MMAE. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RKA-(NH)-(CH ₃ )-OB, where O is the oxygen atom of the ether bond, and B is Hydroxyl-containing toxins. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RKA-(NH)-( _CH3 )-O-camptothecin. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RKA-(NH)-(CH ₃ )-SB, where S is the sulfur atom of the thioether bond, and B It is a thiol-containing toxin. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RKA-(NH)-( _CH3 )-S-DM1.

In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker B-RKA disclosed herein, wherein B is preferably a toxin. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker B-(self-cleaving moiety)-RKA disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the N-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a self-cleaving moiety that includes an ortho-hydroxyl protected aryl sulfate (OHPAS) moiety. In certain embodiments, similar to Figure 9, amine-containing payload B can be coupled to the N-terminal arginine moiety via a dicarboxylic acid linker.

In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker ARK-B disclosed herein, wherein B is preferably a toxin. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker ARK-(self-cleaving moiety)-B disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the C-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a PABC or a methylamine-containing group.

In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker ARK-PABC-B. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker ARK-PABC-MMAE (see Figure 3). In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker ARK-PABC-maytansine. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker ARK-MMAE. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker ARK-PABC-PNP-MMAE. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker ARK-(NH)-(CH ₃ )-OB, where O is the oxygen atom of the ether bond, and B is Hydroxyl-containing toxins. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker ARK-(NH)-( _CH3 )-O-camptothecin. In certain embodiments, the trastuzumab embodiment monoclonal antibody or anti-HER2/neu antibody can be coupled to the linker ARK-(NH)-(CH ₃ )-SB, where S is the sulfur atom of the thioether bond, And B is a hydroxyl-containing toxin. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker ARK-(NH)-( _CH3 )-S-DM1.

In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker B-ARK disclosed herein, wherein B is preferably a toxin. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker B-(self-cleaving moiety)-ARK disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the N-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a self-cleaving moiety that includes an ortho-hydroxyl protected aryl sulfate (OHPAS) moiety.

In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RKR-B disclosed herein, wherein B is preferably a toxin. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RKR-(self-cleaving moiety)-B disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the C-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a PABC or a methylamine-containing group.

In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RKR-PABC-B. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RKR-PABC-MMAE. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RKR-PABC-maytansine. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RKR-MMAE. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RKR-PABC-PNP-MMAE. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RKR-(NH)-(CH ₃ )-OB, where O is the oxygen atom of the ether bond, and B is Hydroxyl-containing toxins. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RKR-(NH)-( _CH3 )-O-camptothecin. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RKR-(NH)-( _CH3 )-SB, where S is the sulfur atom of the thioether bond, and B It is a thiol-containing toxin. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RKR-(NH)-( _CH3 )-S-DM1.

In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker B-RKR disclosed herein, wherein B is preferably a toxin. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker B-(self-cleaving moiety)-RKR disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the N-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a self-cleaving moiety that includes an ortho-hydroxyl protected aryl sulfate (OHPAS) moiety. In certain embodiments, as illustrated in Figure 9, amine-containing payload B can be coupled to an N-terminal arginine moiety via a dicarboxylic acid linker.

In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RK-Val-Cit-B disclosed herein, wherein B is preferably a toxin. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RK-Val-Cit-(self-cleaving moiety)-B disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the C-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a PABC or a methylamine-containing group.

In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RK-Val-Cit-PABC-B. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RK-Val-Cit-PABC-MMAE. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RK-Val-Cit-PABC-maytansine. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RK-Val-Cit-MMAE. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RK-Val-Cit-PABC-PNP-MMAE. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RK-Val-Cit-(NH)-(CH ₃ )-OB, where O is the oxygen atom of the ether bond, And B is a hydroxyl-containing toxin. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RK-Val-Cit-(NH)-( _CH3 )-O-camptothecin. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RK-Val-Cit-(NH)-(CH ₃ )-SB, where S is the sulfur atom of the thioether bond , and B is a thiol-containing toxin. In certain embodiments, trastuzumab or anti-HER2/neu antibodies can be coupled to the linker RK-Val-Cit-(NH)-( _CH3 )-S-DM1.

In a specific embodiment, the present invention relates to an antibody-linker conjugate or an antibody-drug conjugate comprising the antibody ennozumab or alternatively an anti-bindin-4 antibody.

That is, in a specific embodiment, the invention relates to an antibody-linker conjugate comprising:

a) ennosumab or anti-bindingin-4 antibody; and

b) Joint, which includes the following structures:

(Sp ₁ )-RK-(Sp ₂ )-B-(Sp ₃ ) or

(Sp ₁ )-B-(Sp ₂ )-RK-(Sp ₃ ); where

·(Sp ₁ ) is a chemical spacer or does not exist;

·(Sp ₂ ) is a chemical spacer or does not exist;

·(Sp ₃ ) is a chemical spacer or does not exist;

·R is arginine or arginine derivative or arginine mimetic;

·K is lysine or lysine derivative or lysine mimetic;

·B is the connection part or payload;

Wherein, the linker is coupled to ennosumab or anti-conjugin-4 antibody via an isopeptide bond in the γ-carboxylic acid of the glutamine residue contained in ennosumab or anti-connexin-4 antibody. The amide group is formed between a primary amine contained in the side chain of a lysine residue, a lysine derivative or a lysine mimetic contained in the RK motif contained in the linker.

In a specific embodiment, the invention relates to an antibody-payload conjugate comprising ennosumab or an anti-bindingin-4 antibody, wherein the linker is Figure 1, Figure 2, Figure 3, Figure 8, Figure 9, Figure 14, Figure 15, Figure 17, Figure 18, Figure 19, Figure 20, Figure 21, Figure 22, Figure 23, Figure 24, Figure 25, Figure 26, Figure 27, Figure 28, Figure 29, Figure 30, Figure 31 , any of the connectors shown in Figure 32, Figure 33 or Figure 34.

Enfortumab is commercially available as the antibody-drug conjugate Enfortumab vedotin and sold under the name Padcev. Ennosumab Vedotin includes the anti-conjugin-4 antibody ennosumab and the linker maleimidocaproyl-L-valine-L-citrulline-PAC-MMAE (mc-vc-PABC -MMAE), which is commonly known as vedotin. The mc-vc-PABC-MMAE linker of ennosumab vedotin couples to free cysteine residues contained in the antibody. The antibody ennosumab has been disclosed in WO 2012/047724, the entirety of which is incorporated herein by reference in its entirety.

In certain embodiments, the antibody is ennozumab comprising the heavy chain set forth in SEQ ID NO:9 and the light chain set forth in SEQ ID NO:10. However, the present invention also includes variants of ennosumab, wherein the heavy chain and/or the light chain are at least 80%, at least 85%, and at least 90% identical to SEQ ID NO:9 and/or SEQ ID NO:10, respectively. , at least 95% sequence identity. In particular, the antibody may comprise any sequence variant disclosed in WO 2012/047724. In certain embodiments, the light chain of ennozumab can comprise a mutation in residue Q55 of SEQ ID NO:10. In particular, this mutation is Q55N (SEQ ID NO: 11).

It is preferred herein that the ennosumab or anti-conjugin-4 antibody is present in the antibody-linker conjugate in a glycosylated form. That is, ennosumab or anti-bindin-4 antibodies are preferably glycosylated at residue N297 (EU numbering). However, ennosumab or anti-bindin-4 antibodies can also be deglycosylated as described herein.

Ennosumab or anti-bindingin-4 antibodies may be coupled to any of the linkers disclosed herein, particularly for use in methods according to the invention. Preferably, the linker is coupled to glutamine residue Q295 (EU numbering) of the antibody in an MTG-catalyzed manner. However, the linker may also be coupled to an engineered glutamine residue (such as N297Q (EU numbering)) and/or to any of the glutamine-containing tags disclosed herein.

The linker coupled to the ennozumab or anti-bindin-4 antibody may include a single linker moiety or payload B, or may include multiple linker moieties and/or payloads B ₁ , B ₂ , etc.

That is, in certain embodiments, the linker included in the ennosumab-linker conjugate can include one or more linking moieties B. Such ennosumab-linker conjugates can subsequently be functionalized with a suitable payload in the two-step process disclosed herein.

In certain embodiments, the linker included in the ennosumab-payload conjugate can contain one or more Payload Bs. Such ennosumab-payload conjugates can be obtained in a two-step process, where the linker containing the linking moiety has been conjugated to ennosumab in the first step, and the payload has been Connect with the connecting part. Alternatively, ennosumab-payload conjugates can be obtained in a one-step process in which the payload-containing linker is coupled directly to ennosumab.

In certain embodiments, the invention relates to an antibody-drug conjugate comprising ennozumab or an anti-conjugin-4 antibody. That is, the linker included in the antibody-drug conjugate can comprise one or more toxins described herein.

In certain embodiments, an antibody-drug conjugate comprising ennosumab or an anti-bindingin-4 antibody can comprise the amino acid sequence RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO:3), RKR (SEQ ID NO:4) or RK-Val-Cit (SEQ ID NO:54).

That is, in a specific embodiment, the invention relates to an antibody-drug conjugate comprising:

a) ennosumab or anti-bindingin-4 antibody; and

b) A linker comprising drug moiety B, wherein drug moiety B is covalently linked to an amino acid sequence selected from the group consisting of: RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 2) NO:3), RKR (SEQ ID NO:4) or RK-Val-Cit (SEQ ID NO:54);

Among them, the linker is coupled to ennosumab or anti-conjugation 4 antibody via an isopeptide bond at the γ-carboxamide of glutamine residue Q295 (EU numbering) of the _CH 2 domain of the antibody. The group is formed between a primary amine contained in the side chain of a lysine residue contained in the linker.

Preferably, the antibody is ennosumab comprising the heavy chain shown in SEQ ID NO:9 and the light chain shown in SEQ ID NO:10. Therefore, in a specific embodiment, the invention relates to an antibody-drug conjugate according to the invention, wherein the IgG antibody is ennozumab or comprises a heavy chain as set forth in SEQ ID NO: 9 and as set forth in Antibodies to the light chain shown in SEQ ID NO:10.

In certain embodiments, a linker coupled to ennosumab or an anti-conjugin-4 antibody can comprise the structure RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO:4) or RK-Val-Cit (SEQ ID NO:54). In certain embodiments, the structure RKAA (SEQ ID NO:1), RKA (SEQ ID NO:2), ARK (SEQ ID NO:3), RKR (SEQ ID NO:4) or RK-Val-Cit is included. The linker of (SEQ ID NO:54) can be coupled to residue Q295 of ennozumab or anti-conjugin-4 antibody via a primary amine contained in residue K.

In certain embodiments, ennosumab or anti-bindin-4 antibodies can be coupled to the linker RKAA-B disclosed herein, wherein B is preferably a toxin. In certain embodiments, ennosumab or an anti-bindingin-4 antibody can be coupled to the linker RKAA-(self-cleaving moiety)-B disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the C-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a PABC or a methylamine-containing group.

In certain embodiments, ennosumab or anti-bindingin-4 antibodies can be coupled to the linker RKAA-PABC-B. In certain embodiments, ennosumab or anti-bindin-4 antibodies can be coupled to the linker RKAA-PABC-MMAE (see Figure 1). In certain embodiments, ennosumab or anti-bindin-4 antibodies can be coupled to the linker RKAA-PABC-maytansine (see Figure 8). In certain embodiments, ennosumab or anti-bindingin-4 antibodies can be coupled to the linker RKAA-MMAE. In certain embodiments, ennosumab or anti-bindin-4 antibodies can be coupled to the linker RKAA-PABC-PNP-MMAE. In certain embodiments, ennosumab or anti-bindin-4 antibody can be coupled to the linker RKAA-(NH)-(CH ₃ )-OB, where O is the oxygen atom of the ether bond and B is the linker RKAA-(NH)-(CH 3 )-OB. Hydroxy toxin. In certain embodiments, ennosumab or anti-bindin-4 antibodies can be coupled to the linker RKAA-(NH)-( _CH3 )-O-camptothecin. In certain embodiments, ennosumab or anti-bindin-4 antibody can be coupled to the linker RKAA-(NH)-(CH ₃ )-SB, where S is the sulfur atom of the thioether bond and B is Thiol-containing toxins. In certain embodiments, ennosumab or anti-bindingin-4 antibodies can be coupled to the linker RKAA-(NH)-(CH ₃ )-S-DM1.

In certain embodiments, ennosumab or anti-bindin-4 antibodies can be coupled to the linker B-RKAA disclosed herein, wherein B is preferably a toxin. In certain embodiments, ennosumab or anti-bindingin-4 antibodies can be coupled to the linker B-(self-cleaving moiety)-RKAA disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the N-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a self-cleaving moiety that includes an ortho-hydroxyl protected aryl sulfate (OHPAS) moiety. In certain embodiments, similar to Figure 9, amine-containing payload B can be coupled to the N-terminal arginine moiety via a dicarboxylic acid linker.

In certain embodiments, ennosumab or anti-bindin-4 antibodies can be coupled to the linker RKA-B disclosed herein, wherein B is preferably a toxin. In certain embodiments, ennosumab or anti-bindin-4 antibodies can be coupled to the linker RKA-(self-cleaving moiety)-B disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the C-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a PABC or a methylamine-containing group.

In certain embodiments, ennosumab or anti-bindin-4 antibodies can be coupled to the linker RKA-PABC-B. In certain embodiments, ennosumab or anti-bindin-4 antibodies can be coupled to the linker RKA-PABC-MMAE (see Figure 2). In certain embodiments, ennosumab or anti-conbindin-4 antibodies can be coupled to the linker RKA-PABC-maytansine. In certain embodiments, ennosumab or anti-bindingin-4 antibodies can be coupled to the linker RKA-MMAE. In certain embodiments, ennosumab or anti-bindin-4 antibodies can be coupled to the linker RKA-PABC-PNP-MMAE. In certain embodiments, ennosumab or anti-bindin-4 antibody can be coupled to the linker RKA-(NH)-(CH ₃ )-OB, where O is the oxygen atom of the ether bond and B is the linker RKA-(NH)-(CH 3 )-OB. Hydroxy toxin. In certain embodiments, ennosumab or anti-bindin-4 antibodies can be coupled to the linker RKA-(NH)-( _CH3 )-O-camptothecin. In certain embodiments, ennosumab or anti-conjugin-4 antibody can be coupled to the linker RKA-(NH)-(CH ₃ )-SB, where S is the sulfur atom of the thioether bond and B is Thiol-containing toxins. In certain embodiments, ennosumab or anti-conjugin-4 antibody can be coupled to the linker RKA-(NH)-(CH ₃ )-S-DM1.

In certain embodiments, ennosumab or anti-bindin-4 antibodies can be coupled to the linker B-RKA disclosed herein, wherein B is preferably a toxin. In certain embodiments, ennosumab or anti-bindin-4 antibodies can be coupled to the linker B-(self-cleaving moiety)-RKA disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the N-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a self-cleaving moiety that includes an ortho-hydroxyl protected aryl sulfate (OHPAS) moiety. In certain embodiments, similar to Figure 9, amine-containing payload B can be coupled to the N-terminal arginine moiety via a dicarboxylic acid linker.

In certain embodiments, ennosumab or anti-bindingin-4 antibodies can be coupled to the linker ARK-B disclosed herein, wherein B is preferably a toxin. In certain embodiments, ennosumab or anti-bindingin-4 antibodies can be coupled to the linker ARK-(auto-cleaving moiety)-B disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the C-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a PABC or a methylamine-containing group.

In certain embodiments, ennosumab or anti-bindingin-4 antibodies can be coupled to the linker ARK-PABC-B. In certain embodiments, ennosumab or anti-bindingin-4 antibodies can be coupled to the linker ARK-PABC-MMAE (see Figure 3). In certain embodiments, ennosumab or anti-conbindin-4 antibodies can be coupled to the linker ARK-PABC-maytansine. In certain embodiments, ennosumab or anti-bindingin-4 antibodies can be coupled to the linker ARK-MMAE. In certain embodiments, ennosumab or anti-bindingin-4 antibodies can be coupled to the linker ARK-PABC-PNP-MMAE. In certain embodiments, ennosumab or anti-bindingin-4 antibody can be coupled to the linker ARK-(NH)-(CH ₃ )-OB, where O is the oxygen atom of the ether bond, and B is the linker ARK-(NH)-(CH 3 )-OB. Hydroxy toxin. In certain embodiments, ennosumab or anti-conjugin-4 antibodies can be coupled to the linker ARK-(NH)-( _CH3 )-O-camptothecin. In certain embodiments, ennosumab or anti-bindingin-4 antibodies can be coupled to the linker ARK-(NH)-(CH ₃ )-SB, where S is the sulfur atom of the thioether bond and B is Thiol-containing toxins. In certain embodiments, ennosumab or anti-bindingin-4 antibodies can be coupled to the linker ARK-(NH)-(CH ₃ )-S-DM1.

In certain embodiments, ennosumab or anti-bindin-4 antibodies can be coupled to the linker B-ARK disclosed herein, wherein B is preferably a toxin. In certain embodiments, ennosumab or anti-bindin-4 antibodies can be coupled to the linker B-(self-cleaving moiety)-ARK disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the N-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a self-cleaving moiety that includes an ortho-hydroxyl protected aryl sulfate (OHPAS) moiety.

In certain embodiments, ennosumab or anti-bindin-4 antibodies can be coupled to the linker RKR-B disclosed herein, wherein B is preferably a toxin. In certain embodiments, ennosumab or anti-bindingin-4 antibodies can be coupled to the linker RKR-(self-cleaving moiety)-B disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the C-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a PABC or a methylamine-containing group.

In certain embodiments, ennosumab or anti-bindin-4 antibodies can be coupled to the linker RKR-PABC-B. In certain embodiments, ennosumab or anti-bindin-4 antibodies can be coupled to the linker RKR-PABC-MMAE. In certain embodiments, ennosumab or anti-conbindin-4 antibodies can be coupled to the linker RKR-PABC-maytansine. In certain embodiments, ennosumab or anti-bindingin-4 antibodies can be coupled to the linker RKR-MMAE. In certain embodiments, ennosumab or anti-bindin-4 antibodies can be coupled to the linker RKR-PABC-PNP-MMAE. In certain embodiments, ennosumab or anti-bindin-4 antibodies can be coupled to the linker RKR-(NH)-(CH ₃ )-OB, where O is the oxygen atom of the ether bond and B is the linker RKR-(NH)-(CH 3 )-OB. Hydroxy toxin. In certain embodiments, ennosumab or anti-bindin-4 antibodies can be coupled to the linker RKR-(NH)-( _CH3 )-O-camptothecin. In certain embodiments, ennosumab or anti-conjugin-4 antibody can be coupled to the linker RKR-(NH)-(CH ₃ )-SB, where S is the sulfur atom of the thioether bond and B is Thiol-containing toxins. In certain embodiments, ennosumab or anti-bindin-4 antibodies can be coupled to the linker RKR-(NH)-( _CH3 )-S-DM1.

In certain embodiments, ennosumab or anti-bindin-4 antibodies can be coupled to the linker B-RKR disclosed herein, wherein B is preferably a toxin. In certain embodiments, ennosumab or anti-bindin-4 antibodies can be coupled to the linker B-(self-cleaving moiety)-RKR disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the N-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a self-cleaving moiety that includes an ortho-hydroxyl protected aryl sulfate (OHPAS) moiety. In certain embodiments, as illustrated in Figure 9, amine-containing payload B can be coupled to an N-terminal arginine moiety via a dicarboxylic acid linker.

In certain embodiments, ennosumab or anti-bindin-4 antibodies can be coupled to the linker RK-Val-Cit-B disclosed herein, wherein B is preferably a toxin. In certain embodiments, ennosumab or anti-bindingin-4 antibodies can be coupled to the linker RK-Val-Cit-(self-cleaving moiety)-B disclosed herein, wherein B is preferably a toxin. The self-cleaving moiety may be any self-cleaving moiety known in the art and/or disclosed herein that is suitable for coupling a payload to the C-terminus of a peptide. In certain embodiments, the self-cleaving moiety may be a PABC or a methylamine-containing group.

In certain embodiments, ennosumab or anti-bindin-4 antibodies can be coupled to the linker RK-Val-Cit-PABC-B. In certain embodiments, ennosumab or anti-bindingin-4 antibodies can be coupled to the linker RK-Val-Cit-PABC-MMAE. In certain embodiments, ennosumab or anti-conbindin-4 antibodies can be coupled to the linker RK-Val-Cit-PABC-maytansine. In certain embodiments, ennosumab or anti-bindingin-4 antibodies can be coupled to the linker RK-Val-Cit-MMAE. In certain embodiments, ennosumab or anti-bindin-4 antibodies can be coupled to the linker RK-Val-Cit-PABC-PNP-MMAE. In certain embodiments, ennosumab or anti-bindin-4 antibody can be coupled to the linker RK-Val-Cit-(NH)-(CH ₃ )-OB, where O is the oxygen atom of the ether bond, And B is a hydroxyl-containing toxin. In certain embodiments, ennosumab or anti-bindin-4 antibodies can be coupled to the linker RK-Val-Cit-(NH)-(CH ₃ )-O-camptothecin. In certain embodiments, ennosumab or anti-conjugin-4 antibody can be coupled to the linker RK-Val-Cit-(NH)-(CH ₃ )-SB, where S is the sulfur atom of the thioether bond , and B is a thiol-containing toxin. In certain embodiments, ennosumab or anti-conbindin-4 antibodies can be coupled to the linker RK-Val-Cit-(NH)-(CH ₃ )-S-DM1.

Further, the present invention relates to linker constructs comprising the motif RK. The linker construct according to the invention can be used for the coupling of a variety of antibodies. Due to the highly conserved coupling site Q295, the linker conjugates according to the invention can be used "off the shelf" to produce antibody-payload conjugates for essentially any IgG type antibody. Compared to linkers known in the prior art, the RK linkers of the present invention can be used for efficient coupling of glycosylated antibodies and even lead to high coupling efficiencies when bulky payloads (such as toxins) are included.

Thus, in a specific embodiment, the present invention relates to a linker construct comprising the following structure:

(Sp ₁ )-RK-(Sp ₂ )-B-(Sp ₃ ) or

(Sp ₁ )-B-(Sp ₂ )-RK-(Sp ₃ ); where

·(Sp ₁ ) is a chemical spacer or does not exist;

·(Sp ₂ ) is a chemical spacer or does not exist;

·(Sp ₃ ) is a chemical spacer or does not exist;

·R is arginine or arginine derivative or arginine mimetic;

·K is lysine or lysine derivative or lysine mimetic;

·B is the connection part or payload.

It will be understood that the linker construct may have the same linker as disclosed above for the method according to the invention, for the antibody-linker conjugate according to the invention and/or for the antibody-drug conjugate according to the invention. structure and/or characteristics.

In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the chemical spacers (Sp ₁ ), (Sp ₂ ) and (Sp ₃ ) each independently comprise from 0 to 12 amino acid residues.

In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the linker comprises no more than 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 amino acid residues.

In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the net charge of the linker is neutral or positive.

In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the linker does not comprise negatively charged amino acid residues.

In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the linker comprises the amino acid sequence RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3) , RKR (SEQ ID NO:4) or RK-Val-Cit (SEQ ID NO:54).

In a specific embodiment, the invention relates to a linker construct according to the invention, wherein B is the linking moiety.

In a particular embodiment, the invention relates to a linker construct according to the invention, wherein the linking portion B comprises

·Bioorthogonal labeling groups, or

· Non-bioorthogonal entities for cross-linking.

In a particular embodiment, the invention relates to a construct according to the invention, wherein the bioorthogonal labeling group or the non-bioorthogonal entity for cross-linking consists of or contains at least one molecule or moiety , the molecule or moiety is selected from the group consisting of:

·–NN≡N or –N ₃ ;

·Lys(N ₃ );

·Tetrazine;

·Alkynes;

·Strained cyclooctyne;

·BCN;

·Strained olefin;

·Photoreactive groups;

·aldehyde;

·Acyl trifluoroborate;

·Protein degradation agent ('PROTAC');

·Cyclopentadiene/spirocyclopentadiene;

·Thio-selective electrophile;

·-SH; and

·Cysteine.

In a particular embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct comprises or consists of the structure RKAA-B or B-RKAA, in particular wherein B is Lys( _N3 ) or half Cystine.

That is, in specific embodiments, the invention relates to linkers comprising the structure RKAA-B or B-RKAA, where B is the linking moiety. It should be understood that B can be any connecting moiety known in the art and/or disclosed herein. In certain embodiments, B can be a thiol-containing linker (such as cysteine), an azide-containing linker (such as Lys( _N3 )), or a tetrazine-containing linker. It will be appreciated that linkers comprising structure RKAA-B or B-RKAA may contain additional amino acid residues, linking moieties, payloads, and/or other chemical groups, such as, but not limited to, PEG moieties. In certain embodiments, the linker construct consists of structure RKAA-B or B-RKAA.

In a particular embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct comprises or consists of the structure RKA-B or B-RKA, in particular wherein B is Lys( _N3 ) or half Cystine.

That is, in specific embodiments, the invention relates to linkers comprising the structure RKA-B or B-RKA, where B is the connecting moiety. It should be understood that B can be any connecting moiety known in the art and/or disclosed herein. In certain embodiments, B can be a thiol-containing linker (such as cysteine), an azide-containing linker (such as Lys( _N3 )), or a tetrazine-containing linker. It will be appreciated that linkers comprising structure RKA-B or B-RKA may contain additional amino acid residues, linking moieties, payloads, and/or other chemical groups, such as, but not limited to, PEG moieties. In certain embodiments, the linker construct consists of structure RKA-B or B-RKA.

In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct comprises or consists of the structure ARK-B or B-ARK, in particular wherein B is Lys( _N3 ) or half Cystine.

That is, in a specific embodiment, the present invention relates to a joint comprising the structure ARK-B or B-ARK, where B is the connecting portion. It should be understood that B can be any connecting moiety known in the art and/or disclosed herein. In certain embodiments, B can be a thiol-containing linker (such as cysteine), an azide-containing linker (such as Lys( _N3 )), or a tetrazine-containing linker. It will be appreciated that linkers comprising structure ARK-B or B-ARK may contain additional amino acid residues, linking moieties, payloads, and/or other chemical groups, such as, but not limited to, PEG moieties. In certain embodiments, the linker construct consists of structure ARK-B or B-ARK.

In a particular embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct comprises or consists of the structure RKR-B or B-RKR, in particular wherein B is Lys( _N3 ) or half Cystine.

That is, in particular embodiments, the present invention relates to linkers comprising the structure RKR-B or B-RKR, where B is the connecting moiety. It should be understood that B can be any connecting moiety known in the art and/or disclosed herein. In certain embodiments, B can be a thiol-containing linker (such as cysteine), an azide-containing linker (such as Lys( _N3 )), or a tetrazine-containing linker. It will be appreciated that linkers comprising structure RKR-B or B-RKR may contain additional amino acid residues, linking moieties, payloads, and/or other chemical groups, such as, but not limited to, PEG moieties. In certain embodiments, the linker construct consists of structure RKR-B or B-RKR.

In a specific embodiment, the invention relates to a linker construct according to the invention, wherein B is the payload.

In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the payload includes at least one of:

·toxin;

·Cytokines;

·Growth factors;

·Radionuclides;

·hormone;

·Antiviral agents;

·Antibacterial agents;

·Fluorescent dyes;

·Immune modulators/immunostimulants;

·Half-life increased part;

·Solubility increased part;

·Polymer-toxin conjugates;

·Nucleic acid;

·Biotin or streptavidin moiety;

·Vitamins;

·Protein degradation agent (‘PROTAC’);

·Target binding moiety; and/or

·Anti-inflammatory agent.

In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the toxin is selected from at least one of the group consisting of:

·pyrrolobenzodiazepines (e.g., PBD);

·Auristatin (such as MMAE, MMAF);

Maytansinoids (e.g., maytansinoids, DM1, DM4, DM21);

·Becarcinomycin;

·Nicotinamide phosphoribosyltransferase (NAMPT) inhibitor;

·Microtubulysin;

· Enedyne (e.g., calicheamicin);

·Anthracycline derivatives (PNU) (e.g., doxorubicin);

·Pyrrolyl kinesin spindle protein (KSP) inhibitor;

·Nodulin;

·Drug efflux pump inhibitors;

·Sandromycin;

·Amanitanic acid (e.g., alpha-amanitinine); and

· Camptothecins (e.g., ixotecan, drotecan).

In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the chemical spacer ( _Sp2 ) comprises a self-cleaving moiety.

In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the self-cleaving moiety is directly attached to the payload B.

In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the self-cleaving moiety comprises a p-aminobenzylcarbamoyl (PABC) moiety.

In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct comprises or consists of the structure RKAA-(self-cleaving moiety)-B. That is, the linker construct may include any self-cleavable moiety known in the art and/or disclosed herein that is suitable for coupling Payload B to the C-terminus of the peptide. In certain embodiments, payload B may be a toxin. Those skilled in the art are aware of toxins that can be coupled to the C-terminus of the peptide via a suitable self-cleaving moiety. Preferably, the self-cleaving moiety is any one of the self-cleaving moieties disclosed herein, such as, but not limited to, a PABC-based self-cleaving linker, a PABE-based self-cleaving linker, or a methylamine-containing self-cleaving moiety.

In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct comprises or consists of structure B-(self-cleaving moiety)-RKAA. That is, the linker construct may comprise any self-cleavable moiety known in the art and/or disclosed herein that is suitable for coupling payload B to the N-terminus of the peptide. In certain embodiments, payload B may be a toxin. Those skilled in the art are aware of toxins that can be coupled to the N-terminus of the peptide via a suitable self-cleaving moiety. Preferably, the self-cleaving moiety is any of the self-cleaving moieties disclosed herein, such as, but not limited to, a self-cleaving moiety comprising an OHPAS moiety.

In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct comprises or consists of structure B-RKAA or RKAA-B. That is, payload B can be coupled directly to the N-terminus or C-terminus of the peptide. In cases where the functional groups contained in the payload are incompatible with the N-terminus and/or C-terminus of the peptide, linker molecules can be used to couple the payload to the N-terminus and/or C-terminus of the peptide, respectively. Suitable linker molecules for coupling a payload to the N-terminus or C-terminus of a peptide are disclosed herein.

In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct comprises or consists of the structure RKAA-PABC-B, in particular wherein B is auristatin or maytansinoid, In particular, auristatin is MMAE, and maytansinoids are maytansinoids.

That is, in a specific embodiment, the invention relates to a linker comprising the structure RKAA-B, where B is the payload. It should be understood that B may be any payload known in the art and/or disclosed herein. In certain embodiments, B can be a toxin. In certain embodiments, payload B can be separated from the peptide RKAA by a self-cleaving moiety. Thus, the linker may comprise or consist of the structure RKAA-(self-cleaving moiety)-B, where B is the payload. In certain embodiments, the self-cleaving moiety may be a PABC or methylamino group. Thus, in certain embodiments, a linker may include or consist of the structure RKAA-PABC-B. In certain embodiments, the linker can include or consist of the structure RKAA-(NH)-( _CH3 )-OB, where O is an oxygen atom contained in an ether bond and B is a payload containing a hydroxyl group. In certain embodiments, the linker can comprise or consist of the structure RKAA-(NH)-(CH ₃ )-SB, where S is a sulfur atom contained in a thioether bond and B is a thiol-containing molecule. load. In certain embodiments, the payload may be auristatin or maytansinoids. In certain embodiments, auristatin can be MMAE. In such embodiments, the linker may include or consist of the structure RKAA-PABC-MMAE or RKAA-MMAE. In certain embodiments, the maytansinoid may be maytansine. In such embodiments, the linker may comprise or consist of the structure RKAA-PABC-maytansine or RKAA-maytansine. In certain embodiments, the maytansinoid may be DM1 or a DM1 derivative. In such embodiments, the linker may include or consist of the structure RKAA-(NH)-( _CH3 )-S-DM1. In certain embodiments, the payload may be camptothecin. In such embodiments, the linker may include or consist of the structure RKAA-(NH)-( _CH3 )-O-camptothecin.

In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct comprises or consists of the structure RKA-(self-cleaving moiety)-B. That is, the linker construct may include any self-cleavable moiety known in the art and/or disclosed herein that is suitable for coupling Payload B to the C-terminus of the peptide. In certain embodiments, payload B may be a toxin. Those skilled in the art are aware of toxins that can be coupled to the C-terminus of the peptide via a suitable self-cleaving moiety. Preferably, the self-cleaving moiety is any one of the self-cleaving moieties disclosed herein, such as, but not limited to, a PABC-based self-cleaving linker, a PABE-based self-cleaving linker, or a methylamine-containing self-cleaving moiety.

In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct comprises or consists of structure B-(self-cleaving moiety)-RKA. That is, the linker construct may comprise any self-cleavable moiety known in the art and/or disclosed herein that is suitable for coupling payload B to the N-terminus of the peptide. In certain embodiments, payload B may be a toxin. Those skilled in the art are aware of toxins that can be coupled to the N-terminus of the peptide via a suitable self-cleaving moiety. Preferably, the self-cleaving moiety is any of the self-cleaving moieties disclosed herein, such as, but not limited to, a self-cleaving moiety comprising an OHPAS moiety.

In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct comprises or consists of structure B-RKA or RKA-B. That is, payload B can be coupled directly to the N-terminus or C-terminus of the peptide. In cases where the functional groups contained in the payload are incompatible with the N-terminus and/or C-terminus of the peptide, linker molecules can be used to couple the payload to the N-terminus and/or C-terminus of the peptide, respectively. Suitable linker molecules for coupling a payload to the N-terminus or C-terminus of a peptide are disclosed herein.

In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct comprises or consists of the structure RKA-PABC-B, in particular wherein B is auristatin or maytansinoid, In particular, auristatin is MMAE, and maytansinoids are maytansinoids.

That is, in a specific embodiment, the invention relates to a joint comprising structure RKA-B, where B is the payload. It should be understood that B may be any payload known in the art and/or disclosed herein. In certain embodiments, B can be a toxin. In certain embodiments, payload B can be separated from the peptide RKA by a self-cleaving moiety. Thus, the linker may comprise or consist of the structure RKA-(self-cleaving moiety)-B, where B is the payload. In certain embodiments, the self-cleaving moiety may be a PABC or methylamino group. Thus, in certain embodiments, a linker may include or consist of the structure RKA-PABC-B. In certain embodiments, the linker can include or consist of the structure RKA-(NH)-( _CH3 )-OB, where O is an oxygen atom contained in an ether bond and B is a payload containing a hydroxyl group. In certain embodiments, the linker can comprise or consist of the structure RKA-(NH)-(CH ₃ )-SB, where S is a sulfur atom contained in a thioether bond and B is a thiol-containing molecule. load. In certain embodiments, the payload may be auristatin or maytansinoids. In certain embodiments, auristatin can be MMAE. In such embodiments, the linker may include or consist of the structure RKA-PABC-MMAE or RKA-MMAE. In certain embodiments, the maytansinoid may be maytansine. In such embodiments, the linker may comprise or consist of the structure RKA-PABC-maytansine or RKA-maytansine. In certain embodiments, the maytansinoid may be DM1 or a DM1 derivative. In such embodiments, the linker may comprise or consist of the structure RKA-(NH)-( _CH3 )-S-DM1. In certain embodiments, the payload may be camptothecin. In such embodiments, the linker may include or consist of the structure RKA-(NH)-( _CH3 )-O-camptothecin.

In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct comprises or consists of the structure ARK-(auto-cleaving moiety)-B. That is, the linker construct may include any self-cleavable moiety known in the art and/or disclosed herein that is suitable for coupling Payload B to the C-terminus of the peptide. In certain embodiments, payload B may be a toxin. Those skilled in the art are aware of toxins that can be coupled to the C-terminus of the peptide via a suitable self-cleaving moiety. Preferably, the self-cleaving moiety is any one of the self-cleaving moieties disclosed herein, such as, but not limited to, a PABC-based self-cleaving linker, a PABE-based self-cleaving linker, or a methylamine-containing self-cleaving moiety.

In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct comprises or consists of the structure B-(self-cleaving moiety)-ARK. That is, the linker construct may comprise any self-cleavable moiety known in the art and/or disclosed herein that is suitable for coupling payload B to the N-terminus of the peptide. In certain embodiments, payload B may be a toxin. Those skilled in the art are aware of toxins that can be coupled to the N-terminus of the peptide via a suitable self-cleaving moiety. Preferably, the self-cleaving moiety is any of the self-cleaving moieties disclosed herein, such as, but not limited to, a self-cleaving moiety comprising an OHPAS moiety.

In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct comprises or consists of the structure B-ARK or ARK-B. That is, payload B can be coupled directly to the N-terminus or C-terminus of the peptide. In cases where the functional groups contained in the payload are incompatible with the N-terminus and/or C-terminus of the peptide, linker molecules can be used to couple the payload to the N-terminus and/or C-terminus of the peptide, respectively. Suitable linker molecules for coupling a payload to the N-terminus or C-terminus of a peptide are disclosed herein.

In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct comprises or consists of the structure ARK-PABC-B, in particular wherein B is auristatin or maytansinoid, In particular, auristatin is MMAE, and maytansinoids are maytansinoids.

That is, in a specific embodiment, the invention relates to a joint comprising structure ARK-B, where B is the payload. It should be understood that B may be any payload known in the art and/or disclosed herein. In certain embodiments, B can be a toxin. In certain embodiments, payload B can be separated from the peptide ARK via a self-cleaving moiety. Thus, the linker may include or consist of the structure ARK-(auto-cleaving moiety)-B, where B is the payload. In certain embodiments, the self-cleaving moiety may be a PABC or methylamino group. Thus, in certain embodiments, a linker may include or consist of the structure ARK-PABC-B. In certain embodiments, the linker can include or consist of the structure ARK-(NH)-( _CH3 )-OB, where O is an oxygen atom contained in an ether bond and B is a payload containing a hydroxyl group. In certain embodiments, the linker may comprise or consist of the structure ARK-(NH)-(CH ₃ )-SB, where S is a sulfur atom contained in a thioether bond and B is a thiol-containing molecule. load. In certain embodiments, the payload may be auristatin or maytansinoids. In certain embodiments, auristatin can be MMAE. In such embodiments, the linker may include or consist of the structure ARK-PABC-MMAE or ARK-MMAE. In certain embodiments, the maytansinoid may be maytansine. In such embodiments, the linker may comprise or consist of the structure ARK-PABC-maytansine or ARK-maytansine. In certain embodiments, the maytansinoid may be DM1 or a DM1 derivative. In such embodiments, the linker may comprise or consist of the structure ARK-(NH)-( _CH3 )-S-DM1. In certain embodiments, the payload may be camptothecin. In such embodiments, the linker may include or consist of the structure ARK-(NH)-( _CH3 )-O-camptothecin.

In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct consists of or contains the structure RKR-(self-cleaving moiety)-B. That is, the linker construct may include any self-cleavable moiety known in the art and/or disclosed herein that is suitable for coupling Payload B to the C-terminus of the peptide. In certain embodiments, payload B may be a toxin. Those skilled in the art are aware of toxins that can be coupled to the C-terminus of the peptide via a suitable self-cleaving moiety. Preferably, the self-cleaving moiety is any one of the self-cleaving moieties disclosed herein, such as, but not limited to, a PABC-based self-cleaving linker, a PABE-based self-cleaving linker, or a methylamine-containing self-cleaving moiety.

In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct comprises or consists of structure B-(self-cleaving moiety)-RKR. That is, the linker construct may comprise any self-cleavable moiety known in the art and/or disclosed herein that is suitable for coupling payload B to the N-terminus of the peptide. In certain embodiments, payload B may be a toxin. Those skilled in the art are aware of toxins that can be coupled to the N-terminus of the peptide via a suitable self-cleaving moiety. Preferably, the self-cleaving moiety is any of the self-cleaving moieties disclosed herein, such as, but not limited to, a self-cleaving moiety comprising an OHPAS moiety.

In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct consists of or contains structure B-RKR or RKR-B. That is, payload B can be coupled directly to the N-terminus or C-terminus of the peptide. In cases where the functional groups contained in the payload are incompatible with the N-terminus and/or C-terminus of the peptide, linker molecules can be used to couple the payload to the N-terminus and/or C-terminus of the peptide, respectively. Suitable linker molecules for coupling a payload to the N-terminus or C-terminus of a peptide are disclosed herein.

In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct consists of or comprises the structure RKR-PABC-B, in particular wherein B is auristatin or Maytansinoids, especially wherein auristatin is a MMAE, and wherein the maytansinoids are maytansinoids.

That is, in a specific embodiment, the invention relates to a joint comprising structure RKR-B, where B is the payload. It should be understood that B may be any payload known in the art and/or disclosed herein. In certain embodiments, B can be a toxin. In certain embodiments, payload B can be separated from peptide RKR by a self-cleaving moiety. Thus, the linker may comprise or consist of the structure RKR-(self-cleaving moiety)-B, where B is the payload. In certain embodiments, the self-cleaving moiety may be a PABC or methylamino group. Thus, in certain embodiments, a linker may include or consist of the structure RKR-PABC-B. In certain embodiments, the linker can include or consist of the structure RKR-(NH)-( _CH3 )-OB, where O is an oxygen atom contained in an ether bond and B is a payload containing a hydroxyl group. In certain embodiments, the linker may comprise or consist of the structure RKR-(NH)-(CH ₃ )-SB, where S is a sulfur atom contained in a thioether bond and B is a thiol-containing molecule. load. In certain embodiments, the payload may be auristatin or maytansinoids. In certain embodiments, auristatin can be MMAE. In such embodiments, the linker may include or consist of the structure RKR-PABC-MMAE or RKR-MMAE. In certain embodiments, the maytansinoid may be maytansine. In such embodiments, the linker may comprise or consist of the structure RKR-PABC-maytansine or RKR-maytansine. In certain embodiments, the maytansinoid may be DM1 or a DM1 derivative. In such embodiments, the linker may comprise or consist of the structure RKR-(NH)-( _CH3 )-S-DM1. In certain embodiments, the payload may be camptothecin. In such embodiments, the linker may comprise or consist of the structure RKR-(NH)-( _CH3 )-O-camptothecin.

In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct consists of or contains the structure RK-Val-Cit-(self-cleaving moiety)-B Part)-B. That is, the linker construct may include any self-cleavable moiety known in the art and/or disclosed herein that is suitable for coupling Payload B to the C-terminus of the peptide. In certain embodiments, payload B may be a toxin. Those skilled in the art are aware of toxins that can be coupled to the C-terminus of the peptide via a suitable self-cleaving moiety. Preferably, the self-cleaving moiety is any one of the self-cleaving moieties disclosed herein, such as, but not limited to, a PABC-based self-cleaving linker, a PABE-based self-cleaving linker, or a methylamine-containing self-cleaving moiety.

In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct consists of or contains the structure RK-Val-Cit-B. That is, payload B can be directly coupled to the C-terminus of the peptide. In cases where the functional groups contained in the payload are incompatible with the C-terminus of the peptide, linker molecules can be used to couple the payload to the C-terminus of the peptide. Suitable linker molecules for coupling payloads to the C-terminus of peptides are disclosed herein.

In a specific embodiment, the invention relates to a linker construct according to the invention, wherein the linker construct consists of or comprises the structure RK-Val-Cit-PABC-B, in particular wherein B is auristatin or maytansinoid, particularly wherein auristatin is MMAE, and wherein the maytansinoid is maytansine.

That is, in a specific embodiment, the present invention relates to a linker comprising the structure RK-Val-Cit-B, where B is the payload. It should be understood that B may be any payload known in the art and/or disclosed herein. In certain embodiments, B can be a toxin. In certain embodiments, payload B can be separated from the peptide RK-Val-Cit by a self-cleaving moiety. Thus, the linker may comprise or consist of the structure RK-Val-Cit-(self-cleaving moiety)-B, where B is the payload. In certain embodiments, the self-cleaving moiety may be a PABC or methylamino group. Thus, in certain embodiments, a linker may include or consist of the structure RK-Val-Cit-PABC-B. In certain embodiments, the linker can include or consist of the structure RK-Val-Cit-(NH)-(CH ₃ )-OB, where O is an oxygen atom contained in the ether bond and B is a hydroxyl group contained payload. In certain embodiments, the linker can comprise or consist of the structure RK-Val-Cit-(NH)-(CH ₃ )-SB, where S is a sulfur atom contained in a thioether bond and B is a sulfur atom contained in a thioether bond. Thiol payload. In certain embodiments, the payload may be auristatin or maytansinoids. In certain embodiments, auristatin can be MMAE. In such embodiments, the linker may comprise or consist of the structure RK-Val-Cit-PABC-MMAE or RK-Val-Cit-MMAE. In certain embodiments, the maytansinoid may be maytansine. In such embodiments, the linker may comprise or consist of the structure RK-Val-Cit-PABC-maytansine or RK-Val-Cit-maytansine. In certain embodiments, the maytansinoid may be DM1 or a DM1 derivative. In such embodiments, the linker may include or consist of the structure RK-Val-Cit-(NH)-( _CH3 )-S-DM1. In certain embodiments, the payload may be camptothecin. In such embodiments, the linker may include or consist of the structure RK-Val-Cit-(NH)-( _CH3 )-O-camptothecin.

In a specific embodiment, the invention relates to an antibody-linker conjugate or an antibody-drug conjugate according to the invention, wherein the antibody-linker conjugate or antibody-drug conjugate comprises at least one toxin.

That is, the antibody-linker conjugate or antibody-drug conjugate according to the present invention includes an antibody coupled to at least one linker, wherein one linker includes at least one toxin. In certain embodiments, an antibody-linker conjugate or antibody-drug conjugate contains two linkers, wherein each heavy chain of the antibody is coupled to one linker. In certain embodiments, the antibody-linker conjugate or antibody-drug conjugate comprises four linkers, wherein each heavy chain of the antibody is coupled to two linkers. In this case, each linker may contain one or more payloads, such as toxins.

In certain embodiments, antibody-linker conjugates or antibody-drug conjugates according to the invention comprise two linkers, wherein each linker contains a payload, such as a toxin. In other embodiments, antibody-linker conjugates or antibody-drug conjugates according to the invention comprise two linkers, wherein each linker contains two payloads, such as a toxin and a further payload or Two identical or different toxins. In embodiments where the antibody-linker conjugate or antibody-drug conjugate comprises two linkers, preferably the linker is coupled to residue Q295 of both heavy chains of the IgG antibody. Even more preferably, the antibody is an IgG antibody glycosylated at residue N297.

In certain embodiments, an antibody-linker conjugate or antibody-drug conjugate according to the present invention contains four linkers, wherein each linker contains a payload, such as a toxin. In other embodiments, an antibody-linker conjugate or an antibody-drug conjugate according to the invention comprises four linkers, wherein each linker contains two payloads, such as a toxin and a further payload or Two identical or different toxins. In embodiments where the antibody-linker conjugate or antibody-drug conjugate contains four linkers, it is preferred that the linker is coupled to residues Q295 and N297Q of both heavy chains of the IgG antibody.

In a specific embodiment, the invention relates to an antibody-linker conjugate or an antibody-drug conjugate according to the invention, wherein the antibody-linker conjugate or antibody-drug conjugate comprises two different toxins.

In certain embodiments, antibody-linker conjugates or antibody-drug conjugates according to the invention may comprise two different toxins. That is, in certain embodiments, an antibody-linker conjugate or an antibody-drug conjugate can comprise two linkers, wherein each linker comprises two different toxins. An advantage of antibody-linker conjugates or antibody-drug conjugates containing two different toxins is that they can have increased cytotoxic activity. This increased cytotoxic activity can be achieved by combining two toxins that target two different cellular mechanisms. For example, an antibody-linker conjugate or an antibody-drug conjugate according to the invention may comprise a first toxin that inhibits cell division, and a second toxin that interferes with the replication and/or transcription of DNA.

Therefore, in a specific embodiment, the invention relates to an antibody-linker conjugate or an antibody-drug conjugate according to the invention, wherein the first toxin is a toxin that inhibits cell division and the second toxin is DNA-interfering Replicative and/or transcribed toxins.

Cytostatic toxins, such as antimitotic agents or spindle toxins, are agents that have the potential to inhibit or prevent cell mitosis. Spindle toxins are toxins that disrupt cell division by affecting the threads of proteins that connect the centromeric regions of chromosomes, called spindles. Spindle toxins effectively halt the production of new cells by interrupting the mitotic phase of cell division at the spindle assembly checkpoint (SAC). The mitotic spindle is composed of microtubules (polymerized tubulin), which together with regulatory proteins assist each other in the activity of properly segregated replicating chromosomes. Certain compounds that affect the mitotic spindle have been shown to be highly effective against solid tumors and hematological malignancies.

Two specific families of antimitotic agents—vinca alkaloids and taxanes—interrupt cell division through agitation of microtubule dynamics. Vinca alkaloids act by causing inhibition of tubulin polymerization into microtubules, leading to G2/M arrest during the cell cycle and ultimately cell death. In contrast, taxanes arrest the mitotic cell cycle by stabilizing microtubules against depolymerization. Although there are many other spindle proteins that may be targets of novel chemotherapeutic drugs, tubulin-binding agents are the only type used clinically. Agents that affect the motor protein kinesin are beginning to enter clinical trials. Another type of taxol works by attaching to tubulin within existing microtubules. Preferred toxins that inhibit cell division in the present invention are auristatins (such as MMAE and MMAF) and maytansinoids (such as DM1, DM3, DM4 and/or DM21).

In a specific embodiment, the invention relates to an antibody-linker conjugate or an antibody-drug conjugate according to the invention, wherein at least one toxin is auristatin or a maytansinoid.

Several agents that prevent the correct replication and/or transcription of DNA molecules and have been shown to be suitable for cancer treatment are known to those skilled in the art. For example, antimetabolites (such as nucleotides or nucleoside analogs) that are misincorporated into newly formed DNA and/or RNA molecules are known in the art and have been described by Tsesmetzis et al., Cancers (Basel) , 2018, 10(7):240 Summary. Another toxin known to interfere with DNA replication and/or transcription is duoromycin.

Therefore, in certain embodiments, an antibody-linker conjugate or an antibody-drug conjugate according to the invention comprises two different toxins, wherein the first toxin is doromycin, and wherein the second toxin The payload is auristatin or maytansinoids.

In certain embodiments, the invention relates to an antibody-linker conjugate or an antibody-drug conjugate according to the invention, wherein the antibody-linker conjugate comprises two different auristatins.

A major advantage of antibody-linker conjugates or antibody-drug conjugates containing two different toxins is that the antibody-linker conjugate or antibody-drug conjugate can still target targets that have escaped the mechanism of action of one of the toxins. Cell-acting and/or antibody-payload conjugates may have higher efficacy against heterogeneous tumors.

In a specific embodiment, the invention relates to an antibody-linker conjugate or an antibody-drug conjugate according to the invention, wherein the antibody-linker conjugate comprises an inhibitor of a toxin and a drug efflux transporter.

In a specific embodiment, the invention relates to an antibody-linker conjugate or an antibody-drug conjugate according to the invention, wherein the antibody-linker conjugate or antibody-drug conjugate comprises a toxin and a solubility increasing moiety.

That is, the antibody-linker conjugate or antibody-drug conjugate may include two payloads, where the first payload is a toxin and the second payload is a solubility-increasing moiety. Alternatively, antibody-linker conjugates or antibody-drug conjugates can be made by clicking a toxin to the azide-containing linking portion of the linker and by clicking a maleimide-containing solubility-increasing moiety to the same linker. cysteine side chain. Alternatively, the toxin and/or solubility increasing moiety can be attached to the linker by chemical synthesis.

In a specific embodiment, the invention relates to an antibody-linker conjugate or an antibody-drug conjugate according to the invention, wherein the antibody-linker conjugate comprises a toxin and an immunostimulant.

As used herein and depending on the context, the term "immunostimulant" includes compounds that increase a subject's immune response to an antigen. Examples of immunostimulants include immunostimulants and immune cell activating compounds. Antibody-linker conjugates of the invention may contain immunostimulatory agents that help program immune cells to recognize ligands and enhance antigen presentation. Immune cell activating compounds include Toll-like receptor (TLR) agonists. Such agonists include pathogen-associated molecular patterns (PAMPs), e.g., compositions that mimic infection (such as bacterial-derived immunomodulators (also known as danger signals)) and damage-associated molecular patterns (DAMPs) (e.g., mimic stress or injury). composition of cells). TLR agonists include nucleic acids or lipid compositions (eg, monophosphoryl lipid A (MPLA)). In one example, the TLR agonist includes a TLR9 agonist, such as cytosine-guanosine oligonucleotide (CpG-ODN), poly(ethyleneimine) (PEI)-condensed oligonucleotide (ODN) ( Such as, PEI-CpG-ODN), or double-stranded deoxyribonucleic acid (DNA). In another example, TLR agonists include TLR3 agonists, such as polyinosine-polycytidylic acid (poly(I:C)), PEI-poly(I:C), polyadenylic acid-polyuridylic acid (poly(A:U)), PEI-poly(A:U), or double-stranded ribonucleic acid (RNA). Other exemplary vaccine immunostimulatory compounds include lipopolysaccharide (LPS), chemokines/cytokines, fungal beta-glucans (such as lentin), imiquimod, CRX-527, and OM-174.

In a specific embodiment, the invention relates to an antibody-linker conjugate or an antibody-drug conjugate according to the invention, wherein the antibody-linker conjugate or antibody-drug conjugate comprises two different immunostimulatory agents agent.

In a specific embodiment, the invention relates to an antibody-linker conjugate or an antibody-drug conjugate according to the invention, wherein at least one immunostimulatory agent is a TLR agonist.

As used herein, the term "TLR agonist" refers to a molecule capable of eliciting a signaling response through the TLR signaling pathway (either as a direct ligand or indirectly through the production of endogenous or exogenous ones). An agonist ligand for a TLR receptor is (i) a native ligand of the actual TLR receptor or a functionally equivalent variant thereof that retains the ability to bind to the TLR receptor and induce a costimulatory signal thereon; or ( ii) An agonist antibody directed against a TLR receptor, or a functionally equivalent variant thereof, which is capable of specifically binding to a TLR receptor, more specifically to the extracellular domain of said receptor, and inducing a response thereto. Some immune signals controlled by receptors and related proteins. Binding specificity may be for a human TLR receptor or for a TLR receptor homologous to a human TLR from a different species.

In certain embodiments, antibody-linker conjugates according to the invention may comprise one or more imaging agents. Therefore, in a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the antibody-linker conjugate comprises a radionuclide and a fluorescent dye.

In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, wherein the radionuclide is suitable for tomography, in particular single photon emission computed tomography (SPECT) or positron emission tomography (PET)) radionuclide, and wherein the fluorescent dye is a near-infrared fluorescent dye.

The term "radionuclide" as used herein has the same meaning as radioactive nuclide, radioisotope or radioactive isotope.

The radionuclides are preferably detectable by nuclear medicine molecular imaging techniques such as positron emission tomography (PET), single photon emission computed tomography (SPECT), a mixture of SPECT and/or PET, or a combination thereof. Single photon emission computed tomography (SPECT) herein includes planar scintigraphy (PS).

Mixtures of SPECT and/or PET are, for example, SPECT/CT, PET/CT, PET/IRM or SPECT/IRM.

SPECT and PET obtain information about the concentration (or uptake) of radionuclides introduced into the subject's body. PET generates images by detecting pairs of gamma rays emitted indirectly by positron-emitting radionuclides. PET analysis produces a series of thin-slice images of the body over a region of interest (e.g., brain, breast, liver, etc.). These thin-section images can be assembled into a three-dimensional representation of the area being examined. SPECT is similar to PET, but the radioactive material used in SPECT has a longer decay time than that used in PET and emits single gamma rays instead of double gamma rays. Although SPECT images exhibit lower sensitivity and less detail than PET images, SPECT technology is much cheaper than PET and offers the advantage of not requiring access to a particle accelerator. Actual clinical PET exhibits higher sensitivity and better spatial resolution than SPECT, and exhibits the advantage of precise attenuation correction due to the high energy of photons; therefore PET provides more accurate quantitative data than SPECT. Planar scintigraphy (PS) is similar to SPECT in that it uses the same radionuclides. However, PS only generates 2D information.

SPECT produces computer-generated images of local radioactive tracer uptake, while CT produces 3D anatomical images of the body's X-ray density. Combined SPECT/CT imaging sequentially provides functional information from SPECT and anatomical information from CT, acquired during a single examination. CT data are also used for fast and optimal attenuation correction of single-photon emission data. By pinpointing areas of abnormal and/or physiological tracer uptake, SPECT/CT improves sensitivity and specificity, but can also help achieve accurate dosimetric estimates as well as guide interventional procedures or better define targets for external beam radiotherapy volume. Gamma camera imaging using single-photon emitting radiotracers represents the majority of surgical procedures.

The radionuclide can be selected from Technetium-99m ( ^99m Tc), Gallium-67 ( ⁶⁷ Ga), Gallium-68 ( ⁶⁸ Ga), Yttrium-90 ( ⁹⁰ Y), Indium-111 ( ¹¹¹ In), Rhenium-186 ( ¹⁸⁶ Re), fluorine-18 ( ¹⁸ F), copper-64 ( ⁶⁴ Cu), terbium-149 ( ¹⁴⁹ Tb) or thallium-201 ( ²⁰¹ TI). Radionuclides can be contained in molecules or combined with chelating agents.

In a specific embodiment, the invention relates to the use of a linker construct according to the invention for the production of antibody-linker conjugates by microbial transglutaminase.

That is, the linker constructs described above can be used to produce the antibody-linker conjugates described herein. Preferably, the antibody is an IgG antibody containing endogenous glutamine residue Q295 (EU numbering). In certain embodiments, linkers according to the invention are used to produce antibody-linker conjugates by applying any of the reaction conditions disclosed herein.

Thus, in a specific embodiment, the invention relates to the use according to the invention, wherein the antibody is an IgG antibody, in particular an IgGl antibody.

In a more preferred embodiment, the invention relates to the use according to the invention, wherein the antibody is polotuzumab or trastuzumab or ennozumab.

Further, the present invention relates to a pharmaceutical composition comprising an antibody-linker conjugate or an antibody-drug conjugate according to the present invention.

Thus, in a specific embodiment, the invention relates to a pharmaceutical composition comprising:

a) an antibody-linker conjugate according to the invention, in particular wherein the antibody-linker conjugate comprises at least one payload;

or

b) antibody drug conjugates according to the invention; and

Pharmaceutical compositions contain at least one pharmaceutically acceptable ingredient.

It is understood that pharmaceutical compositions may include antibody-payload conjugates that have been prepared using the one- or two-step methods disclosed herein.

Antibody-Payload Constructs to be Included in Pharmaceutical Compositions The type of payload included in the antibody-payload construct depends on the use of the pharmaceutical composition. In embodiments where the pharmaceutical composition is used to treat a disease, the payload is preferably a drug. If the disease is a neoplastic disease, the payload is preferably a toxin. In embodiments where the pharmaceutical composition is used for diagnosis, the payload is preferably an imaging agent.

Alternatively, the drug may include an antibody-drug conjugate disclosed herein. Pharmaceutical compositions containing antibody-drug conjugates are preferably used to treat diseases.

In a specific embodiment, the invention relates to a pharmaceutical composition according to the invention, comprising at least one additional therapeutically active agent.

Pharmaceutical compositions according to the present invention may comprise at least one pharmaceutically acceptable ingredient.

Pharmaceutically acceptable ingredients are those ingredients in a pharmaceutical preparation that are not toxic to the subject, other than the active ingredients. Pharmaceutically acceptable ingredients include, but are not limited to, buffers, excipients, stabilizers or preservatives.

Pharmaceutical formulations of antibody-linker conjugates described herein are prepared by combining the conjugate with the desired purity with one or more optional pharmaceutically acceptable ingredients (Flemington's Pharmaceutical Sciences 16th edition, Oslo, A. Ed. (1980)) are mixed and prepared as lyophilized preparations or aqueous solutions. Pharmaceutically acceptable ingredients are generally nontoxic to the receptor at the doses and concentrations employed and include, but are not limited to: buffering agents (such as phosphates, citrates, and other organic acids); antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethylammonium chloride; benzalkonium chloride; benzyl chloride; phenol, butanol or benzyl alcohol; p-hydroxybenzene Alkyl formates, such as methyl- or propyl-paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins (such as serum albumin, gelatin or immunoglobulins); hydrophilic polymers (such as polyvinylpyrrolidone); amino acids (such as glycine, glutamine, asparagine, histidine , arginine or lysine); monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; chelating agents (such as EDTA); sugars (such as sucrose, mannitol, trehalose or sorbate) alcohol); a salt-forming counterion (such as sodium); a metal complex (such as a Zn protein complex); and/or a nonionic surfactant (such as polyethylene glycol (PEG)). Exemplary pharmaceutically acceptable ingredients herein also include insterstitial pharmaceutical dispersants, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronic acid glycoprotein, such as rHuPH20 ( Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968. For example, sHASEGP can be combined with one or more additional glycosaminoglycanases, such as chondroitinase.

In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, in particular wherein the antibody-linker conjugate comprises at least one payload, an antibody-drug conjugate according to the invention or an antibody-linker conjugate according to the invention. Pharmaceutical compositions for treatment and/or diagnosis of the present invention.

That is, the antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition according to the present invention can be used to treat a subject or diagnose a disease or condition in a subject. The individual or subject is preferably a mammal. Mammals include, but are not limited to, domesticated animals (eg, cattle, sheep, cats, dogs, and horses), primates (eg, humans and non-human primates (such as macaques)), rabbits, and rodents (such as , mice and rats). In certain embodiments, the individual or subject is a human. When an antibody-linker conjugate or a pharmaceutical composition comprising an antibody-linker conjugate according to the invention is used for therapy, it is preferred that the linker comprises a drug. When an antibody-linker conjugate or a pharmaceutical composition comprising an antibody-linker conjugate according to the invention is used for diagnosis, it is preferred that the linker contains at least one imaging agent.

In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, in particular wherein the antibody-linker conjugate comprises at least one payload, an antibody-drug conjugate according to the invention, or Pharmaceutical composition according to the present invention for use in the treatment of

●Patients suffering from neoplastic diseases, neurological diseases, autoimmune diseases, inflammatory diseases or infectious diseases,

●Patients at risk of developing neoplastic, neurological, autoimmune, inflammatory or infectious diseases, and/or

●Patients diagnosed with neoplastic diseases, neurological diseases, autoimmune diseases, inflammatory diseases or infectious diseases.

In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, in particular wherein the antibody-linker conjugate comprises at least one payload, an antibody-drug conjugate according to the invention or an antibody-linker conjugate according to the invention. The pharmaceutical composition of the present invention is used to treat patients suffering from tumor diseases.

As used herein, the term "neoplastic disease" refers to a disease characterized by uncontrolled, abnormal growth of cells. Neoplastic diseases include cancer. Examples of cancers include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers include breast cancer, prostate cancer, colon cancer, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, ovarian cancer, cervical cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma , liver cancer, bladder cancer, hepatoma, colorectal cancer, cervical cancer, endometrial cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, primary liver cancer ), skin cancer, melanoma, brain cancer, ovarian cancer, neuroblastoma, myeloma, various head and neck cancers, acute lymphoblastic leukemia, acute myeloid leukemia, Ewing sarcoma, and peripheral neuroepithelium tumor. Preferred cancers include liver cancer, lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, Ewing sarcoma, and peripheral neuroepithelial tumors.

That is, the antibody-linker conjugate or antibody-drug conjugate according to the present invention is preferably used for treating cancer. Thus, in certain embodiments, antibody-linker conjugates or antibody-drug conjugates according to the present invention comprise antibodies that specifically bind to antigens present on tumor cells. In certain embodiments, the antigen may be an antigen on the surface of tumor cells. In certain embodiments, when the antibody-linker conjugate binds to the antigen, the antigen on the surface of the tumor cell can be internalized into the cell along with the antibody-linker conjugate.

If the antibody-linker conjugate or antibody-drug conjugate according to the invention is used for the treatment of cancer, it is preferred that the antibody-linker conjugate or antibody-drug conjugate contains at least one payload which has the ability to kill or the potential to inhibit the proliferation of tumor cells to which the antibody-linker conjugate or antibody-drug conjugate binds. In certain embodiments, at least one payload exhibits its cytotoxic activity after the antibody-linker conjugate or antibody-drug conjugate has been internalized into the tumor cell. In certain embodiments, at least one payload is a toxin.

Inflammatory diseases can be autoimmune diseases. Infectious diseases can be bacterial or viral infections.

In certain embodiments, antibody-linker conjugates, antibody-drug conjugates and/or pharmaceutical compositions according to the invention can be used to treat B cell-associated cancers.

Thus, in certain embodiments, the invention relates to an antibody-linker conjugate, an antibody-drug conjugate or a pharmaceutical composition for use according to the invention, wherein the antibody-linker comprised in the pharmaceutical composition The conjugate or antibody-drug-linker conjugate comprises polotuzumab, and wherein the neoplastic disease is a B cell-associated cancer.

Accordingly, preferred antibody-linker conjugates or antibody-drug conjugates include an anti-CD79b antibody disclosed herein, preferably wherein the anti-CD79b antibody is internalized into the target cell upon binding to CD79b. In certain embodiments, the anti-CD79b antibody is polotuzumab having a heavy chain as set forth in SEQ ID NO:5 and a light chain as set forth in SEQ ID NO:6. Further, it is preferred that the antibody-linker conjugate or antibody-drug conjugate contains at least one toxin.

In certain embodiments, an anti-CD79b antibody included in an antibody-linker conjugate, an antibody-drug conjugate, or a pharmaceutical composition can be combined with Figure 1, Figure 2, Figure 3, Figure 8, Figure 9, Figure 14 or any of the linkers shown in Figure 15 or any of the linkers disclosed herein.

The B-cell associated cancer may be any one selected from the group consisting of: high-, intermediate-, and low-grade lymphomas (including B-cell lymphomas such as, for example, mucosa-associated lymphoid tissue B-cell lymphoma and non-Hodgkin Lymphoma (NHL), mantle cell lymphoma, Burkitt lymphoma, small lymphocytic lymphoma, marginal zone lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, and Hodgkin lymphoma and T cell lymphoma) and leukemias (including secondary leukemias, chronic lymphocytic leukemia (CLL) (such as B-cell leukemia (CD5+B lymphocytes)), myeloid leukemias (such as acute myeloid leukemia, chronic myeloid leukemia) ), lymphoid leukemias, such as acute lymphoblastic leukemia (ALL) and myeloid dysplasia), and other hematological and/or B-cell or T-cell related cancers (including cancers of other hematopoietic cells, including polymorphonuclear leukocytes, such as basophils granulocytes, eosinophils, neutrophils and monocytes, dendritic cells, platelets, red blood cells, and natural killer cells). Also included are cancerous B-cell proliferative disorders selected from: lymphoma, non- Hodgkin lymphoma (NHL) aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, Hairy cell leukemia (HCL), acute lymphoblastic leukemia (ALL), and mantle cell lymphoma.

In a specific embodiment, the invention relates to an antibody-linker conjugate, an antibody-drug conjugate or a pharmaceutical composition for use, wherein the B-cell associated cancer is non-Hodgkin's lymphoma, in particular wherein, B The cell-associated cancer is diffuse large B-cell lymphoma.

Further, anti-CD79b antibody-linker conjugates, anti-CD79b antibody-drug conjugates and/or pharmaceutical compositions comprising anti-CD79b antibody-linker conjugates or anti-CD79b antibody-drug conjugates may be used with Used in combination with other therapies to treat B cell-related cancers.

Therefore, in a specific embodiment, the invention relates to an antibody-linker conjugate, an antibody-drug conjugate or a pharmaceutical composition for use according to the invention, wherein the antibody-linker conjugate, antibody-drug The conjugate or pharmaceutical composition is administered in combination with bendamustine and/or rituximab.

It will be appreciated that the antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition does not necessarily have to be administered simultaneously with an additional therapeutic agent, such as bendamustine and/or rituximab. In contrast, the antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition may be administered on a different dosing schedule, and thus on different days, as other therapeutic agents used to treat the same disease.

In certain embodiments, antibody-linker conjugates, antibody-drug conjugates and/or pharmaceutical compositions according to the invention can be used to treat HER2-positive cancers.

Thus, in a specific embodiment, the invention relates to an antibody-linker conjugate, an antibody-drug conjugate or a pharmaceutical composition for use according to the invention, wherein the antibody-linker comprised in the pharmaceutical composition The conjugate or antibody-drug conjugate comprises trastuzumab, and wherein the neoplastic disease is a HER2-positive cancer, in particular HER2-positive breast cancer, gastric cancer, ovarian cancer or lung cancer.

Thus, preferred antibody-linker conjugates or antibody-drug conjugates include anti-HER2/neu antibodies disclosed herein, preferably wherein the anti-HER2/neu antibodies internalize to the target upon binding to HER2/neu in cells. In certain embodiments, the anti-HER2/neu antibody is trastuzumab having a heavy chain set forth in SEQ ID NO:7 and a light chain set forth in SEQ ID NO:8. Further, it is preferred that the antibody-linker conjugate or antibody-drug conjugate contains at least one toxin.

In certain embodiments, anti-HER2/neu antibodies included in antibody-linker conjugates, antibody-drug conjugates, or pharmaceutical compositions can be combined with Figures 1, 2, 3, 8, 9, 14, or 15 Coupled with any of the linkers shown in or any of the linkers disclosed herein.

HER2-positive cancer as used herein may be, but is not limited to, HER2-positive breast cancer, gastric cancer, ovarian cancer, or lung cancer. Technicians can determine whether the cancer is HER2-positive. For example, tumor cells can be isolated in a biopsy and the presence of HER2/neu can be determined using any method known in the art.

Further, anti-HER2/neu antibody-linker conjugates, anti-HER2/neu antibody-drug conjugates and/or comprise anti-HER2/neu antibody-linker conjugates or anti-HER2/neu antibody-drug conjugates The pharmaceutical composition may be used in combination with other therapies suitable for the treatment of HER2-positive cancers.

Therefore, in a specific embodiment, the invention relates to an antibody-linker conjugate, an antibody-drug conjugate or a pharmaceutical composition for use according to the invention, wherein the antibody-linker conjugate, antibody-drug The conjugate or pharmaceutical composition is administered in combination with lapatinib, capecitabine and/or a taxane.

It is understood that the antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition does not necessarily have to be administered simultaneously with additional therapeutic agents (such as lapatinib, capecitabine and/or taxanes) . In contrast, the antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition may be administered on a different dosing schedule, and thus on different days, as other therapeutic agents used to treat the same disease.

In certain embodiments, antibody-linker conjugates, antibody-drug conjugates and/or pharmaceutical compositions according to the present invention can be used to treat bindin-4 positive cancers.

Thus, in a specific embodiment, the invention relates to an antibody-linker conjugate, an antibody-drug conjugate or a pharmaceutical composition for use according to the invention, wherein the antibody-linker conjugate comprised in the pharmaceutical composition The conjugate or antibody-drug conjugate comprises ennosumab or an ennosumab variant, and wherein the neoplastic disease is integrin-4 positive cancer, in particular integrin-4 positive pancreatic cancer, lung cancer, bladder cancer cancer or breast cancer.

Thus, preferred antibody-linker conjugates or antibody-drug conjugates include an anti-Conductin-4 antibody disclosed herein, preferably wherein the anti-Conductin-4 antibody is internalized upon binding to Conbindin-4. in target cells. In certain embodiments, the anti-bindin-4 antibody is ennozumab having a heavy chain as set forth in SEQ ID NO:9 and a light chain as set forth in SEQ ID NO:10. Further, it is preferred that the antibody-linker conjugate or antibody-drug conjugate contains at least one toxin.

In certain embodiments, an anti-bindin-4 antibody included in an antibody-linker conjugate, antibody-drug conjugate, or pharmaceutical composition can be combined with Figure 1, 2, 3, 8, 9, 14, or 15 Coupled with any of the linkers shown in or any of the linkers disclosed herein.

As used herein, integrin-4-positive cancer may be, but is not limited to, integrin-4-positive pancreatic cancer, cancer, bladder cancer, or breast cancer. Technicians can determine whether the cancer is integrin-4-positive. For example, tumor cells can be isolated in a biopsy and the presence of integrin-4 can be determined using any method known in the art.

Further, the anti-conbindin-4 antibody-linker conjugate, the anti-conbindin-4 antibody-drug conjugate and/or the anti-conductin-4 antibody-linker conjugate or the anti-conductin-4 antibody-drug conjugate The pharmaceutical composition of the conjugate may be used in combination with other therapies suitable for the treatment of integrin-4 positive cancers.

Therefore, in a specific embodiment, the invention relates to an antibody-linker conjugate, an antibody-drug conjugate or a pharmaceutical composition for use according to the invention, wherein the antibody-linker conjugate, antibody-drug The conjugate or pharmaceutical composition is administered in combination with a cisplatin-based chemotherapeutic agent and/or pembrolizumab.

It is understood that the antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition does not necessarily have to be administered simultaneously with an additional therapeutic agent, such as a cisplatin-based chemotherapeutic agent and/or pembrolizumab. In contrast, the antibody-linker conjugate, antibody-drug conjugate, or pharmaceutical composition may be administered on a different schedule, and thus on different days, as other therapeutic agents used to treat the same disease.

In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention (especially wherein the antibody-linker conjugate comprises at least one payload), an antibody-drug conjugate according to the invention, or Use of a pharmaceutical composition according to the invention for the manufacture of a medicament for the treatment of,

●Patients suffering from neoplastic diseases, neurological diseases, autoimmune diseases, inflammatory diseases or infectious diseases,

●Patients at risk of developing neoplastic, neurological, autoimmune, inflammatory or infectious diseases, and/or

●Patients diagnosed with neoplastic diseases, neurological diseases, autoimmune diseases, inflammatory diseases or infectious diseases.

In a specific embodiment, the invention relates to a method of treating or preventing a neoplastic disease, comprising administering to a patient in need thereof an antibody-linker conjugate according to the invention, in particular wherein the antibody-linker conjugate The conjugate comprises at least one payload, an antibody-drug conjugate according to the invention, or a pharmaceutical composition according to the invention.

In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention (especially wherein the antibody-linker conjugate comprises at least one payload), an antibody-drug conjugate according to the invention or The pharmaceutical composition of the present invention is used for preoperative, intraoperative or postoperative imaging.

That is, the antibody-linker conjugate according to the present invention can be used in medical imaging. To this end, antibody-linker conjugates can be visualized upon binding to specific target molecules, cells, or tissues. Different techniques are known in the art to visualize specific payloads. For example, if the payload is a radionuclide, the molecules, cells, or tissues bound by the antibody-linker conjugate can be visualized by PET or SPECT. If the payload is a fluorescent dye, the molecules, cells, or tissues bound by the antibody-linker conjugate can be visualized by fluorescence imaging. In certain embodiments, antibody-linker conjugates according to the invention include two different payloads, such as a radionuclide and a fluorescent dye. In this case, two different and/or complementary imaging techniques (e.g., PET/SPECT and fluorescence imaging) can be used to visualize the molecules, cells, or tissues bound by the antibody-linker conjugate.

Antibody-linker conjugates can be used for preoperative, intraoperative and/or postoperative imaging.

Preoperative imaging includes all imaging techniques that can be performed before surgery to make specific target molecules, cells, or tissues visible when diagnosing a disease or condition, and optionally to provide guidance for surgery. Preoperative imaging may include the step of visualizing the tumor by PET or SPECT prior to surgery through the use of an antibody-linker conjugate that specifically binds to an antigen on the tumor and is coupled to a radionuclide including payload of antibodies.

Intraoperative imaging includes all imaging techniques that can be performed during surgery to make specific target molecules, cells, or tissues visible to guide surgery. In certain embodiments, antibody-linker conjugates containing near-infrared fluorescent dyes can be used to visualize tumors by near-infrared fluorescence imaging during surgery. Intraoperative imaging allows surgeons to identify specific tissue, such as tumor tissue, during surgery so that the tumor tissue can be completely removed.

Postoperative imaging includes all imaging techniques that can be performed after surgery to visualize specific target molecules, cells, or tissues and to assess the outcome of the surgery. Postoperative imaging can be performed similarly to preoperative procedures.

In certain embodiments, the invention relates to antibody-linker conjugates comprising two or more different payloads. For example, antibody-linker conjugates can include radionuclides and near-infrared fluorescent dyes. This antibody-payload conjugate can be used for imaging via PET/SPECT and near-infrared fluorescence. The advantage of this antibody is that it can be used to visualize target tissues (such as tumors) before and after surgery via PET or SPECT. At the same time, tumors can be visualized during surgery through near-fluorescence infrared imaging.

In a specific embodiment, the invention relates to an antibody-linker conjugate according to the invention, in particular wherein the antibody-linker conjugate comprises at least one payload, an antibody-drug conjugate according to the invention or an antibody-linker conjugate according to the invention. The pharmaceutical composition of the present invention is used for cancer surgery guided by intraoperative imaging.

As described above, the antibody-linker conjugates of the invention can be used to visualize target molecules, cells or tissues and guide a surgeon or robot during surgery. That is, antibody-linker conjugates can be used to visualize tumor tissue during surgery, for example by near-infrared imaging, and allow complete removal of tumor tissue.

The antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition according to the invention can be administered to a human or animal subject in an amount or dose effective to treat a disease or sufficient for diagnostic purposes.

The antibody-linker conjugates, antibody-drug conjugates or pharmaceutical compositions according to the invention may be administered by any suitable means, including parenteral, intrapulmonary and intranasal administration, and if local treatment is required, Administration can be intralesional, intrauterine, or intravesical. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration may be by any suitable route, such as by injection (such as intravenous or subcutaneous injection), depending in part on whether the administration is transient or long-term. Various dosing regimens are contemplated herein, including, but not limited to, single or multiple dosing at various time points, bolus dosing, and pulse infusion.

Antibody-linker conjugates, antibody-drug conjugates or pharmaceutical compositions according to the invention may be formulated, administered and administered in a manner consistent with the invention and will be formulated, administered in a manner consistent with good medical practice and administration. Factors considered herein include the specific condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of delivery of the agent, the method of administration, the timing of administration, and other factors known to the physician . The antibody-linker conjugates, antibody-drug conjugates or pharmaceutical compositions according to the invention need not be, but are optionally formulated with one or more agents currently used for the prevention or treatment of the condition in question. The effective amount of such other agents depends on the amount of antibody-linker conjugate present in the formulation, the type of condition or treatment, and other factors discussed above. These are generally used at the same dosages and routes of administration as described herein, or from about 1% to 99% of the dosages described herein, or at any dosage and by any route that is empirically/clinically determined to be appropriate.

Appropriate dosages of antibody-linker conjugates, antibody-drug conjugates or pharmaceutical compositions according to the invention (when used alone or in combination with one or more other additional therapeutic agents) for the purpose of preventing or treating disease Will depend on the type of disease to be treated, the type of antibody-payload conjugate, the severity and course of the disease, whether the antibody-linker conjugate is administered for prophylactic or therapeutic purposes, previous treatments, the patient's clinical history and Reaction to antibody-linker conjugates, and the judgment of the attending physician. The antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition according to the present invention is suitable for administration to a patient in one session or in a series of treatments.

Description of the drawings

Figure 1 shows the chemical structure of the RKAA-MMAE linker-payload complex according to the present invention, in which the N-terminally protected Ac-RKAA peptide is covalently linked to PABC and MMAE.

Figure 2 shows the chemical structure of the RKA-MMAE linker-payload complex according to the invention, in which the N-terminally protected Ac-RKA peptide is covalently linked to PABC and MMAE.

Figure 3 shows the chemical structure of the ARK-MMAE linker-payload complex according to the present invention, in which the N-terminally protected Ac-ARK peptide is covalently linked to PABC and MMAE.

Figure 4 shows the chemical structure of the KRA-MMAE linker-payload complex (not according to the invention), in which the N-terminally protected Ac-KRA peptide is covalently linked to PABC and MMAE.

Figure 5 shows the chemical structure of the AKR-MMAE linker-payload complex (not according to the invention), in which the N-terminally protected Ac-AKR peptide is covalently linked to PABC and MMAE.

Figure 6 shows the chemical structure of a KAAR-MMAE linker-payload complex according to the invention (not according to the invention), in which the N-terminally protected Ac-KAAR peptide is covalently linked to PABC and MMAE.

Figure 7 shows the chemical structure of the KARA-MMAE linker-payload complex (not according to the invention) in which the N-terminally protected Ac-KARA peptide is covalently linked to PABC and MMAE.

Figure 8 shows the chemical structure of the RKAA-maytansine linker-payload complex according to the present invention, in which the N-terminally protected Ac-RKAA peptide is covalently linked to PABC and maytansine.

Figure 9 shows the chemical structure of a maytansine-RKR linker-payload complex according to the present invention, wherein maytansine is covalently linked to a C4 alkyl amide spacer attached to the RKR peptide (with a C-terminal amide protecting group) things.

Figure 10 shows the size exclusion chromatogram (SEC) of the antibody-drug conjugate ARA-01-RKAA-PABC-MMAE of the present invention.

Figure 11 depicts the dose-dependent in vitro cytotoxicity of the antibody-drug conjugate ARA-01-RKAA-PABC-MMAE of the present invention on three different CD79b overexpressing cell lines (a-c) and CD79b non-expressing cell lines (d) The result of the action.

Figure 12 shows the levels of polotuzumab (SEQ ID NO: 5 and 6) and the antibody-drug conjugate of the present invention (ARA01-RKAA-PABC) at different time points after a single intravenous injection of 5 mg/kg into CD1 Swiss mice. -MMAE) and polotuzumab vedotin plasma concentration. Determine the concentration in plasma using ELISA. Mean plasma concentrations from 5 mice are plotted versus time, and error bars represent the standard error of the mean (SEM). It should be understood that the abbreviation ARA01-RKAA-MMAE in Figure 12 refers to the antibody-drug conjugate ARA01-RKAAC-PABC-MMAE.

Figure 13 schematically shows the one-step coupling process.

Figure 14 shows the chemical structure of the ARK-PEG2-PABC-MMAE linker-payload complex according to the present invention, in which the N-terminally protected Ac-ARK peptide is covalently linked to the PEG2 spacer and the PABC-MMAE payload.

Figure 15 shows the chemical structure of the ARK-PEG2-(NH)-( _CH3 )-S-C4-maytansinoid linker-payload complex according to the present invention, in which the N-terminally protected Ac-ARK peptide co- The valency is attached to the PEG2-(NH)-(CH ₃ )-S-C4 alkyl spacer and maytansinoid payload.

Figure 16 depicts the Granta 519 mouse tumor model. Human B-cell lymphoma tumor cells (Granta 519) were inoculated subcutaneously into CB17 SCID mice (n=8 per treatment group). When tumors reached a size of approximately 200 ^mm , animals received 0.53 mg/kg or 2.1 mg/kg polotuzumab vedotin and a single injection of ARA01-RKAA-PABC-MMAE at 0.53 mg/kg, 1 mg/kg, or 2.1 mg/kg. ARA01-RKAA-PABC-MMAE provided equivalent tumor growth inhibition and survival relative to polotuzumab vedotin at approximately half the payload dose (Figure 16A, 0.53 mg/kg in Figure 16B kg dose). At approximately equivalent payload doses relative to polozumab vedotin, treatment with ARA01-RKAA-PABC-MMAE resulted in greater tumor response than polozumab vedotin with 0/8 complete tumor responses. Antitumor efficacy and considerable survival advantage, with 6/8 complete tumor responses (Fig. 16B comparing polozumab vedotin at a dose of 0.53 mg/kg and ARA01-RKAA-PABC-MMAE at a dose of 1 mg/kg Compare). Mean tumor volumes are shown as standard error of the mean (SEM).

Figure 17 shows the chemical structure of the RKAA-payload complex according to the present invention, in which the N-terminally protected Ac-RKAA peptide is covalently linked to the ixotecan derivative Dxd via a self-cleaving methylamine linker.

Figure 18 shows the chemical structure of the RKAA-payload complex according to the present invention, in which the N-terminally protected Ac-RKAA peptide is covalently linked to the payload PNU-159682 via the self-cleaving moiety PABC-EDA.

Figure 19 shows the chemical structure of the RKAA-payload complex according to the present invention, in which the N-terminally protected Ac-RKAA peptide is covalently linked to the payload beclomycin GA via the self-cleaving moiety PABC-EDA.

Figure 20 shows the chemical structure of the RKAA-payload complex according to the present invention, in which the N-terminally protected Ac-RKAA peptide is covalently linked to the payload PBD via the self-cleaving moiety PABE.

Figure 21 shows the chemical structure of the RKAA-payload complex according to the present invention, in which the N-terminally protected Ac-RKAA peptide is covalently linked to the payload maytan via a self-cleavable methylamine linker and an additional alkyl linker molecule. white.

Figure 22 shows the chemical structure of the RKR-payload complex according to the present invention, in which the N-terminally protected Ac-RKR peptide is directly coupled to the payload MMAE.

Figure 23 shows the chemical structure of the RKAA-payload complex according to the present invention, in which the N-terminally protected Ac-RKAA peptide is covalently linked to the payload becarmycin via a self-cleaving p-methylaniline (PMA) moiety G.A.

Figure 24 shows the chemical structure of the RKR-payload complex according to the present invention, in which the C-terminally protected RKR amide peptide is covalently linked to the payload PNU-159682 via a carbamate.

Figure 25 shows the chemical structure of the RKR-payload complex according to the present invention, in which the C-terminally protected RKR amide peptide is covalently linked to the payload via a self-cleavable moiety OHPAS-PHB-EDA and an additional (PEG) ₂ moiety. Payload PNU-159682.

Figure 26 shows the chemical structure of a RKR-payload complex according to the invention, in which the C-terminally protected RKR amide peptide is covalently linked to the payload PBD via the self-cleavable moiety OHPAS and an additional (PEG) ₂ moiety.

Figure 27 shows the chemical structure of the RKR-payload complex according to the present invention, in which the C-terminally protected RKR-amide peptide is covalently linked to the payload PBD via the self-cleaving moiety OHPAS.

Figure 28 shows the chemical structure of the RKR-payload complex according to the present invention, in which the C-terminally protected RKR-amide peptide is directly coupled to the payload DM21.

Figure 29 shows the chemical structure of the RKR-payload complex according to the present invention, in which the C-terminally protected RKR amide peptide is covalently linked to the payload DM4 via an alkyl linker molecule containing carboxyl and thiol groups.

Figure 30 shows the chemical structure of a RKR-payload complex according to the present invention, in which the C-terminally protected RKR-amide peptide is covalently linked to the payload MMAE via a dicarboxylic acid linker molecule.

Figure 31 shows the chemical structure of the RKR-payload complex according to the present invention, in which the C-terminally protected RKR amide peptide is covalently linked to the payload MMAE via the self-cleavable moiety OHPAS-PHB and an additional (PEG) ₂ moiety .

Figure 32 shows the chemical structure of the RKR-payload complex according to the present invention, in which the C-terminally protected RKR-amide peptide is covalently linked to the payload MMAE via the self-cleaving moiety OHPAS-PHB.

Figure 33 shows the chemical structure of a RKR-payload complex according to the present invention, in which a C-terminally protected RKR amide peptide is covalently linked to the payload via a self-cleaving moiety OHPAS quaternary ammonium and an additional (PEG) ₂ moiety Oncomycin GA.

Figure 34 shows the chemical structure of a RKR-payload complex according to the present invention, in which the C-terminally protected RKR-amide peptide is covalently linked to the payload becanomycin GA via the self-cleaving moiety OHPAS quaternary ammonium.

Figure 35 depicts the dose-dependent in vitro effects of the antibody-drug conjugate trastuzumab-RKAA-PABC-MMAE or trastuzumab-RKAA-PABC-maytansine on the HER2-positive cell line SKBR-3 of the present invention. Result of cytotoxic effects.

Figure 36 depicts the dose dependence of the anti-CD79b antibody-drug conjugates ARA01-ARK-PABC-MMAE, ARA01-RKA-PABC-MMAE and ARA01-RKValCit-PABC-MMAE of the present invention on the CD79b positive cell line Granta-519 in vitro. Result of cytotoxic effects.

Figure 37 depicts the binding of the anti-bindin-4 antibody-drug conjugates ARA04-RKAA-PABC-MMAE, ARA04-ARK-PABC-MMAE, ARA04-RKA-PABC-MMAE and ARA04-RKValCit-PABC-MMAE of the invention. Results of the dose-dependent in vitro cytotoxicity of SUM190PT, a positive cell line of SUM-4.

Figure 38 depicts the binding of anti-bindin-4 antibody-drug conjugates ARA04-RKAA-PABC-MMAE, ARA04-ARK-PABC-MMAE, ARA04-RKA-PABC-MMAE and ARA04-RKValCit-PABC-MMAE of the invention. Results of the dose-dependent in vitro cytotoxicity of the protein-4-negative cell line A549.

Figure 39 shows the antibody-drug conjugate of the present invention (ARA01-ARK) of polotuzumab/ARA01 (SEQ ID NO: 5 and 6) at different time points after a single intravenous injection of 5 mg/kg into CD1 Swiss mice. -PABC-MMAE, ARA01-RKA-PBC-MMAE, ARA01-RKValCit-PABC-MAE) and polotuzumab vedotin plasma concentration. The ADC concentration in plasma was determined by ELISA. Mean plasma concentrations from 5 mice are plotted versus time, and error bars represent the standard error of the mean (SEM). It should be understood that the abbreviation ARA01-ARK/RKA/RKValCit-PABC-MMAE in Figure 39 refers to the antibody-drug conjugate ARA01-ARK/RKA/RKValCit-PABC-MMAE.

Figure 40 shows ennosumab/ARA04 (SEQ ID NO: 9 and 11) antibody-drug conjugates of the present invention (ARA04-RKAA-PABC) at different time points after a single intravenous injection of 5 mg/kg into CD1 Swiss mice. -MMAE, ARA04-ARK-PABC-MMAE, ARA04-RKA-PBC-MMAE, ARA04-RKValCit-PABC-MMAE) and ennosumab vedotin plasma concentration. The ADC concentration in plasma was determined by ELISA. Mean plasma concentrations from 5 mice are plotted versus time, and error bars represent the standard error of the mean (SEM). It should be understood that the abbreviation ARA04-ARK/RKA/RKValCit-PABC-MMAE in Figure 39 refers to the antibody-drug conjugate ARA04-ARK/RKA/RKValCit-PABC-MMAE.

Figure 41 depicts the Ramos mouse tumor model. Human Burkitt's lymphoma tumor cells (Ramos) were inoculated subcutaneously into CB17SCID mice (n=6 per treatment group). When tumors reached a size of approximately 200 ^mm , animals received 1.43 mg/kg polotuzumab vedotin or a single injection of 1.25 mg/kg (payload-adjusted dose of polotuzumab vedotin) ARA01-RKAA-PABC-MMAE or ARA01-ARK-PABC-MMAE. Relative to polotuzumab vedotin, both ADCs of the invention provided equivalent tumor growth inhibition and survival and durable antitumor responses at approximately half the payload dose. In contrast, polotuzumab vedotin administered at the same payload dose showed only transient tumor elimination. Mean tumor volumes are shown as standard error of the mean (SEM).

Figure 42 depicts the SUM190PT mouse tumor model. Breast cancer-tumor cells (SUM190PT) were inoculated into the mammary fatpat of CB17SCID mice (n=6 per treatment group). ^When tumor size reached approximately 200 mm, animals received 1.5 mg/kg ennosumab vedotin or a single injection of 3 mg/kg (payload-adjusted dose of ennozumab vedotin) ARA04-RKAA-PABC-MMAE or ARA04-ARK-PABC-MMAE. The two ADCs of the invention provided a complete and durable anti-tumor response lasting over 103 days at the same payload dose as ennosumab vedotin. In contrast, ennosumab vedotin administered at the same payload dose showed a transient antitumor response. When observing Figures 42 and 43, it is obvious that the ADC, ARA04-RKAA-PABC-MMAE or ARA04-ARK-PABC-MMAE of the present invention, even if only 1/4 (= 4 times or less) of the payload dose is administered ), also showed excellent efficacy. Non-binding mAb-RKAA-PABC-MMAE did not show any effect on tumor growth. Mean tumor volumes are shown as standard error of the mean (SEM).

Figure 43 depicts the SUM190PT mouse tumor model. Breast cancer-tumor cells (SUM190PT) were inoculated into the mammary fat of CB17SCID mice (n=6 per treatment group). ^When tumor size reached approximately 200 mm, animals received 0.5 mg/kg ennosumab vedotin or a single injection of 1 mg/kg (payload-adjusted dose of ennozumab vedotin) ARA04-RKAA-PABC-MMAE or ARA04-ARK-PABC-MMAE. The two ADCs of the invention provided a complete and durable anti-tumor response lasting over 103 days at the same payload dose as ennosumab vedotin. In contrast, ennosumab vedotin administered at the same payload dose resulted in only a slight tumor growth retardation. Mean tumor volumes are shown as standard error of the mean (SEM).

Example

Example 1: Coupling of peptide MMAE linker to two different antibodies

method

The antibody trastuzumab is commercially available ( Roche, purchased from pharmacies), and all linker-payload constructs (custom synthesized by Levena Biopharma). Polotuzumab with heavy and light chains consisting of the sequences of SEQ ID NO: 5 and 6 was transiently transfected into suspension-adapted CHO-K1 cells and cultured in serum-free/animal component-free expression in the base. Protein was purified from the supernatant by protein A affinity chromatography (Mab Select Sure column; GE Healthcare).

For 1-step coupling (see Figure 13), use 5 mg/ml native glycosylated monoclonal antibody in 50 mM Tris pH 7.6, microbial transglutaminase at a concentration of 5 U/mg in 50 mM Tris pH 7.6 or water ( MTG, Zedira) and 5 molar equivalents of the indicated linker-payloads and incubated in a rotary thermomixer at 37°C for 24 hours. The coupling efficiency was evaluated by LC-MS under DTT reducing conditions. Reduction of samples was achieved by incubating antibody-drug conjugates (ADC) in 50mM DTT/50mM Tris buffer for 10 minutes at 37°C. Probes were analyzed in a Xevo G2-XS QTOF (Waters) connected to an Acquity UPLC Class H system (Waters) and an Acquity UPLC BEH C18 column. The coupling efficiency was calculated from the deconvoluted spectra and expressed as % linker-payload conjugated antibody (=ADC). The calculation of the overall coupling efficiency takes into account the intensity produced by the two glycoforms (G1F and G0F), i.e.

Total coupling efficiency (%) = total intensity – intensity % of uncoupled antibody, the following formula is obtained:

Coupling efficiency (%) = 100*(1-(strength (G1F) + strength (G0F))/total strength)

result

The coupling efficiency varied depending on the linker structure and antibody used, however, it was observed that the highest binding efficiency was achieved when the lysine-containing peptide linker contained an RK motif (Tables 3 and 4).

Table 3. Shows the coupling efficiency of linker-payload complexes (according to the invention)

Table 4. Shows the coupling efficiency of linker-payload (not according to the invention) complexes

Example 2: Coupling of peptide-maytansine with two different antibodies

To demonstrate that high conjugation efficiencies could also be achieved with another payload, i.e. one other than MMAE, a maytansine-containing linker-payload construct was used to conjugate two different antibodies.

method

Coupling was performed in exactly the same manner as described in Example 1. The corresponding maytansinoid-linker construct was custom synthesized by Levena Biopharm.

result

When using a different payload than MMAE, in this example maytansine, the coupling efficiency was also very high when the lysine-containing peptide linker contained an RK motif (Table 4). This example shows that when the lysine-containing peptide linker contains an RK motif, the coupling efficiency is high regardless of the payload.

Table 5. Shows the coupling efficiency of linker-payload complexes (according to the invention)

Example 3: Coupling of linker-payload (according to the invention) to a third antibody

To further demonstrate the high coupling efficiency obtained with the linker-payload construct (according to the present invention), a third antibody was selected and successfully conjugated with high efficiency (for two different payloads, further demonstrating its general applicability).

method

Coupling was performed in exactly the same manner as described in Example 1. The antibody ennozumab with a heavy chain consisting of the sequence SEQ ID NO:9 and a light chain variant consisting of the sequence SEQ ID NO:10 was transiently transfected into suspension-adapted CHO-K1 cells and incubated in the absence of Expressed in serum/animal component-free media. Protein was purified from the supernatant by protein A affinity chromatography (MabSelect Sure column; GE Healthcare).

result

High coupling efficiencies were obtained with the antibody ennozumab using two different linker-payload constructs according to the invention.

Table 6. Shows the coupling efficiency of linker-payload complexes (according to the invention)

Example 4: Coupling of linker-payload (according to the invention) containing non-amino acid spacers

To further demonstrate the high coupling efficiency obtained with the linker-payload construct (according to the present invention), a linker with a polyethylene glycol (PEG) spacer was used and coupled to two different antibodies with high efficiency.

method

Coupling was performed in exactly the same manner as described in Example 1. All linker payload constructs are custom synthesized by Levena Biopharma.

result

High coupling efficiencies for two different antibodies were obtained with linker-payloads containing PEG spacers (according to the invention).

Table 7. Shows the coupling efficiency of linker-payload complexes (according to the invention)

Example 5: The ADC of the present invention is monomeric and does not aggregate

The linker-payload RKAA-PABC-MMAE (Figure 1) was coupled to the antibody polotuzumab (SEQ ID NO: 5 and 6) as described above in Example 1. The resulting ADC termed ARA-01-RKAA-PABC-MMAE has a drug-to-antibody ratio (DAR) of 1.9 (essentially as in Richard Y.C. Huang and Guodong Chen (2016) Characterization of Antibody-Drug Conjugates by Mass Spectrometry : Characterization of antibody-drug conjugates by mass spectrometry: advances and future trends Drug Discover Today Volume 21, Issue 5 (Determined using standard mass spectrometry) and analyzed by size exclusion chromatography.

method

Using a ^SuperdexTM 200Increase 10/300 (Amersham Pharmacia Biotech) column Size exclusion chromatography (SEC) was performed by FPLC (Amersham-Pharmacia Biotech). Proteins were detected using UV/VIS at a wavelength of 280 nm. Samples were analyzed in running buffer 50mM phosphate, 100mM NaCl, pH 7.4 at a flow rate of 1 mL/min.

result

The size exclusion chromatography (SEC) pattern after purification showed that ARA-01-RKAA-MMAE eluted as a single monomer peak, indicating that the ADC has excellent biophysical properties (Figure 10).

Example 6: The ADC of the present invention shows powerful anti-tumor effect in vitro

method

The growth inhibitory effect of ARA01-RKAA-PABC-MMAE on the following three CD79b overexpressing cell lines was studied in vitro: Granta-519 (DSMZ, Acc No: 342), BJAB (CLS) and WSU-DLCL2 (DSMZ, Acc 575) . The CD79 negative cell line HT (ATCC, reference number: CRL-2260) was used as a negative control. 4000 cells were seeded into a 96-well culture plate and incubated with ARA-01-RKAA-PABC-MMAE in a humidified chamber at 37 °C and 5% CO _for 72 h.

The viability of treated cultures was determined by ATP quantification in the CellTiterGloLuminescence Assay as described by the supplier (Promega). Calculate the % viability relative to untreated cells according to the following formula:

The mean % activity was plotted against log10 (concentration), and the resulting dose-response curve was analyzed by nonlinear regression using the software Prism8 using a four-parameter dose-response curve equation.

result

Figure 11 shows that ARA01-RKAA-PABC-MMAE has very high cytotoxic activity against CD79b overexpressing cells, with EC ₅₀ values comparable to traditional ADCs. The cytotoxic activity was highly selective for cells overexpressing CD79b, as there was essentially no reduction in cell viability as expected in the HT cell line. In conclusion, ARA01-RKAA-PABC-MMAE showed antigen-specific and significant anti-proliferative activity in vitro.

Example 7: The ADC of the present invention shows good pharmacokinetic parameters in vivo

The pharmacokinetic characteristics of the anti-CD79b ADC ARA01-RKAA-MMAE according to the invention were studied in mice and compared with the commercially available anti-CD79b-ADC polotuzumab vedotin A comparison was made. Polotuzumab Vedotin is an ADC composed of the anti-CD79b antibody Polotuzumab, in which MMAE is conjugated to the cysteine of the antibody, resulting in an average of 3.5 linked MMAE moieties per antibody ( European Medicines Agency, Assessment Report on/> Procedure number: EMEA/H/C/004879/0000, available at https://www.ema.europa.eu/en/medicines/human/EPAR/polivy).

method

ARA01-RKAA-PABC-MMAE (produced in-house as described in Example 5 above), (Roche, purchased from pharmacies) and the naked anti-CD79b antibody polotuzumab (SEQ ID NO: 5 and 6; expressed and purified as described above) were injected intravenously into 5 females at a dose of 5 mg/kg ADC or antibody, respectively. in mice (CD1 Swiss, Janvier). After 10 minutes, 5.5 hours, 24 hours, 48 hours, 96 hours, 144 hours, 168 hours and 360 hours, approximately 20 μl of blood was withdrawn from the saphenous vein and placed into an EDTA-coated microreactor CB 300 (Sarstedt) . Blood samples were centrifuged at 9500xg for 10 min, and plasma was stored at -80°C until ELISA analysis. Concentrations in plasma were determined by ELISA using a dilution series of corresponding samples with known concentrations using His-tagged human CD79b as capture reagent: 125 ng of HisCD79b (SinoBiological, Ref: 29750-H08H) diluted in PBS was added to Nickel plates (Ni-NTA HisSorb, Qiagen) and after blocking with 200 μl PBS, 4% milk (Rapilait, Migros, Switzerland), 50 μl of diluted plasma sample (4% milk in PBS) was added. After incubation for 1 hour and washing with PBS, total antibodies were detected by adding donkey anti-human IgG-HRP (Biolegend, Poly24109) to the wells, or for total ADC detection, rabbit anti-MMAE antibody (Levena, Ref: LEV -PAE1) for 1 hour, washed and detected via anti-rabbit IgG-HRP. Peroxidase activity was detected by the addition of 3,3',5,5'-tetramethylbenzidine (Sigma) and stopped by the addition of acid. Readings were taken at 450 nm after 1 to 5 minutes. Based on the concentration of the sample in the plasma measured by ELISA at different time points after injection and the slope k (plotted on a semi-log scale) of the elimination phase (time points 24h-360h), it was calculated using the formula t _1/2 =ln2/-k Half-life of the sample (t _1/2 ).

result

Plasma concentrations measured in samples collected at different time points after injection are shown in Figure 12. ARA01-RKAA-PABC-MMAE and The half-lives of are given in Table 4 below. It can be seen that the half-life of the ADC according to the present invention, ARA01-RKAA-PABC-MMAE, in vivo is that of the approved ADC/> at least 2 times. Improved plasma stability may lead to a better safety profile, as the payload does not appear to be released prematurely.

Table 8. Plasma half-life

Example 8: The anti-CD79b ADC of the invention inhibits tumor growth in vivo more effectively than the approved anti-CD89b ADC polotuzumab vedotin

The tumor growth inhibitory effect of the anti-CD79b ADC ARA01-RKAA-PABC-MMAE was studied in vivo and compared with the commercially available polotuzumab vedotin.

method

CB17 SCID mice (Janvier) were implanted subcutaneously with 20 x 10 ⁶ human B-cell lymphoma tumor cells Granta 519 (DSMZ, Acc No: 342). Tumor size and body weight were recorded three times per week. Tumor volume was calculated according to the formula volume = (width) ² x length x 0.5. ^When the average tumor size reaches approximately 200 mm, mice are assigned to treatment groups using a nonrandom stratified scheme, with each group including 8 mice. On Day 0 (randomization day), ARA01-RKAA-PABC-MMAE (in-house as described in Example 5 above) was administered as a single intravenous injection at doses of 0.53 mg/kg, 1 mg/kg, and 2.1 mg/kg. Produced) and polotuzumab vedotin at doses of 0.53 mg/kg and 2.1 mg/kg. Mice in the control group were injected with PBS. All mouse experiments were performed in accordance with Swiss guidelines and approved by the Veterinarian Office of Zürich, Switzerland. According to these guidelines, all mice in the PBS group and the 0.53 mg/kg dose must be sacrificed on day 10, while the 2 mice in the 1 mg/kg group must be sacrificed on days 6 and 30 (tumor ulceration).

result:

The in vivo efficacy of ARA01-RKAA-PABC-MMAE (DAR 1.9) compared with polotuzumab vedotin (DAR 3.5) was evaluated in the Granta 519 tumor model. Specifically, these animals received 0.53 mg/kg or 2.1 mg/kg polotuzumab vedotin and a single injection of ARA01-RKAA-PABC-MMAE at 0.53 mg/kg, 1 mg/kg, or 2.1 mg/kg. Importantly, ARA01-RKAA-PABC-MMAE provided the same tumor growth inhibition and efficacy as polotuzumab vedotin at approximately half the payload dose. Survival (see Figure 16A for comparison at 2 mg/kg dose and Figure 16B for comparison at 0.53 mg/kg dose). Relative to polozumab vedotin at approximately equivalent payload doses, ARA01-RKAA-PABC-MMAE treatment resulted in greater than 0/8 complete tumor responses with polotuzumab vedotin Antitumor efficacy and considerable survival advantage, with 6/8 complete tumor responses (Fig. 16B comparing polozumab vedotin at a dose of 0.53 mg/kg and ARA01-RKAA-PABC-MMAE at a dose of 1 mg/kg Compare). In summary, ARA01-RKAA-PABC-MMAE treatment produced greater antitumor efficacy than polotuzumab vedotin at approximately equivalent payload doses and was associated with a considerable survival advantage.

Example 9: Conjugation of various RK-motif-peptides to the antibody trastuzumab

All RK-containing peptides tested were coupled with high efficiency.

method

The coupling reaction was carried out according to the conditions described in Example 1. Briefly, 5 mg/ml native glycosylated trastuzumab antibody, 1.5 U/mg concentration of MTG and 20 molar equivalents of the indicated peptide-linker containing the RK-motif were mixed in Tris 50 mM pH 7.6 at 37 °C in a rotary thermomixer for 24 hours. Coupling efficiency was evaluated by LCMS as follows: Coupling efficiency (CE) was calculated from the deconvoluted spectra and expressed in %. The calculation takes into account the intensity produced by the two glycoforms (G1F and G0F) according to the following formula:

where cj=coupled, ncj=non-coupled result

All tested RK-motif-linkers coupled well to native fully glycosylated trastuzumab with >50% efficiency, as shown in Table 9.

Table 9. Conjugation efficiency of RK-motif-containing peptide linkers to trastuzumab

RK-motif peptide linker Coupling efficiency (%) HRKHA(SEQ ID NO:55) 98% HRKAH(SEQ ID NO:56) 91% RKAH(SEQ ID NO:57) 91% RKH(SEQ ID NO:58) 87% RKAA(SEQ ID NO:1) 86% RKA(SEQ ID NO:2) 86% RKHA(SEQ ID NO:59) 86% RKHH(SEQ ID NO:60) 85% ARKAH(SEQ ID NO:61) 82% ARKHA(SEQ ID NO:62) 82% HRK(SEQ ID NO:63) 81% RKAAH(SEQ ID NO:64) 81% ARKHH(SEQ ID NO:65) 80% RKAAA(SEQ ID NO:66) 80%

Example 10: Coupling of RK-motif-peptides

method

Reaction conditions: 5 mg/ml of native glycosylated trastuzumab antibody, 5 U/mg concentration of MTG and 5 molar equivalents of the indicated peptide-linker in Tris 50mM pH 7.6 at 37°C with rotary thermomixing Mix in a container for 24 hours. Coupling efficiency was assessed by LCMS as described in Example 9.

result

Peptides containing RK-motifs were coupled with significant coupling efficiencies, as shown in Table 10.

Table 10. Conjugation efficiency of peptide linkers containing RK motifs to trastuzumab

RK-motif peptide linker Coupling efficiency (%) RKAAR(SEQ ID NO:67) 95% RRKAY(SEQ ID NO:68) 100% RRK(SEQ ID NO:69) 99% ARKRA(SEQ ID NO:70) 98%

Example 11: Conjugation of RK-motif-linker-payload to MMAE

To show that the RK-motif-linker-payload is also suitable for one-step antibody conjugation, an additional linker-payload containing the RK-motif was used for conjugation with trastuzumab using MMAE as the payload.

method

By combining 5 mg/ml native glycosylated trastuzumab antibody, MTG at a concentration of 5 U/mg and 5 molar equivalents of the indicated linker-payload in Tris 50 mM pH 7.6 in a 37°C rotary thermomixer. Mix for 24 hours to perform the coupling reaction. Coupling efficiency was assessed by LCMS as described in Example 9.

result

Surprisingly, as shown in Table 11A, excellent conjugation efficiencies were obtained using various RK-motif-linker-payloads containing MMAE conjugated with naturally glycosylated trastuzumab antibodies ( more than 85%). Surprisingly, as shown in Tables 11A and 11B, significantly lower coupling efficiency was observed when the linker-payload did not contain the RK-motif.

Table 11A. Conjugation efficiency of MMAE-containing RK-motif linker-payloads to trastuzumab (according to the invention)

RK-joint-payload with MMAE Coupling efficiency (%) RKAA-PABC-MMAE(SEQ ID NO:1) 100% RKA-PABC-MMAE(SEQ ID NO:2) 100% ARK-PABC-MMAE(SEQ ID NO:3) 100% RKAAR-PABC-MMAE(SEQ ID NO:67) 99% RRKAY-PABC-MMAE(SEQ ID NO:68) 100% RRKAY-PABC-MMAE(SEQ ID NO:69) 96% ARKRA-PABC-MMAE(SEQ ID NO:70) 89% ARKRA-PABC-MMAE(SEQ ID NO:54) 91% ARK-PEG2-PABC-MMAE(SEQ ID NO:3) 99%

Table 11B. Coupling efficiency of non-RK-motif-linker-payloads to MMAE (not according to the invention)

non-rk-joint-payload with MMAE Coupling efficiency (%) ARKRA-PABC-MMAE(SEQ ID NO:50) 43% KRA-PABC-MMAE(SEQ ID NO:51) 68% KR-PABC-MMAE(SEQ ID NO:71) 46% KAAR-PABC-MMAE(SEQ ID NO:52) 64% KARA-PABC-MMAE(SEQ ID NO:53) 77% KAA-PABC-MMAE(SEQ ID NO:72) 57%

Example 12: Conjugation of RK-motif linker-payloads using alternative payload classes

To demonstrate the generality of the present linker technology, various RK-motif linker-payloads were used to conjugate trastuzumab. Payloads were selected from the following payload categories: Cytotoxicants, steroids (cortisol=CS) and immunomodulators (i.e. STING agonists) were evaluated.

method

By combining 5 mg/ml of native glycosylated trastuzumab antibody, MTG at a concentration of 5-10 U/mg and 5-10 molar equivalents of the indicated linker-payload in Tris in a rotary thermomixer at 37°C. The coupling reaction was carried out by mixing in 50mM pH 7.6 for 24 hours. Coupling efficiency was assessed by LCMS as described in Example 9.

result

Surprisingly, excellent coupling efficiencies (over 80%) were obtained using various RK-motif linker versions and payload classes, as shown in Table 4. Also surprising was the observation that payloads located at the N-terminal position were well tolerated (as demonstrated for May-C5-RKR).

Table 12. Conjugation efficiency of linker-payloads containing RK-motif linkers to 3 different payload classes.

RK-linker-payload containing multiple toxins and drugs Coupling efficiency (%) RKAA-PABC-May(SEQ ID NO:1) 92% RKAA-PABC-Exa(SEQ ID NO:1) 98% RKAA-PABC-EDA-PNU(SEQ ID NO:1) 99% RKAA-PABE-amanitine (SEQ ID NO: 1) 90% RKAA-EDA-cortisol (SEQ ID NO:1) 88% RKAA-PABC-EDA-STING(SEQ ID NO:1) 96% RKAAR-PABC-Exa(SEQ ID NO:67) 99% RKAAR-EDA-CS(SEQ ID NO:67) 98% ARK-S-C5-May(SEQ ID NO:3) 98% ARK-PABC-Exa(SEQ ID NO:3) 94% ARK-PEG2-S-C5-May(SEQ ID NO:3) 99% May-C5-RKR(SEQ ID NO:4) 96% RRK-PABC-Exa(SEQ ID NO:69) 83%

May: maytansine; Exa: ixotecan derivatives; STING (stimulator of interferon genes; immunostimulatory factors); PNU (anthracycline analogues).

Example 13: Conjugation of RK-motif linker-payload to three different antibodies

To demonstrate the general applicability of this reaction, RK-motif-linker-payloads containing MMAE or maytansine (May) were chosen to be conjugated to three different antibodies: trastuzumab, polotuzumab and ennosumab variants (heavy chain SEQ ID NO: 9 and light chain SEQ ID NO: 11).

method

By combining 5 mg/ml of the indicated native glycosylated antibody, a concentration of 5-10 U/mg of MTG and 5-10 molar equivalents of the indicated linker-payload in Tris 50mM pH 7.6 in a rotary thermomixer at 37°C. Mix for 24 hours to carry out the coupling reaction. Coupling efficiency was assessed by LCMS as described in Example 9.

result

Surprisingly, as shown in Table 13, high coupling efficiencies were obtained for all three tested antibodies with all tested RK-motif-MMAE or May linker payloads.

Table 13. Linker-payload conjugation efficiency with three different antibodies

NT: Not tested

Example 14: Coupling of RK-motif linker-payload under different reaction conditions

To demonstrate that conjugation to RK-linker-payload is tolerant to multiple reaction conditions, linker-payload conjugation to polotuzumab was performed using a series of reaction conditions with different parameters.

method

As standard conditions, the following parameters were used: 5 mg/ml naturally glycosylated polotuzumab antibody, 5 U/mg concentration of MTG and 5 molar equivalents of RKAA-PABC-MMAE in Tris 50 mM, pH 7.6 in 37°C in a rotary thermomixer for 24 hours. Coupling efficiency was assessed by LCMS as described in Example 9 above.

The variable parameters are shown in Table 14.

result

RKAA-PABC-MMAE linker-payload conjugates with very high coupling efficiencies over a very wide range of reaction conditions: using antibody concentrations between 5 and 17 mg/ml, between 2 and 10 U/mg relative to the antibody Concentration of MTG (U/mg) achieved >80% coupling efficiency. Further, high coupling efficiencies were also obtained with molar concentrations of the linker relative to the antibody (2 to 8 equivalents) and a very wide range of pH (coupling efficiency from 67% at pH 6.0 to 86% at pH 8).

Surprisingly, higher coupling efficiencies were obtained using less linker-payload excess compared to the antibody, i.e. 2-20 equiv of linker-payload yielded higher coupling than using 80 equiv. efficiency, which is contrary to expectations (Table 14).

Table 14. Coupling efficiency of RK-linker-payload and polotuzumab under different reaction conditions

Example 15: ADC containing RK-motif PABC-payload linker payload is effective in vitro using three different antibodies

To demonstrate that ADCs according to the invention (i.e. generated using RK-motif-MMAE/maytansinoid linker payloads) produce efficient release and target-specific toxicity to cancer cell lines, the linkers RKAA-PABC-MMAE, RKAA-PABC were used - Maytansine, ARK-PABC-MMAE, RKA-PABC-MMAE and RKValCit-MMAE were tested on targeted expressing cells based on trastuzumab-, polozumab-(ARA01) and ennozumab Anti-(SEQ ID 9 and SEQ ID 11; ARA04) ADC.

method

To study the growth inhibitory effects of trastuzumab-RKAA-PABC-MMAE and trastuzumab-RKAA-PABC-maytansine on HER-2-positive SKBR-3 (ATCC HTB-30) cells, ARA01 was tested -The inhibitory effect of ARK-PABC-MAE, ARA01-RKA-PABC-MMAE and ARA01-RKValCit-PABC-MMAE on CD79b-positive Granta-519 lymphoma cells, and ARA04-RKAA-PABC-MMAE, ARA04-ARK were studied - Cytotoxic effects of PABC-MMAE, ARA04-RKA-PABC-MMAE and ARA04-KValCit-PABC-MMAE on integrin-4 positive breast cancer cells SUM190PT (BIOIVT, 28068A16284). Target dependence and specificity were tested on bindin-4 negative lung cancer cells A549 (ATCC CCL-185). Under all conditions, 4000 cells were seeded into 96-well culture plates and incubated with respective ADCs at 37 °C in a humidified chamber with 5% CO _for 72 h.

result

Figure 35 shows that both trastuzumab-RKAA-PABC-MMAE and trastuzumab-RKAA-PABC-maytansine of the present invention exert very high cytotoxic activity on HER-2 overexpressing cells, and their EC _{The 50} value is comparable to a traditional ADC.

Among CD79b and integrin-4 targeting ADCs, ARA01 and ARA04 ADCs containing different linkers of the invention showed very high targeting specificity to target-positive Granta-519 (Figure 36) and SUM190PT (Figure 37) cells, respectively. Toxic activity. EC ₅₀ values are within the range of conventional ADCs. In contrast, the same ADC did not affect target-negative A549 cells (Figure 38) and showed comparable efficacy to conventional ADCs.

In summary, all ADCs using RK-motif linker-payloads according to the invention (coupled to trastuzumab, polotuzumab or ennozumab as parent antibodies) showed target specificity in vitro and significant antiproliferative activity.

Example 16: ADC containing RK-motif-PABC-MMAE linker-payload shows good pharmacokinetic properties in vivo

To evaluate the in vivo stability of the RK-motif MMAE ADC, mouse pharmacokinetic studies were performed using different RK-motif linker payloads conjugated to polotuzumab and ennosumab.

Anti-CD79b ADCs ARA01-ARK-PABC-MMAE, ARA01-RKA-PABC-MMAE and ARA01-RKValCit-PABC-MMAE generated with the linker-payloads of the invention; and with different linkers ARA04-ARK-PABC-MMAE, The pharmacokinetic profiles of anti-conjugin-4 ADCs generated by ARA04-RKA-PABC-MMAE and ARA04-RKValCit-PABC-MMAE were studied in mice and compared with the commercially available anti-CD79b ADC polostatin anti-vedotin and the commercially available anti-binding protein-4ADC ennosumab-vedodin/> A comparison was made.

method

Pharmacokinetic studies were performed as described in Example 7, with adjusted sampling time points: blood samples were drawn from the saphenous vein after 10 minutes, 4 hours, 48 hours, 96 hours, 168 hours, 264 hours, 336 hours and 504 hours. For anti-CD79bADC detection, the method described in Example 7 was used. Anti-conjugatein-4 ADC detection was performed simply as follows: ADC concentration in plasma was determined by ELISA using His-tagged human conbindin-4 as a capture reagent: 125 ng of His-conjugatein-4 (SinoBiological, Ref.: 19771- HO8H) was diluted in PBS and added to nickel plates (Ni-NTA HisSorb, Qiagen). After blocking with 200 μl of PBS, 4% milk (Rapilait, Migros, Switzerland), 50 μl of diluted plasma sample (4% milk in PBS) was added. After incubation for 1 hour and washing with PBS, total ADC was detected using rabbit anti-MMAE antibody (Levena, Ref: LEV-PAE1), which was added for an additional hour at room temperature, washed and detected by anti-rabbit IgG HRP. Peroxidase activity was detected by the addition of 3,3',5,5'-tetramethylbenzidine (Sigma) and stopped by the addition of acid. Readings were taken at 450 nm after 1 to 5 minutes. Half-life was calculated using ADC concentration plotted against time in the sample in plasma (semi-log scale). The resulting slope k of the elimination phase at time points 48-504h was used to determine the half-life (t _1/2 ) using the following formula: t _1/2 =ln2/-k.

result

Plasma concentrations of intact ADC measured in samples collected at different time points after injection are shown for anti-CD79b ADC (Figure 39) and Binbin-4 ADC (Figure 40).

The half-lives of ARA01-ARK-PABC-MMAE, ARA01-RKA-PABC-MMAE and ARA01-RKValCit-PABC-MMAE are shown in Table 15 below. Surprisingly, an approximately 2-fold prolongation of the half-life of all ADCs according to the invention was observed compared to the calculated polotuzumab vedotin.

ARA04-RKAA-MMAE, ARA04-ARK-MMAE, ARA04-RKA-MMAE, ARA04-RKValCit-MMAE and The half-lives are shown in Table 16 below, showing that the average half-life of these ADCs is 2 to 2.5 times longer compared to the approved ennosumab vedotin.

In summary, all ADCs generated according to the present invention showed a 2-2.5-fold improved half-life compared to the vedotin baseline, using either polotuzumab or ennozumab as parent antibodies. This indicates that ADCs generated with linker-payloads according to the present invention lead to an increase in ADC stability in vivo, which can lead to an overall better safety profile and therapeutic index (TI) since the payload is not premature freed.

Table 15. Plasma half-life of anti-CD79b ADC

Table 16. Plasma half-life of anti-conjugatein-4ADC

Example 17: ADC containing RK-motif-PABC-MMAE linker payload shows more effective tumor growth inhibition in vivo compared to baseline in CD79b-positive liquid tumor model and integrin-4-positive solid tumor model

Tumor growth inhibition by anti-CD79b ADCARA01-RKAA-PABC-MMAE and ARA01-ARK-PABC-MMAE according to the invention was studied in vivo in the Ramos (CD79b positive, liquid tumor) model. The anti-tumor properties of anti-bindin-4 ADCs ARA04-RKAA-PABC-MMAE and ARO4-ARK-PAPBC-MMAE according to the present invention were tested in the SUM190PT (bindin-4 positive, solid tumor) xenograft model. A non-binding mAb-RKAA-PABC-MMAE control ADC was included to exclude non-specific ADC activity.

method

For SUM190PT xenografts, ^2x10 cells were injected into mammary fat; for Ramos, ^20x10 cells were injected subcutaneously into CB17 SCID mice (Janvier). Tumor size and body weight were recorded three times per week. Tumor volume was calculated according to the formula volume = (width) ² x length x 0.5. ^When the average tumor size reaches approximately 200 mm, mice are assigned to treatment groups using a nonrandom stratified scheme, with each group including 6 mice. ADC was injected intravenously once on the day of randomization.

As described in Example 5, all ADCs were produced in-house.

ARA01-RKAA-PABC-MMAE and ARA01-RKAA-PABC-MMAE (both DAR1.9) were injected at a dose of 1.25 mg/kg (equivalent to a payload of 25ug per kg of body weight). Polotuzumab vedotin (PV, DAR 3.6) was injected at a dose of 1.43 mg/kg (equivalent to a 50ug/kg payload or twice the ARA01-ADC payload dose).

ARA04-RKAA-PABC-MMAE and ARA04-RKAA-PABC-MMAE (both DAR 1.9) were injected at ADC doses of 1 mg/kg and 3 mg/kg (corresponding to 10 μg and 30 μg payload per kg body weight) and combined with 0.5 mg /kg and 1.5 mg/kg doses of ennosumab vedotin (EV, DAR 3.8). Non-conjugated mAb-RKAA-PABC-MMAE ADC (with the same linker-payload and DAR as ARA04 ADC) was injected at 3 mg/kg. Mice in the control group were injected with PBS. All mouse experiments were performed in accordance with Swiss guidelines and approved by the Veterinary Office in Zurich, Switzerland.

result:

The ARA01-RKAA-PABC-MMAE and ARA01-ARK-PABC-MMAE ADCs of the invention were compared to polotuzumab vedotin (PV) in the fast-growing Ramos xenograft model. Figure 41 shows that a single intravenous injection of 1.25 mg/kg (or 25 ug payload per kg of mouse body weight) produced a highly effective anti-tumor response in all mice. In contrast, PV administered at 1.43mg/kg or 50ug payload/kg body weight showed transient tumor elimination only 20 days after treatment in tumor-free individuals. In contrast, and very surprisingly, ARA01-RKAA-PABC-MMAE and ARA01-ARK-PABC-MMAE showed durable complete antitumor responses at half the payload dose.

In solid tumor models, ARA04-RKAA-PABC-MMAE and ARA04-ARK-PABC-MMAE were compared to ennosumab vedotin (EV) in the SUM190PT breast cancer model. Importantly, as shown in Figures 42 and 43, the two ADCs of the present invention, ARA04-RKAA-PABC-MMAE and ARA04-ARK-PABC-MMAE, are extremely effective at 1 mg/kg and 3 mg/kg , and resulted in complete tumor eradication and durable response throughout the study (103 days post-injection).

At approximately equivalent payload doses relative to ennosumab vedotin, treatment with ARA04-RKAA-PABC-MMAE and ARA04-ARK-PABC-MMAE (3 mg/kg) resulted in greater and more durable antitumor efficacy and Considerable survival advantage, with 4/6 complete tumor responses compared with 0/6 complete responses for EV tumors (Comparison of EV at 1.5 mg/kg dose and ARA04-RKAA-PABC-MMAE at 3 mg/kg dose in Figure 42) . Non-binding mAb-RKAA-PABC-MMAE did not have any effect on tumor growth and demonstrated high target specificity for targeting the ADC. Strikingly, ARA04-RKAA-PABC-MMAE and ARA04-ARK-PABC-MMAE ADC performed approximately three-thirds better than ennosumab vedotin (1.5 mg/kg = 30ug/kg payload dose comparison). A dose of one and a dose of 1 mg/kg (=10ug/kg payload dose) ARA04-RKAA/ARK-MMAE provided the same tumor growth inhibition and survival, see Figure 42 and Figure 43. Overall, ARA04-RKAA-PABC-MMAE and ARA04-ARK-PABC-MMAE treatments produced greater and more durable antitumor effects relative to ennosumab vedotin at approximately one-third of the payload dose. efficacy, and a considerable survival advantage.

Overall, we conclude that anti-CD79b and anti-conjugated ADCs (consisting of the same antibodies and payloads as their corresponding baseline ADCs) generated with RK-motif linker-payloads according to the invention are highly potent in vivo active and showed 2-3 times superior efficacy, providing a considerable survival advantage over vedotin-based ADCs, which was very surprising.

sequence list

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35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 7

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> Trastuzumab heavy chain

<400> 7

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 8

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> Trastuzumab light chain

<400> 8

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 9

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> Enfortumab heavy chain

<400> 9

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asn Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Ser Ser Ser Ser Ser Thr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Ser

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 10

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> Ennosumab light chain

<400> 10

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Gly Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Pro

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 11

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> Ennosumab heavy chain

<400> 11

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asn Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Ser Ser Ser Ser Ser Thr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Ser

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 12

<211> 335

<212> PRT

<213> Artificial sequence

<220>

<223> Streptomyces mobaraensis MTG Zedira

<400> 12

Phe Arg Ala Pro Asp Ser Asp Asp Arg Val Thr Pro Pro Ala Glu Pro

1 5 10 15

Leu Asp Arg Met Pro Asp Pro Tyr Arg Pro Ser Tyr Gly Arg Ala Glu

20 25 30

Thr Val Val Asn Asn Tyr Ile Arg Lys Trp Gln Gln Val Tyr Ser His

35 40 45

Arg Asp Gly Arg Lys Gln Gln Met Thr Glu Glu Gln Arg Glu Trp Leu

50 55 60

Ser Tyr Gly Cys Val Gly Val Thr Trp Val Asn Ser Gly Gln Tyr Pro

65 70 75 80

Thr Asn Arg Leu Ala Phe Ala Ser Phe Asp Glu Asp Arg Phe Lys Asn

85 90 95

Glu Leu Lys Asn Gly Arg Pro Arg Ser Gly Glu Thr Arg Ala Glu Phe

100 105 110

Glu Gly Arg Val Ala Lys Glu Ser Phe Asp Glu Glu Lys Gly Phe Gln

115 120 125

Arg Ala Arg Glu Val Ala Ser Val Met Asn Arg Ala Leu Glu Asn Ala

130 135 140

His Asp Glu Ser Ala Tyr Leu Asp Asn Leu Lys Lys Glu Leu Ala Asn

145 150 155 160

Gly Asn Asp Ala Leu Arg Asn Glu Asp Ala Arg Ser Pro Phe Tyr Ser

165 170 175

Ala Leu Arg Asn Thr Pro Ser Phe Lys Glu Arg Asn Gly Gly Asn His

180 185 190

Asp Pro Ser Arg Met Lys Ala Val Ile Tyr Ser Lys His Phe Trp Ser

195 200 205

Gly Gln Asp Arg Ser Ser Ser Ala Asp Lys Arg Lys Tyr Gly Asp Pro

210 215 220

Asp Ala Phe Arg Pro Ala Pro Gly Thr Gly Leu Val Asp Met Ser Arg

225 230 235 240

Asp Arg Asn Ile Pro Arg Ser Pro Thr Ser Pro Gly Glu Gly Phe Val

245 250 255

Asn Phe Asp Tyr Gly Trp Phe Gly Ala Gln Thr Glu Ala Asp Ala Asp

260 265 270

Lys Thr Val Trp Thr His Gly Asn His Tyr His Ala Pro Asn Gly Ser

275 280 285

Leu Gly Ala Met His Val Tyr Glu Ser Lys Phe Arg Asn Trp Ser Glu

290 295 300

Gly Tyr Ser Asp Phe Asp Arg Gly Ala Tyr Val Ile Thr Phe Ile Pro

305 310 315 320

Lys Ser Trp Asn Thr Ala Pro Asp Lys Val Lys Gln Gly Trp Pro

325 330 335

<210> 13

<211> 335

<212> PRT

<213> Artificial sequence

<220>

<223> Streptomyces mohara MTG

<400> 13

Phe Arg Ala Pro Asp Ser Asp Glu Arg Val Thr Pro Pro Ala Glu Pro

1 5 10 15

Leu Asp Arg Met Pro Asp Pro Tyr Arg Pro Ser Tyr Gly Arg Ala Glu

20 25 30

Thr Ile Val Asn Asn Tyr Ile Arg Lys Trp Gln Gln Val Tyr Ser His

35 40 45

Arg Asp Gly Arg Lys Gln Gln Met Thr Glu Glu Gln Arg Glu Trp Leu

50 55 60

Ser Tyr Gly Cys Val Gly Val Thr Trp Val Asn Ser Gly Gln Tyr Pro

65 70 75 80

Thr Asn Arg Leu Ala Phe Ala Phe Phe Asp Glu Asp Lys Tyr Lys Asn

85 90 95

Glu Leu Lys Asn Gly Arg Pro Arg Ser Gly Glu Thr Arg Ala Glu Phe

100 105 110

Glu Gly Arg Val Ala Lys Asp Ser Phe Asp Glu Ala Lys Gly Phe Gln

115 120 125

Arg Ala Arg Asp Val Ala Ser Val Met Asn Lys Ala Leu Glu Asn Ala

130 135 140

His Asp Glu Gly Ala Tyr Leu Asp Asn Leu Lys Lys Glu Leu Ala Asn

145 150 155 160

Gly Asn Asp Ala Leu Arg Asn Glu Asp Ala Arg Ser Pro Phe Tyr Ser

165 170 175

Ala Leu Arg Asn Thr Pro Ser Phe Lys Asp Arg Asn Gly Gly Asn His

180 185 190

Asp Pro Ser Lys Met Lys Ala Val Ile Tyr Ser Lys His Phe Trp Ser

195 200 205

Gly Gln Asp Arg Ser Gly Ser Ser Asp Lys Arg Lys Tyr Gly Asp Pro

210 215 220

Glu Ala Phe Arg Pro Asp Arg Gly Thr Gly Leu Val Asp Met Ser Arg

225 230 235 240

Asp Arg Asn Ile Pro Arg Ser Pro Thr Ser Pro Gly Glu Ser Phe Val

245 250 255

Asn Phe Asp Tyr Gly Trp Phe Gly Ala Gln Thr Glu Ala Asp Ala Asp

260 265 270

Lys Thr Val Trp Thr His Gly Asn His Tyr His Ala Pro Asn Gly Ser

275 280 285

Leu Gly Ala Met His Val Tyr Glu Ser Lys Phe Arg Asn Trp Ser Asp

290 295 300

Gly Tyr Ser Asp Phe Asp Arg Gly Ala Tyr Val Val Thr Phe Val Pro

305 310 315 320

Lys Ser Trp Asn Thr Ala Pro Asp Lys Val Thr Gln Gly Trp Pro

325 330 335

<210> 14

<211> 407

<212> PRT

<213> Artificial sequence

<220>

<223> Streptomyces mohara MTG P81453

<400> 14

Met Arg Ile Arg Arg Arg Ala Leu Val Phe Ala Thr Met Ser Ala Val

1 5 10 15

Leu Cys Thr Ala Gly Phe Met Pro Ser Ala Gly Glu Ala Ala Ala Asp

20 25 30

Asn Gly Ala Gly Glu Glu Thr Lys Ser Tyr Ala Glu Thr Tyr Arg Leu

35 40 45

Thr Ala Asp Asp Val Ala Asn Ile Asn Ala Leu Asn Glu Ser Ala Pro

50 55 60

Ala Ala Ser Ser Ala Gly Pro Ser Phe Arg Ala Pro Asp Ser Asp Asp

65 70 75 80

Arg Val Thr Pro Pro Ala Glu Pro Leu Asp Arg Met Pro Asp Pro Tyr

85 90 95

Arg Pro Ser Tyr Gly Arg Ala Glu Thr Val Val Asn Asn Tyr Ile Arg

100 105 110

Lys Trp Gln Gln Val Tyr Ser His Arg Asp Gly Arg Lys Gln Gln Met

115 120 125

Thr Glu Glu Gln Arg Glu Trp Leu Ser Tyr Gly Cys Val Gly Val Thr

130 135 140

Trp Val Asn Ser Gly Gln Tyr Pro Thr Asn Arg Leu Ala Phe Ala Ser

145 150 155 160

Phe Asp Glu Asp Arg Phe Lys Asn Glu Leu Lys Asn Gly Arg Pro Arg

165 170 175

Ser Gly Glu Thr Arg Ala Glu Phe Glu Gly Arg Val Ala Lys Glu Ser

180 185 190

Phe Asp Glu Glu Lys Gly Phe Gln Arg Ala Arg Glu Val Ala Ser Val

195 200 205

Met Asn Arg Ala Leu Glu Asn Ala His Asp Glu Ser Ala Tyr Leu Asp

210 215 220

Asn Leu Lys Lys Glu Leu Ala Asn Gly Asn Asp Ala Leu Arg Asn Glu

225 230 235 240

Asp Ala Arg Ser Pro Phe Tyr Ser Ala Leu Arg Asn Thr Pro Ser Phe

245 250 255

Lys Glu Arg Asn Gly Gly Asn His Asp Pro Ser Arg Met Lys Ala Val

260 265 270

Ile Tyr Ser Lys His Phe Trp Ser Gly Gln Asp Arg Ser Ser Ser Ala

275 280 285

Asp Lys Arg Lys Tyr Gly Asp Pro Asp Ala Phe Arg Pro Ala Pro Gly

290 295 300

Thr Gly Leu Val Asp Met Ser Arg Asp Arg Asn Ile Pro Arg Ser Pro

305 310 315 320

Thr Ser Pro Gly Glu Gly Phe Val Asn Phe Asp Tyr Gly Trp Phe Gly

325 330 335

Ala Gln Thr Glu Ala Asp Ala Asp Lys Thr Val Trp Thr His Gly Asn

340 345 350

His Tyr His Ala Pro Asn Gly Ser Leu Gly Ala Met His Val Tyr Glu

355 360 365

Ser Lys Phe Arg Asn Trp Ser Glu Gly Tyr Ser Asp Phe Asp Arg Gly

370 375 380

Ala Tyr Val Ile Thr Phe Ile Pro Lys Ser Trp Asn Thr Ala Pro Asp

385 390 395 400

Lys Val Lys Gln Gly Trp Pro

405

<210> 15

<211> 395

<212> PRT

<213> Artificial sequence

<220>

<223> Streptoverticillium ladakanum MTG

<400> 15

Met His Arg Arg Ile His Ala Val Gly Gln Ala Arg Pro Pro Pro Thr

1 5 10 15

Met Ala Arg Gly Lys Glu Thr Lys Ser Tyr Ala Glu Thr Tyr Arg Leu

20 25 30

Thr Ala Asp Asp Val Ala Asn Ile Asn Ala Leu Asn Glu Ser Ala Pro

35 40 45

Ala Ala Ser Ser Ala Gly Pro Ser Phe Arg Ala Pro Asp Ser Asp Asp

50 55 60

Arg Val Thr Pro Pro Ala Glu Pro Leu Asp Arg Met Pro Asp Pro Tyr

65 70 75 80

Arg Pro Ser Tyr Gly Arg Ala Glu Thr Val Val Asn Asn Tyr Ile Arg

85 90 95

Lys Trp Gln Gln Val Tyr Ser His Arg Asp Gly Arg Lys Gln Gln Met

100 105 110

Thr Glu Glu Gln Arg Glu Trp Leu Ser Tyr Gly Cys Val Gly Val Thr

115 120 125

Trp Val Asn Ser Gly Gln Tyr Pro Thr Asn Arg Leu Ala Phe Ala Ser

130 135 140

Phe Asp Glu Asp Arg Phe Lys Asn Glu Leu Lys Asn Gly Arg Pro Arg

145 150 155 160

Ser Gly Glu Thr Arg Ala Glu Phe Glu Gly Arg Val Ala Lys Glu Ser

165 170 175

Phe Asp Glu Glu Lys Gly Phe Gln Arg Ala Arg Glu Val Ala Ser Val

180 185 190

Met Asn Arg Ala Leu Glu Asn Ala His Asp Glu Ser Ala Tyr Leu Asp

195 200 205

Asn Leu Lys Lys Glu Leu Ala Asn Gly Asn Asp Ala Leu Arg Asn Glu

210 215 220

Asp Ala Arg Ser Pro Phe Tyr Ser Ala Leu Arg Asn Thr Pro Ser Phe

225 230 235 240

Lys Glu Arg Asn Gly Gly Asn His Asp Pro Ser Arg Met Lys Ala Val

245 250 255

Ile Tyr Ser Lys His Phe Trp Ser Gly Gln Asp Arg Ser Ser Ser Ala

260 265 270

Asp Lys Arg Lys Tyr Gly Asp Pro Asp Ala Phe Arg Ser Ala Pro Gly

275 280 285

Thr Gly Leu Val Asp Met Ser Arg Asp Arg Asn Ile Pro Arg Ser Pro

290 295 300

Thr Ser Pro Gly Glu Gly Phe Val Asn Phe Asp Tyr Gly Trp Phe Gly

305 310 315 320

Ala Gln Thr Glu Ala Asp Ala Asp Lys Thr Val Trp Thr His Gly Asn

325 330 335

His Tyr His Ala Pro Asn Gly Ser Leu Gly Cys His Ala Cys Leu Thr

340 345 350

Arg Ala Ser Ser Ala Thr Gly Ser Glu Gly Tyr Ser Asp Phe Asp Arg

355 360 365

Gly Glu Pro Tyr Val Val Ser Pro Ser Pro Ser Pro Arg Met Leu Glu

370 375 380

His Arg Pro Arg Gln Gly Lys Ala Gly Leu Ala

385 390 395

<210> 16

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Q-Tag 1

<400> 16

Leu Leu Gln Gly Gly

1 5

<210> 17

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> Q-Tag 2

<400> 17

Leu Leu Gln Gly

1

<210> 18

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Q-Tag 3

<400> 18

Leu Ser Leu Ser Gln Gly

1 5

<210> 19

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Q-Tag 4

<400> 19

Gly Gly Gly Leu Leu Gln Gly Gly

1 5

<210> 20

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Q-Tag 5

<400> 20

Gly Leu Leu Gln Gly

1 5

<210> 21

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Q-Tag 6

<400> 21

Leu Leu Gln

1

<210> 22

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Q-Tag 7

<400> 22

Gly Ser Pro Leu Ala Gln Ser His Gly Gly

1 5 10

<210> 23

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Q-Tag 8

<400> 23

Gly Leu Leu Gln Gly Gly Gly

1 5

<210> 24

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Q-Tag 9

<400> 24

Gly Leu Leu Gln Gly Gly

1 5

<210> 25

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> Q-Tag 10

<400> 25

Gly Leu Leu Gln

1

<210> 26

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Q-Tag 11

<400> 26

Leu Leu Gln Leu Leu Gln Gly Ala

1 5

<210> 27

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Q-Tag 12

<400> 27

Leu Leu Gln Gly Ala

1 5

<210> 28

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Q-Tag 13

<400> 28

Leu Leu Gln Tyr Gln Gly Ala

1 5

<210> 29

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Q-Tag 14

<400> 29

Leu Leu Gln Gly Ser Gly

1 5

<210> 30

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Q-Tag 15

<400> 30

Leu Leu Gln Tyr Gln Gly

1 5

<210> 31

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Q-Tag 16

<400> 31

Leu Leu Gln Leu Leu Gln Gly

1 5

<210> 32

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Q-Tag 17

<400> 32

Ser Leu Leu Gln Gly

1 5

<210> 33

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Q-Tag 18

<400> 33

Leu Leu Gln Leu Gln

1 5

<210> 34

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Q-Tag 19

<400> 34

Leu Leu Gln Leu Leu Gln

1 5

<210> 35

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Q-Tag 20

<400> 35

Leu Leu Gln Gly Arg

1 5

<210> 36

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Q-Tag 21

<400> 36

Glu Glu Gln Tyr Ala Ser Thr Tyr

1 5

<210> 37

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Q-Tag 22

<400> 37

Glu Glu Gln Tyr Gln Ser Thr Tyr

1 5

<210> 38

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Q-Tag 23

<400> 38

Glu Glu Gln Tyr Asn Ser Thr Tyr

1 5

<210> 39

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Q-Tag 24

<400> 39

Glu Glu Gln Tyr Gln Ser

1 5

<210> 40

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Q-Tag 25

<400> 40

Glu Glu Gln Tyr Gln Ser Thr

1 5

<210> 41

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Q-Tag 26

<400> 41

Glu Gln Tyr Gln Ser Thr Tyr

1 5

<210> 42

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> Q-Tag 27

<400> 42

Gln Tyr Gln Ser

1

<210> 43

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Q-Tag 28

<400> 43

Gln Tyr Gln Ser Thr Tyr

1 5

<210> 44

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Q-Tag 29

<400> 44

Tyr Arg Tyr Arg Gln

1 5

<210> 45

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Q-Tag 30

<400> 45

Asp Tyr Ala Leu Gln

1 5

<210> 46

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Q-Tag 31

<400> 46

Phe Gly Leu Gln Arg Pro Tyr

1 5

<210> 47

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Q-Tag 32

<400> 47

Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu

1 5 10

<210> 48

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Q-Tag 33

<400> 48

Leu Gln Arg

1

<210> 49

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Q-Tag 34

<400> 49

Tyr Gln Arg

1

<210> 50

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Connector 5

<400> 50

Lys Arg Ala

1

<210> 51

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Connector 6

<400> 51

Ala Lys Arg

1

<210> 52

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> Connector 7

<400> 52

Lys Ala Ala Arg

1

<210> 53

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> Connector 8

<400> 53

Lys Ala Arg Ala

1

<210> 54

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> Connector 9

<220>

<221> MOD_RES

<222> 4

<223> Xaa is L-citrulline

<400> 54

Arg Lys Val Xaa

1

<210> 55

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Connector 10

<400> 55

His Arg Lys His Ala

1 5

<210> 56

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Connector 11

<400> 56

His Arg Lys Ala His

1 5

<210> 57

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> Connector 12

<400> 57

Arg Lys Ala His

1

<210> 58

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Connector 13

<400> 58

Arg Lys His

1

<210> 59

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> Connector 14

<400> 59

Arg Lys His Ala

1

<210> 60

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> Connector 15

<400> 60

Arg Lys His His

1

<210> 61

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Connector 16

<400> 61

Ala Arg Lys Ala His

1 5

<210> 62

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Connector 17

<400> 62

Ala Arg Lys His Ala

1 5

<210> 63

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Connector 18

<400> 63

His Arg Lys

1

<210> 64

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Connector 19

<400> 64

Arg Lys Ala Ala His

1 5

<210> 65

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Connector 20

<400> 65

Ala Arg Lys His His

1 5

<210> 66

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Connector 21

<400> 66

Arg Lys Ala Ala Ala

1 5

<210> 67

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Connector 22

<400> 67

Arg Lys Ala Ala Arg

1 5

<210> 68

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Connector 23

<400> 68

Arg Arg Lys Ala Tyr

1 5

<210> 69

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Connector 24

<400> 69

Arg Arg Lys

1

<210> 70

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Connector 25

<400> 70

Ala Arg Lys Arg Ala

1 5

<210> 71

<211> 2

<212> PRT

<213> Artificial sequence

<220>

<223> Connector 26

<400> 71

Lys Arg

1

<210> 72

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Connector 27

<400> 72

Lys Ala Ala

1

Claims (116)

1. A method for producing an antibody-linker conjugate by Microbial Transglutaminase (MTG), comprising reacting a polypeptide comprising (as shown in the N.fwdarw.C direction) (Sp ₁ )-RK-(Sp ₂ )-B-(Sp ₃ ) Or (Sp) ₁ )-B-(Sp ₂ )-RK-(Sp ₃ ) A step of coupling a linker of the structure to a Gln residue contained in the antibody, wherein,

·(Sp ₁ ) Is a chemical spacer or is absent;

·(Sp ₂ ) Is a chemical spacer or is absent;

·(Sp ₃ ) Is a chemical spacer or is absent;

r is arginine or an arginine derivative or arginyl mimetic arginine mimetic;

k is lysine or a lysine derivative or a lysine mimetic;

B is a linking part or payload;

and wherein the linker is coupled to a Gln residue comprised in the antibody via a primary amine comprised in a side chain of a lysine residue, the lysine derivative or the lysine mimetic.

2. The method according to claim 1, wherein the chemical spacer (Sp ₁ )、(Sp ₂ ) Sum (Sp) ₃ ) Each independently comprises 0 to 12 amino acid residues.

3. The method of claim 1 or 2, wherein the linker comprises no more than 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 amino acid residues.

4. A method according to any one of claims 1 to 3, wherein the net charge of the linker is neutral or positive.

5. The method of any one of claims 1 to 4, wherein the linker does not comprise negatively charged amino acid residues.

6. The method according to any one of claims 1 to 5, wherein the linker comprises the amino acid sequence RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4) or RK-Val-Cit (SEQ ID NO: 54).

7. The method according to any one of claims 1 to 6, wherein the linker comprises the amino acid sequence RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3) or RK-Val-Cit (SEQ ID NO: 54).

8. The method according to any one of claims 1 to 7, wherein the linker comprises the amino acid sequence RKAA (SEQ ID NO: 1).

9. The method of any one of claims 1 to 8, wherein B is a linking moiety.

10. The method of claim 9, wherein the connecting portion B comprises

Bio-orthogonal labelling group, or

Non-bioorthogonal entities for cross-linking.

11. The method of claim 10, wherein the bio-orthogonal labeling group or the non-bio-orthogonal entity for cross-linking consists of or comprises at least one molecule or moiety selected from the group consisting of:

-N-N≡N or-N ₃ ；

·Lys(N ₃ )；

Tetrazine;

alkynes;

strained cyclooctyne;

·BCN；

strained olefins;

photoreactive groups;

aldehyde;

acyl trifluoroborates;

protein degrading agent ('PROTAC');

cyclopentadiene/spirocyclopentadiene;

a thio-selective electrophile;

-SH; and

cysteine.

12. The method according to any one of claims 9 to 11, comprising the further step of coupling one or more payloads to the linking moiety B.

13. The method of claim 12, wherein the one or more payloads are coupled to the linking moiety B via a click reaction.

14. The method of any one of claims 1 to 8, wherein B is a payload.

15. The method of any of claims 12 to 14, wherein the payload comprises at least one of:

toxins;

cytokines;

growth factors;

radionuclides;

hormones;

antiviral agents;

an antimicrobial agent;

fluorescent dye;

immunomodulators/immunostimulants;

half-life increasing moiety;

a solubility-increasing moiety;

polymer-toxin conjugate;

nucleic acid;

biotin or streptavidin moiety;

vitamins;

protein degrading agent ('PROTAC');

a target binding moiety; and/or

Anti-inflammatory agents.

16. The method of claim 15, wherein the toxin is at least one selected from the group consisting of:

pyrrolobenzodiazepines (e.g., PBDs);

auristatin (e.g., MMAE, MMAF);

maytansinoids (e.g., maytansine, DM1, DM4, DM 21);

sesquicomycin;

nicotinamide phosphoribosyl transferase (NAMPT) inhibitors;

Microtubule lysin;

enediyne (e.g., calicheamicin);

anthracycline derivatives (PNUs) (e.g., doxorubicin);

inhibitors of the pyrrolyl Kinesin Spindle Protein (KSP);

candidiasis;

drug efflux pump inhibitors;

mountain Zhuo Meisu;

amanitine (e.g., α -amanitine); and

camptothecins (e.g., irinotecan, delutinacon).

17. The method according to any one of claims 14 to 16, wherein the chemical spacer (Sp ₂ ) Including self-cleaving moieties.

18. The method of claim 17, wherein the self-cleaving portion is directly attached to the payload B.

19. The method of claim 17 or 18, wherein the self-cleaving moiety comprises a p-aminobenzylcarbamoyl (PABC) moiety.

20. The method according to any one of claims 1 to 19, wherein the antibody is an IgG antibody, in particular an IgG1 antibody.

21. The method according to claim 20, wherein the linker-coupled Gln residue is comprised in the Fc domain of the antibody, in particular wherein the linker-coupled Gln residue is C of an IgG antibody _H Gln residue Q295 of the 2 domain (EU numbering).

22. The method of claim 20, wherein the linker-coupled Gln residues are introduced into the heavy or light chain of the antibody by molecular engineering.

23. The method of claim 22, wherein the Gln residues introduced into the heavy or light chain of the antibody by molecular engineering are C of a non-glycosylated IgG antibody _H 2 domain N297Q (EU numbering).

24. The method of claim 22, wherein the Gln residues introduced into the heavy or light chain of the antibody by molecular engineering are contained in a peptide that has been (a) integrated into the heavy or light chain of the antibody, or (b) fused to the N-terminus or C-terminus of the heavy or light chain of the antibody.

25. The method of claim 24, wherein the peptide comprising a Gln residue has been fused to the C-terminus of the heavy chain of the antibody.

26. The method according to any one of claims 20 to 22 or 24 to 25, wherein the IgG antibody is a glycosylated IgG antibody, in particular wherein the IgG antibody is at C _H Glycosylation at residue N297 (EU numbering) of the 2 domain.

27. The method of any one of claims 1 to 26, wherein the antibody is selected from the group consisting of: vibutuzumab, trastuzumab, gemtuzumab, oxtuzumab, avermectin, cetuximab, rituximab, up Lei Tuoyou mab, pertuzumab, vedolizumab, oreuzumab, tolizumab, wu Sinu mab, golimumab, otouzumab, sha Xituo mab, bei Lantuo mab, polotouzumab and enrolment mab.

28. The method of any one of claims 1 to 27, wherein the antibody is selected from the group consisting of: velutinab, gemtuzumab, trastuzumab, oxtuzumab, poltuzumab, enrolment mab, sha Xituo bead mab, bei Lantuo mab.

29. The method of any one of claims 1 to 28, wherein the antibody is poloxamer or trastuzumab or enrolment mab.

30. The method of any one of claims 1 to 29, wherein the linker is coupled to a γ -carboxamide group of a Gln residue comprised in the antibody.

31. The method of any one of claims 1 to 30, wherein the linker is adapted to couple to a glycosylated antibody with a coupling efficiency of at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95%.

32. The method according to any one of claims 1 to 31, wherein the microbial transglutaminase is derived from a streptomyces species, in particular streptomyces mobaraensis.

33. An antibody-linker conjugate prepared by the method of any one of claims 1 to 32.

34. An antibody-linker conjugate comprising:

a) An antibody; and

b) A joint, the joint comprising the following structure:

(Sp ₁ )-RK-(Sp ₂ )-B-(Sp ₃ ) Or (b)

(Sp ₁ )-B-(Sp ₂ )-RK-(Sp ₃ ) The method comprises the steps of carrying out a first treatment on the surface of the Wherein the method comprises the steps of

·(Sp ₁ ) Is a chemical spacer or is absent;

·(Sp ₂ ) Is a chemical spacer or is absent;

·(Sp ₃ ) Is a chemical spacer or is absent;

r is arginine or an arginine derivative or arginine mimetic;

k is lysine or a lysine derivative or a lysine mimetic;

b is a linking part or payload;

wherein the linker is coupled to the antibody via an isopeptide bond formed between a γ -carboxamide group of a glutamine residue contained in the antibody and a primary amine contained in a side chain of a lysine residue, a lysine derivative or a lysine mimetic contained in an RK motif contained in the linker.

35. The antibody-linker conjugate of claim 34 wherein the chemical spacer (Sp ₁ )、(Sp ₂ ) Sum (Sp) ₃ ) Each independently comprises 0 to 12 amino acid residues.

36. The antibody-linker conjugate of claim 34 or 35 wherein the linker comprises no more than 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 amino acid residues.

37. The antibody-linker conjugate of any one of claims 34 to 36 wherein the net charge of the linker is neutral or positive.

38. The antibody-linker conjugate of any one of claims 34 to 37 wherein the linker does not comprise a negatively charged amino acid residue.

39. The antibody-linker conjugate of any one of claims 34 to 38 wherein said linker comprises an amino acid sequence selected from the group consisting of: RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4), RK-Val-Cit (SEQ ID NO: 54).

40. The antibody-linker conjugate of any one of claims 34 to 39, wherein said linker comprises an amino acid sequence selected from the group consisting of: RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RK-Val-Cit (SEQ ID NO: 54).

41. The antibody-linker conjugate according to any one of claims 34 to 40, wherein said linker comprises the amino acid sequence RKAA (SEQ ID NO: 1).

42. The antibody-linker conjugate of any one of claims 34 to 41 wherein B is a linking moiety.

43. The antibody-linker conjugate according to claim 42, wherein the linking moiety B comprises

Bio-orthogonal labelling group, or

Non-bioorthogonal entities for cross-linking.

44. The antibody-linker conjugate according to claim 43, wherein the bio-orthogonal labeling group or the non-bio-orthogonal entity for cross-linking consists of or comprises at least one molecule or moiety selected from the group consisting of:

-N-N≡N or-N ₃ ；

·Lys(N ₃ )；

Tetrazine;

alkynes;

strained cyclooctyne;

·BCN；

strained olefins;

photoreactive groups;

aldehyde;

acyl trifluoroborates;

protein degrading agent ('PROTAC');

cyclopentadiene/spirocyclopentadiene;

a thio-selective electrophile;

-SH; and

cysteine.

45. The antibody-linker conjugate of any one of claims 42 to 44 wherein one or more payloads are coupled to the linking moiety B.

46. The antibody-linker conjugate according to claim 45, wherein one or more payloads are coupled to the linking moiety B via a click reaction.

47. The antibody-linker conjugate of any one of claims 34 to 41 wherein B is a payload.

48. The antibody-linker conjugate of any one of claims 45 to 47, wherein the payload comprises at least one of:

Toxins;

cytokines;

growth factors;

radionuclides;

hormones;

antiviral agents;

an antimicrobial agent;

fluorescent dye;

immunomodulators/immunostimulants;

half-life increasing moiety;

a solubility-increasing moiety;

polymer-toxin conjugate;

nucleic acid;

biotin or streptavidin moiety;

vitamins;

protein degrading agent ('PROTAC');

a target binding moiety; and/or

Anti-inflammatory agents.

49. The antibody-linker conjugate according to claim 48, wherein the toxin is at least one selected from the group consisting of:

pyrrolobenzodiazepines (e.g., PBDs);

auristatin (e.g., MMAE, MMAF);

maytansinoids (e.g., maytansine, DM1, DM4, DM 21);

sesquicomycin;

nicotinamide phosphoribosyl transferase (NAMPT) inhibitors;

microtubule lysin;

enediyne (e.g., calicheamicin);

anthracycline derivatives (PNUs) (e.g., doxorubicin);

inhibitors of the pyrrolyl Kinesin Spindle Protein (KSP);

candidiasis;

drug efflux pump inhibitors;

mountain Zhuo Meisu;

amanitine (e.g., α -amanitine); and

camptothecins (e.g., irinotecan, delutinacon).

50. The antibody-linker conjugate of any one of claims 47 to 49 wherein the chemical spacer (Sp ₂ ) Including self-cleaving moieties.

51. The antibody-linker conjugate according to claim 50, wherein the self-cleaving moiety is directly attached to the payload B.

52. The antibody-linker conjugate of claim 50 or 51 wherein the self-cleaving moiety comprises a p-aminobenzylcarbamoyl (PABC) moiety.

53. The antibody-linker conjugate of any one of claims 34 to 52 wherein the antibody is an IgG antibody, particularly an IgG1 antibody.

54. The antibody-linker conjugate according to claim 53, wherein theThe linker-conjugated Gln residues are comprised in the Fc domain of said antibody, in particular wherein the linker-conjugated Gln residues are C of an IgG antibody _H Gln residue Q295 of the 2 domain (EU numbering).

55. The antibody-linker conjugate according to claim 53, wherein the linker-conjugated Gln residue has been introduced into the heavy or light chain of the antibody by molecular engineering.

56. The antibody-linker conjugate of claim 55, wherein Gln residues introduced into the heavy or light chain of the antibody by molecular engineering are C of a non-glycosylated IgG antibody _H 2 domain N297Q (EU numbering).

57. The antibody-linker conjugate according to claim 55, wherein the Gln residue introduced into the heavy or light chain of the antibody by molecular engineering is contained in a peptide that has been (a) integrated into the heavy or light chain of the antibody, or (b) fused to the N-terminus or C-terminus of the heavy or light chain of the antibody.

58. The antibody-linker conjugate according to claim 57, wherein the peptide comprising a Gln residue has been fused to the C-terminus of the heavy chain of the antibody.

59. The antibody-linker conjugate of any one of claims 53 to 55 or 57 to 58, wherein the IgG antibody is a glycosylated IgG antibody, particularly wherein the IgG antibody is at C _H Glycosylation at residue N297 (EU numbering) of the 2 domain.

60. The antibody-linker conjugate of any one of claims 34 to 59 wherein said antibody is selected from the group consisting of: vibutuzumab, trastuzumab, gemtuzumab, oxtuzumab, avermectin, cetuximab, rituximab, up Lei Tuoyou mab, pertuzumab, vedolizumab, oreuzumab, tolizumab, wu Sinu mab, golimumab, otouzumab, sha Xituo mab, bei Lantuo mab, polotouzumab and enrolment mab.

61. The antibody-linker conjugate of any one of claims 34 to 60 wherein said antibody is selected from the group consisting of: velutinab, gemtuzumab, trastuzumab, oxtuzumab, poltuzumab, enrolment mab, sha Xituo bead mab, bei Lantuo mab.

62. The antibody-linker conjugate of any one of claims 34 to 61, wherein the antibody is either pertuzumab or trastuzumab or enrolment mab.

63. An antibody-drug conjugate comprising:

a) IgG antibodies; and

b) A linker comprising a drug moiety B, wherein the drug moiety B is covalently linked to an amino acid sequence selected from the group consisting of: RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4) or RK-Val-Cit (SEQ ID NO: 54);

wherein the linker is via a C-terminus of the antibody _H An isopeptide bond formed between the gamma-carboxamide group of glutamine residue Q295 (EU numbering) of the 2 domain and the primary amine contained in the side chain of the lysine residue contained in the linker is coupled to the IgG antibody.

64. The antibody-drug conjugate of claim 63 wherein drug moiety B is linked to the N-terminus or C-terminus of the amino acid sequence contained in the linker via a self-cleaving moiety.

65. The antibody-drug conjugate of claim 64 wherein the self-cleaving moiety comprises a p-aminobenzylcarbamoyl (PABC) moiety.

66. The antibody-drug conjugate of any of claims 63 to 65 wherein the IgG antibody is a glycosylated IgG antibody, particularly wherein the antibody is at C _H Glycosylation at residue N297 (EU numbering) of the 2 domain.

67. The antibody-drug conjugate of any of claims 63-66 wherein the IgG antibody is an IgG1 antibody.

68. The antibody-drug conjugate of any of claims 63 to 67 wherein the IgG antibody is a poloxamer or an antibody comprising a heavy chain as set forth in SEQ ID No. 5 and a light chain as set forth in SEQ ID No. 6.

69. The antibody-drug conjugate of any of claims 63-67 wherein the IgG antibody is trastuzumab or enrolment mab or an antibody comprising a heavy chain as set forth in SEQ ID No. 7 and a light chain as set forth in SEQ ID No. 8.

70. The antibody-drug conjugate of any of claims 63-67 wherein the IgG antibody is enrolment mab or an antibody comprising a heavy chain as set forth in SEQ ID No. 9 and a light chain as set forth in SEQ ID No. 10 or 11.

71. The antibody-drug conjugate of any one of claims 63 to 70 wherein the drug is selected from the group consisting of toxins of:

pyrrolobenzodiazepines (e.g., PBDs);

auristatin (e.g., MMAE, MMAF);

maytansinoids (e.g., maytansine, DM1, DM4, DM 21);

sesquicomycin;

nicotinamide phosphoribosyl transferase (NAMPT) inhibitors;

microtubule lysin;

enediyne (e.g., calicheamicin);

anthracycline derivatives (PNUs) (e.g., doxorubicin);

inhibitors of the pyrrolyl Kinesin Spindle Protein (KSP);

candidiasis;

drug efflux pump inhibitors;

mountain Zhuo Meisu;

amanitine (e.g., α -amanitine); and

camptothecins (e.g., irinotecan, delutinacon).

72. Antibody-drug conjugate according to any one of claims 63 to 71, wherein the linker has the structure RKAA-PABC-B, in particular wherein B is an auristatin or a maytansinoid, in particular wherein the auristatin is MMAE, and wherein the maytansinoid is DM1 or maytansinoid.

73. Antibody-drug conjugate according to any one of claims 63 to 71, wherein the linker has the structure RKA-PABC-B, in particular wherein B is an auristatin or a maytansinoid, in particular wherein the auristatin is MMAE, and wherein the maytansinoid is DM1 or maytansinoid.

74. The antibody-drug conjugate of any one of claims 63 to 71, wherein the linker has the structure ARK-PABC-B, in particular wherein B is an auristatin or a maytansinoid, in particular wherein the auristatin is MMAE, and wherein the maytansinoid is DM1 or maytansinoid.

75. Antibody-drug conjugate according to any one of claims 63 to 71, wherein the linker has the structure RKR-PABC-B, in particular wherein B is an auristatin or a maytansinoid, in particular wherein the auristatin is MMAE, and wherein the maytansinoid is DM1 or maytansinoid.

76. Antibody-drug conjugate according to any one of claims 63 to 71, wherein the linker has the structure RK-Val-Cit-PABC-B, in particular wherein B is auristatin or maytansinoid, in particular wherein the auristatin is MMAE, and wherein the maytansinoid is DM1 or maytansinoid.

77. A linker construct comprising the structure:

(Sp ₁ )-RK-(Sp ₂ )-B-(Sp ₃ ) Or (b)

(Sp ₁ )-B-(Sp ₂ )-RK-(Sp ₃ ) The method comprises the steps of carrying out a first treatment on the surface of the Wherein the method comprises the steps of

·(Sp ₁ ) Is a chemical spacer or is absent;

·(Sp ₂ ) Is a chemical spacer or is absent;

·(Sp ₃ ) Is a chemical spacer or is absent;

r is arginine or an arginine derivative or arginine mimetic;

K is lysine or a lysine derivative or a lysine mimetic;

b is the connection portion or payload.

78. The linker construct according to claim 77, wherein said chemical spacer (Sp ₁ )、(Sp ₂ ) Sum (Sp) ₃ ) Each independently comprises 0 to 12 amino acid residues.

79. The linker construct of claim 77 or 78, wherein said linker comprises no more than 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 amino acid residues.

80. The linker construct of any one of claims 77 to 79, wherein the net charge of said linker is neutral or positive.

81. The linker construct of any one of claims 77 to 80, wherein said linker does not comprise a negatively charged amino acid residue.

82. The linker construct according to any one of claims 77 to 81, wherein said linker comprises the amino acid sequence RKAA (SEQ ID NO: 1), RKA (SEQ ID NO: 2), ARK (SEQ ID NO: 3), RKR (SEQ ID NO: 4) or RK-Val-Cit (SEQ ID NO: 54).

83. The linker construct of any one of claims 77 to 82, wherein B is a linking moiety.

84. The linker construct of claim 83, wherein said linking moiety B comprises

Bio-orthogonal labelling group, or

Non-bioorthogonal entities for cross-linking.

85. The linker construct according to claim 84, wherein said bio-orthogonal labeling group or said non-bio-orthogonal entity for cross-linking consists of or comprises at least one molecule or moiety selected from the group consisting of:

-N-N≡N or-N ₃ ；

·Lys(N ₃ )；

Tetrazine;

alkynes;

strained cyclooctyne;

·BCN；

strained olefins;

photoreactive groups;

aldehyde;

acyl trifluoroborates;

protein degrading agent ('PROTAC');

cyclopentadiene/spirocyclopentadiene;

a thio-selective electrophile;

-SH; and

cysteine.

86. The linker construct according to any one of claims 77 to 85, wherein said linker construct consists of or comprises the structure RKAA-B, in particular wherein B is Lys (N ₃ ) Or cysteine.

87. The linker construct according to any one of claims 77 to 85, wherein said linker construct consists of or comprises the structure RKA-B, in particular wherein B is Lys (N ₃ ) Or cysteine.

88. The linker construct according to any one of claims 77 to 85, wherein said linker construct consists of or comprises structure ARK-B, in particular wherein B is Lys (N ₃ ) Or cysteine.

89. The linker construct according to any one of claims 77 to 85, wherein said linker construct consists of or comprises structure B-RKR, in particular wherein B is Lys (N ₃ ) Or cysteine.

90. The joint construct according to any one of claims 77 to 82, wherein B is a payload.

91. The joint construct of claim 90, wherein the payload comprises at least one of:

toxins;

cytokines;

growth factors;

radionuclides;

hormones;

antiviral agents;

an antimicrobial agent;

fluorescent dye;

immunomodulators/immunostimulants;

half-life increasing moiety;

a solubility-increasing moiety;

polymer-toxin conjugate;

nucleic acid;

biotin or streptavidin moiety;

vitamins;

protein degrading agent ('PROTAC');

a target binding moiety; and/or

Anti-inflammatory agents.

92. The linker construct of claim 91, wherein said toxin is at least one selected from the group consisting of:

pyrrolobenzodiazepines (e.g., PBDs);

auristatin (e.g., MMAE, MMAF);

maytansinoids (e.g., maytansine, DM1, DM4, DM 21);

Sesquicomycin;

nicotinamide phosphoribosyl transferase (NAMPT) inhibitors;

microtubule lysin;

enediyne (e.g., calicheamicin);

anthracycline derivatives (PNUs) (e.g., doxorubicin);

inhibitors of the pyrrolyl Kinesin Spindle Protein (KSP);

candidiasis;

drug efflux pump inhibitors;

mountain Zhuo Meisu;

amanitine (e.g., α -amanitine); and

camptothecins (e.g., irinotecan, delutinacon).

93. The linker construct according to any one of claims 90 to 92, wherein said chemical spacer (Sp ₂ ) Including self-cleaving moieties.

94. The linker construct of claim 93, wherein said self-cleaving moiety is directly attached to said payload B.

95. The linker construct of claim 93 or 94, wherein said self-cleaving moiety comprises a p-aminobenzylcarbamoyl (PABC) moiety.

96. The linker construct according to any one of claims 90 to 95, wherein the linker construct consists of or comprises the structure RKAA-PABC-B, in particular wherein B is auristatin or maytansinoid, in particular wherein the auristatin is MMAE, and wherein the maytansinoid is DM1 or maytansinoid.

97. The linker construct according to any one of claims 90 to 95, wherein the linker construct consists of or comprises the structure RKA-PABC-B, in particular wherein B is an auristatin or maytansinoid, in particular wherein the auristatin is MMAE, and wherein the maytansinoid is DM1 or maytansinoid.

98. The linker construct according to any one of claims 90 to 95, wherein the linker construct consists of or comprises the structure ARK-PABC-B, in particular wherein B is auristatin or maytansinoid, in particular wherein the auristatin is MMAE, and wherein the maytansinoid is DM1 or maytansinoid.

99. The linker construct according to any one of claims 90 to 95, wherein the linker construct consists of or comprises the structure B-PABC-RKR, in particular wherein B is auristatin or maytansinoid, in particular wherein the auristatin is MMAE, and wherein the maytansinoid is DM1 or maytansinoid.

100. The linker construct according to any one of claims 90 to 95, wherein said linker construct consists of or comprises the structure RK-Val-Cit-PABC-B, in particular wherein B is an auristatin or maytansinoid, in particular wherein said auristatin is MMAE, and wherein said maytansinoid is DM1 or maytansinoid.

101. Use of the linker construct according to any one of claims 77 to 100 in the production of an antibody-linker conjugate by microbial transglutaminase.

102. The use of claim 99, wherein the antibody is an IgG antibody, in particular an IgG1 antibody.

103. The use of claim 101 or 102, wherein the antibody is either pertuzumab or trastuzumab or enrolment mab.

104. A pharmaceutical composition comprising:

a) The antibody-linker conjugate of any one of claims 34 to 63, in particular wherein the antibody-linker conjugate comprises at least one payload;

or (b)

b) The antibody-drug conjugate of any one of claims 64 to 76; and is also provided with

The pharmaceutical composition comprises at least one pharmaceutically acceptable ingredient.

105. The pharmaceutical composition of claim 104, comprising at least one additional therapeutically active agent.

106. The antibody-linker conjugate of any one of claims 34 to 63, particularly wherein said antibody-linker conjugate comprises at least one payload, the antibody-drug conjugate of any one of claims 64 to 76, or the pharmaceutical composition of claim 104 or 105 for use in therapy and/or diagnosis.

107. The antibody-linker conjugate of any one of claims 34 to 63, particularly wherein said antibody-linker conjugate comprises at least one payload, the antibody-drug conjugate of any one of claims 64 to 76, or the pharmaceutical composition of claim 104 or 105, for use in therapy

● Patients suffering from neoplastic diseases, neurological diseases, autoimmune diseases, inflammatory diseases or infectious diseases,

● Patients at risk of developing neoplastic, neurological, autoimmune, inflammatory or infectious diseases, and/or

● Patients diagnosed with neoplastic disease, neurological disease, autoimmune disease, inflammatory disease or infectious disease.

108. The antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition for use according to claim 107, wherein the antibody-linker conjugate or antibody-drug-linker conjugate comprised in the pharmaceutical composition comprises poloxamer and wherein the neoplastic disease is B cell-related cancer.

109. The antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition for use according to claim 108, wherein the B cell-related cancer is non-hodgkin's lymphoma, in particular wherein the B cell-related cancer is diffuse large B cell lymphoma.

110. The antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition for use according to claim 108 or 109, wherein said antibody-linker conjugate, said antibody-drug conjugate or said pharmaceutical composition is administered in combination with bendamustine and/or rituximab.

111. The antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition for use according to claim 107, wherein the antibody-linker conjugate or antibody-drug conjugate comprised in the pharmaceutical composition comprises trastuzumab, and wherein the neoplastic disease is a HER2 positive cancer, in particular HER2 positive breast cancer, gastric cancer, ovarian cancer or lung cancer.

112. The antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition for use according to claim 111, wherein said antibody-linker conjugate, said antibody-drug conjugate or said pharmaceutical composition is administered in combination with lapatinib, capecitabine, and/or a taxane.

113. The antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition for use according to claim 107, wherein the antibody-linker conjugate or antibody-drug conjugate comprised in the pharmaceutical composition comprises enrolment mab or an enrolment mab variant, and wherein the neoplastic disease is a binding-4 positive cancer, in particular a binding-4 positive pancreatic cancer, lung cancer, bladder cancer or breast cancer.

114. The antibody-linker conjugate, antibody-drug conjugate or pharmaceutical composition for use according to claim 113, wherein said antibody-linker conjugate, said antibody-drug conjugate or said pharmaceutical composition is administered in combination with a cisplatin-based chemotherapeutic agent and/or palbociclib.

115. The antibody-linker conjugate of any one of claims 34 to 63, particularly wherein said antibody-linker conjugate comprises at least one payload, the antibody-drug conjugate of any one of claims 64 to 76 or the pharmaceutical composition of claim 104 or 105 for use in the manufacture of a medicament for use in the treatment of

● Patients suffering from neoplastic diseases, neurological diseases, autoimmune diseases, inflammatory diseases or infectious diseases,

● Patients at risk of developing neoplastic, neurological, autoimmune, inflammatory or infectious diseases, and/or

● Patients diagnosed with neoplastic disease, neurological disease, autoimmune disease, inflammatory disease or infectious disease.

116. A method of treating or preventing a neoplastic disease, the method comprising administering the antibody-linker conjugate of any one of claims 34 to 63 to a patient in need thereof, particularly wherein the antibody-linker conjugate comprises at least one payload, the antibody-drug conjugate of any one of claims 64 to 76, or the pharmaceutical composition of claim 104 or 105.