Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/54—Interleukins [IL]
  • C07K14/55—IL-2
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/54—Interleukins [IL]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/555—Interferons [IFN]
  • C07K14/56—IFN-alpha
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2318/00—Antibody mimetics or scaffolds
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
  • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor

Definitions

• the present invention belongs to the field of immunology and relates to proteins and polypeptides comprising an immunoglobulin single variable domain (ISVD) fused with a cytokine, wherein the fusion is obtained via insertion of the cytokine in the ISVD at a fusion site in a loop or turn of the ISVD which is not a complementarity determining region and/or wherein the cytokine is a circularly permuted variant of the wild-type cytokine.
• the binding of the cytokine-ISVD chimeric protein to the cytokine receptor or receptor subunit allows for modulation of cytokine receptor activity and/or (downstream) signalling.
• Cytokines are small signalling proteins and are known to play a critical role in the body's response to inflammation and immune attack. Pro-inflammatory cytokines alert the immune system to the presence of potential infection or danger. However, unregulated cytokine production can lead to autoinflammatory disease states.
• Cytokines have been widely used as therapy against cancer, infection or other diseases.
• Interleukin-2 IL-2
• Cytokine receptors are cell-surface glycoproteins that bind specifically to cytokines and transduce their signals. The biologic response can vary between cytokine receptors and from cell to cell but in general it involves gene expression, changes in the cell cycle, and release of mediators such as cytokines themselves. Cytokine receptors generally function as oligomeric complexes consisting of typically two to four subunits that may be the same or different. Once cytokines bind to surface receptors, they induce receptor clustering or oligomerization (e.g., heterodimerization or heterotrimerization) followed by receptor activation and the generation of intracellular signals (downstream signalling) (Christopher J.
• receptor clustering or oligomerization e.g., heterodimerization or heterotrimerization
• a heterotrimeric receptor is, for example, the IL-2 receptor (IL-2R), which has three forms: IL- 2Ra (or CD25), IL-2R (or CD122) and I L-2Ry (or CD132).
• the a chain receptor binds IL-2 with low affinity, the combination of
• the intermediate and high affinity receptor forms are functional and cause changes in the cell when IL-2 binds to them (Liao W, Lin JX and Leonard WJ, "IL-2 family cytokines: new insights into the complex roles of IL-2 as a broad regulator of T helper cell differentiation", Curr Opin Immunol., 2011, 23(5):598-604).
• IFNAR IFN-alpha receptor
• IFNAR1 IFNAR1
• IFNAR2 IFN-alpha receptor 2
• the biological actions of type I IFNs include different ranges of subtypes in different cells (van Boxel-Dezaire et al., "Complex modulation of cell type-specific signalling in response to type I interferons", Immunity, 2006, 25(3):361-72).
• IL-18 receptor is another example of a heterodimeric receptor. It consists of two distinct but structurally related immunoglobulin-like domains that are members of the IL-1 receptor family: IL-18Ra and IL-18RP. Secreted, mature IL-18 interacts with IL-18Ra. This complex heterodimerizes with the signal-transducing I L-18RP accessory protein that facilitates a conformational change in the receptor to allow high-affinity binding of ligand (Stylianou E., "Interleukins IL-1 and IL-18", Encyclopedia of Respiratory Medicine, 2006, 350-354).
• 3 does not directly interact with IL-18, and it is IL-18Ra that is solely responsible for IL-18 binding.
• IL-18Ra that is solely responsible for IL-18 binding.
• 3 complex may present different contact sites for IL-18. These differences may involve conformational changes, leading to different orientations as well as different numbers of contacting sites (Chengbin Wu., et al., "IL-18 receptor ⁇ -induced changes in the presentation of IL-18 binding sites affect ligand binding and signal transduction", J Immunol, 2003, 170 (11): 5571-5577).
• cytokine's receptor-binding functionality to, e.g., modulate the response triggered by the cytokines upon binding the receptor/receptor subunit
• modulation of cytokine's receptor-binding functionality to, e.g., modulate the response triggered by the cytokines upon binding the receptor/receptor subunit
• being able to steer activation to a certain cell population could be beneficial for the treatment of certain conditions such as, e.g., cancer.
• Lopes et al. (“ALKS 4230: a novel engineered IL-2 fusion protein with an improved cellular selectivity profile for cancer immunotherapy", Journal for ImmunoTherapy of Cancer, 2020, 8:e000673) describes an engineered fusion protein comprised of a circularly-permuted IL-2 with the extracellular domain of IL-2Ra, to selectively activate effector lymphocytes bearing the intermediate-affinity IL-2R. According to the authors, the extracellular domain of IL-2Ra of the fusion protein would sterically inhibit the interaction of the IL-2 of the fusion protein with endogenous IL-2Ra subunit.
• the IL-2 of the fusion protein would be able to retain the ability to signal through the intermediate-affinity IL-2R (subunits
• Memory CD8 + T cells and NK cells have been shown to be required for protective anticancer immune responses.
• the present invention solves the above problem and provides a chimeric protein which comprises an immunoglobulin single variable domain (ISVD) fused with a cytokine, wherein an internal fusion site of the ISVD is linked to the cytokine, wherein the internal fusion site is located in a loop or turn between two secondary structure elements, and wherein the cytokine is preferably a circularly permuted cytokine.
• ISVD immunoglobulin single variable domain
• the chimeric protein provided by the present invention is more rigid as compared to /V/C-terminal end-to-end fusions (i.e., it is a "Mega body" -type fusion, see, e.g., WO 2019/086548). Hence, the chimeric protein of the present invention is less prone to proteasomal degradation or flexible movement as compared to /V/C-terminal end-to-end fusion proteins.
• the ISVD present in the chimeric protein provided by the present invention may bind to its target, so the chimeric protein may thus comprise a further target-binding moiety that may be selected depending on the specific properties that are desired for the chimeric protein/polypeptide comprising the same (extension of half-life, labelling, specific localizations or any other functional requirement).
• chimeric protein of the present invention may be linked to further moieties with different functionalities (see the polypeptide of the present invention).
• the chimeric proteins provided by the present invention allow for directed modulation of cytokine's receptor/receptor's subunit-binding functionality and may also have further advantages, as described above.
• the invention provides a polypeptide comprising the chimeric protein of the present invention, optionally wherein the polypeptide further comprises one or more further groups, residues, moieties or binding units, preferably wherein the polypeptide further comprises one or more ISVDs.
• the chimeric protein and/or polypeptide of the present invention is able to modulate the activity (or downstream consequences of the binding of the cytokine comprised in the chimeric protein to at least one of its receptors or receptor subunits) of the cytokine comprised in the chimeric protein and/or protein of the present invention.
• nucleic acid molecule encoding the chimeric protein or the polypeptide of the present invention
• vectors comprising the nucleic acid molecule of the present invention
• host cells comprising the chimeric protein and/or polypeptide of the invention
• nucleic acid molecule or vector encoding the chimeric protein of the invention are provided.
• the present invention further provides methods for producing the chimeric protein and/or the peptide of the present invention.
• a method for modulating the signaling and/or affinity of a cytokine to at least one of its receptors or receptor subunits by fusing the cytokine and an ISVD, preferably wherein the cytokine and the ISVD are fused to create the chimeric protein of the present invention.
• the invention further provides for a fusion protein comprising a cytokine fused to an ISVD, directly or by means of a linker, for modulating the binding affinity of the cytokine comprised in the fusion protein to its receptor.
• the invention also provides the use of the chimeric protein and/or polypeptide of the present invention in medicine, in particular in the treatment of cancer and/or in the treatment of inflammatory diseases.
• Figure 1 Engineering principles of an antigen-binding chimeric protein built from a circularly permuted variant of a scaffold protein that is inserted into the first (3-turn connecting P- strands A and B of an ISVD.
• This scheme shows how an immunoglobulin single variable domain (ISVD) can be grafted onto a large scaffold protein via two peptide bonds or two short linkers that connect the antigenbinding domain to the scaffold.
• Scissors indicate which exposed turns have to be cut in the ISVD and the scaffold.
• Dashed lines indicate how the remaining parts of the ISVD and the scaffold have to be concatenated by use of peptide bonds or short peptide linkers to build the antigen-binding chimeric protein.
• CDRs, framework residues and p-turn regions of the ISVD are defined according to IMGT (Lefranc MP, "Immunoglobulin and T Cell Receptor Genes: IMGT(®) and the birth and Rise of Immunoinformatics", Front Immunol., 2014, 5:22).
• Figure 2 Engineering principles of an antigen-binding chimeric protein built from a circularly permuted variant of IL-2 that is inserted into the first P-turn connecting P-strands A and B of an ISVD.
• This scheme shows how an ISVD can be grafted to IL-2 via two peptide bonds with two short linkers that connect the antigen-binding domain to circularly permuted IL-2.
• Scissors indicate which exposed turns are cut in the ISVD and the scaffold.
• Dashed lines indicate how the remaining parts of the ISVD and the scaffold are concatenated by use of peptide bonds or short peptide linkers to build the antigen-binding chimeric protein.
• CDRs, framework residues and p-turn regions of the ISVD are defined according to IMGT.
• FIG. 4 shows different sites where an ISVD can be grafted onto IL-2 via two peptide bonds.
• Figure 4. A schematic view of the different IL-2 Megabody proteins.
• This figure shows the design of the different IL-2(K35E,C125S) "Megabody proteins".
• the amino acid (AA) sequence of the circularly permuted IL-2(K35E,C125S) is given as a 'collier-de- perle' starting at amino acid at position 1 and ending at amino acid at position 133.
• the small GG linker (grey with white letters) connects the C-terminus of IL-2(K35E,C125S) to the N- terminal part of IL-2.
• the first 3 amino acids of IL-2(K35E,C125S) are deleted, depicted as strikethrough in the figure.
• the point mutations K35E and C125S have circles light grey background in the sequence.
• the insertion place of IL-2(K35E,C125S) for construct SA17667 is enlarged at the bottom of the figure showing where the circularly permuted IL- 2(K35E,C125S) is interrupted (the amino acids between the scissors are deleted in this construct) and how it is fused to ISVD207.
• Construct SA17667 starts with residues 1-12 of ISVD207 followed by a 4 amino acid linker (GSGG), is fused to amino acid at position 62 of IL- 2(K35E,C125S) till amino acid at position 133 which is linked via the GG linker to amino acid at position 4 of IL-2(K35E,C125S), ends at amino acid at position L59 and is connected via a 4 amino acid linker (GGSG) to residues 16-126 of ISVD207.
• the GSG linker between the circularly permuted IL-2(K35E,C125S) and the ISVD207 is given in dotted lined circles, as is the extra Glycine (G).
• a 4 amino acid linker is present between the circularly permuted IL-2(K35E,C125S) and the ISVD207 on either site.
• FIG. 5 An AlphaFold model of a 29 kPa GFP-binding chimeric protein built from a circularly permuted variant of IL-2 inserted into the first (3-turn connecting
• A Model of an antigen-binding chimeric protein made by fusion of an anti-GFP ISVD (top) and a circularly permuted variant of the human IL-2 (bottom) via two peptide bonds or linkers that connect the ISVD to the scaffold.
• B A circularly permuted gene encoding the circularly permuted IL-2(K35E,C125S) (bottom) was inserted in the first p-turn of an anti-GFP ISVD (top, SEQ ID NO: 1) connecting p-strand A to p-strand B (P-turn AB).
• Figure 6 An AlphaFold model of a 29 kPa GFP-binding chimeric protein built from a circularly permuted variant of IL-2 inserted into the first P-turn connecting P-strands A and B of an anti- GFP ISVD.
• A Model of an antigen-binding chimeric protein made by fusion of an anti-GFP ISVD (topright) and a circularly permuted variant of the human IL-2 (bottom-left) via two peptide bonds or linkers that connect the ISVD to the scaffold.
• B A circularly permuted gene encoding the circularly permuted IL-2(K35E,C125S) (bottom) was inserted in the first p-turn of an anti-GFP ISVD (top, SEQ ID NO: 1) connecting p-strand A to p-strand B (P-turn AB).
• Figure 7 An AlphaFold model of a 29 kPa GFP-binding chimeric protein built from a circularly permuted variant of IL-2 inserted into the first P-turn connecting P-strands A and B of an anti- GFP ISVD.
• A Model of an antigen-binding chimeric protein made by fusion of an anti-GFP ISVD (topleft) and a circularly permuted variant of the human interleukine 2 (IL-2, bottom-right) via two peptide bonds or linkers that connect the ISVD to the scaffold.
• B A circularly permuted gene encoding the circularly permuted IL-2(K35E,C125S) (bottom) was inserted in the first p-turn of an anti-GFP ISVD (top, SEQ ID NO: 1) connecting p-strand A to p-strand B (P-turn AB).
• Figure 8 An AlphaFold model of a 29 kPa GFP-binding chimeric protein built from a circularly permuted variant of IL-2 inserted into the first (3-turn connecting
• A Model of an antigen-binding chimeric protein made by fusion of an anti-GFP ISVD (bottom) and a circularly permuted variant of the human IL-2 (top) via two peptide bonds or linkers that connect the ISVD to the scaffold.
• B A circularly permuted gene encoding the circularly permuted IL-2(K35E,C125S) (bottom) was inserted in the first p-turn of an anti-GFP ISVD (top, SEQ ID NO: 1) connecting -strand A to -strand B (P-turn AB).
• Figure 9 Overview of the properties of the different IL-2(K35E,C125S) ISVD207 chimeric Megabody proteins analyzed by yeast display and FACS.
• Figure 10 shows phosphorylation of STAT5 for different immune cell subtypes upon treatment with IL-2-containing compounds.
• CD8+CD25- cells are evaluated in A and B, and CD4+CD25+ cells are evaluated in C and D. Treatment is performed in the absence (A and C) and presence of Human Serum Albumin (HSA) (B and D).
• HSA Human Serum Albumin
• Figure 11 shows proliferation of different immune cell subtypes upon treatment with IL-2- containing compounds.
• CD8+CD25- cells are evaluated in A and B, and CD4+CD25+ cells are evaluated in C and D. Treatment is performed in the absence (A and C) and presence of HSA (B and D).
• Figure 13 Structure of the quaternary complex of IL-2 receptor alpha, beta, and gamma with the AlphaFold model of the IL-2(K35E,C125S)[GF42-M39G]_ISVD207 Mega body protein.
• Figure 14 Structure of the quaternary complex of IL-2 receptor alpha, beta, and gamma with the AlphaFold model of the I L-2(K35E,C125S)[L132-1129] JSVD207 Megabody protein (SA17678).
• Figure 15 Structure of the quaternary complex of IL-2 receptor alpha, beta, and gamma with the AlphaFold model of the IL-2(K35E,C125S)[GL85-P82G]_ISVD207 Megabody protein (SA17659).
• Figure 16 shows IFNy production in a Tetanus Toxoid recall assay that interrogates functionality of anti-PD-Ll-IL-2 compounds in Donor D1688 (A and B) and Donor ABL-0341-02 (C and D).
• Figure 17 Structure of IFNA2a This figure shows on the structure of IFNA2a (PDB 1ITF) the sites where the IFNA2a will be opened (between position 76 & 77) to create a new N- and C- terminus and where the N- and C-terminus will be linked together by peptide linkers to make circularly permuted variants of IFNA2a.
• PDB 1ITF the sites where the IFNA2a will be opened (between position 76 & 77) to create a new N- and C- terminus and where the N- and C-terminus will be linked together by peptide linkers to make circularly permuted variants of IFNA2a.
• Figure 18 Flow cytometric analysis of the expression level of the differently circularly permuted variants of IFNA2a compared to IFNA2a wild-type, each construct is separately displayed on the surface of EBY100 yeast cells; flow cytometric analysis of binding of the antihuman IFNA2a monoclonal Antibody to the same constructs, each separately displayed on the surface of EBY100 yeast cells.
• IFNA2a monoclonal Antibody mAb93452
• mAb93452 an anti-human IFNA2a monoclonal Antibody
• mAb93452 an anti-human IFNA2a monoclonal Antibody
• Phycoerythrin-conjugated Anti-mouse-IgG-Fc an anti-human IFNA2a monoclonal Antibody
• FIG. 19 Flow cytometric analysis of the expression level of the different circularly permuted variants of IFNA2a compared to IFNA2a wild-type, each construct is separately displayed on the surface of EBY100 yeast cells; flow cytometric analysis of binding of IFNAR2 to the same constructs, each separately displayed on the surface of EBY100 yeast cells.
• IFNAR2 Human IFN-alpha/beta R2 protein, His tag
• IFNAR2 Human IFN-alpha/beta R2 protein, His tag
• FIG. 20 An AlphaFold model of a 31 kPa HSA-binding chimeric protein built from a circularly permuted variant of interferon alpha-2a (IFNA2a) inserted into the first (3-turn connecting P- strands A and B of an anti-HSA ISVD.
• IFNA2a interferon alpha-2a
• A Model of an antigen-binding chimeric protein made by fusion of an anti-human serum albumin (HSA) ISVD (bottom) and a circularly permuted variant of the human IFNA2a(top) via two peptide bonds or linkers that connect the ISVD to the scaffold.
• B A circularly permuted gene encoding the circularly permuted interferon alpha-2a (bottom) was inserted in the first P-turn of an anti-HSA ISVD (top, SEQ ID NO: 55) connecting p-strand A to p-strand B (P-turn AB).
• Figure 21 An AlphaFold model of a 31 kPa HSA-binding chimeric protein built from a circularly permuted variant of interferon alpha-2a (IFNA2a) inserted into the first P-turn connecting P- strands A and B of an anti-HSA ISVD.
• IFNA2a interferon alpha-2a
• A Model of an antigen-binding chimeric protein made by fusion of an anti-HSA ISVD (left) and a circularly permuted variant of the IFNA2a (right) via two peptide bonds or linkers that connect the ISVD to the scaffold.
• B A circularly permuted gene encoding the circularly permuted interferon alpha-2a (bottom) was inserted in the first p-turn of an anti-HSA ISVD (top, SEQ ID NO: 55) connecting p-strand A to p-strand B (P-turn AB).
• Figure 22 An AlphaFold model of a 31 kPa HSA-binding chimeric protein built from a circularly permuted variant of interferon alpha-2a inserted into the first P-turn connecting
• A Model of an antigen-binding chimeric protein made by fusion of an anti-HSA ISVD (left) and a circularly permuted variant of the human interferon alpha-2a (I FNA2a)(right) via two peptide bonds or linkers that connect the ISVD to the scaffold.
• B A circularly permuted gene encoding the circularly permuted interferon alpha-2a (bottom) was inserted in the first p-turn of an anti-HSA ISVD (top, SEQ ID NO: 55) connecting -strand A to P-strand B (P-turn AB).
• Figure 23 An AlphaFold model of a 31 kPa HSA-binding chimeric protein built from a circularly permuted variant of interferon alpha-2a inserted into the first P-turn connecting P-strands A and B of an anti-HSA ISVD.
• A Model of an antigen-binding chimeric protein made by fusion of an anti-HSA ISVD (right) and a circularly permuted variant of the human interferon alpha-2a (left) via two peptide bonds or linkers that connect the ISVD to the scaffold.
• B A circularly permuted gene encoding the circularly permuted interferon alpha-2a (bottom) was inserted in the first p-turn of an anti-HSA ISVD (top, SEQ ID NO: 55) connecting P-strand A to P-strand B (P-turn AB).
• Figure 24 Structure of the human ternary complex IFNA2a-IFNAR aligned with the AlphaFold model of IFNA2a[L9-T6] _ALB23002 protein.
• Figure 25 Structure of the human ternary complex IFNA2a-IFNAR aligned with the AlphaFold model of IFNA2a[P109-T6]_ALB23002 protein.
• Figure 26 Structure of the human ternary complex IFNA2a-IFNAR aligned with the AlphaFold model of IFNA2a[S25-K23]_ALB23002 protein.
• Figure 27 Structure of the human ternary complex IFNA2a-IFNAR aligned with the AlphaFold model of IFNA2a[D32-L30]_ALB23002 protein.
• Figure 28 shows phosphorylation of STAT1 in A549 cells upon treatment with IFNA2a- containing compounds.
• Treatment is performed in the absence (A) and presence of HSA (B).
• Figure 29 shows proliferation of RPMI 8226 (A and B) and NCI-H929 (C and D) cells upon treatment with IFNA2a-containing compounds.
• Treatment is performed in the absence (A and C) and presence of HSA (B and D).
• Figure 30 Design of IL-18 circularly permuted variants. This figure shows the position of the sites on the structure of IL-18 (PDB 3F62) where I L18 is opened (between position 69 and 70) to create a new /V- and C-terminus and where the /V- and C-terminus are linked together when designing circularly permuted variants of IL18.
• FIG. 31 Flow cytometric analysis of the expression level of the differently circularly permuted variants of I L18 compared to I L18 wild-type, each construct is separately displayed on the surface of EBY100 yeast cells.
• Figure 32 Schematic view of the genetic fusion to obtain the chimeric proteins of the present invention.
• FIG. 33 mAb D044-3 binds circularly permuted variants of IL18.
• FIG. 34 IL18BP binds some circularly permuted variants of IL18. Flow cytometry analysis of the binding of IL18-BP to IL18 and the circularly permuted variants of IL18 displayed on the cell surface of yeast cells.
• Figure 35 An AlphaFold model of a 33 kD GFP-binding chimeric protein built from a circularly permuted variant of IL18 inserted into the first (3-turn connecting
• A Model of an antigen-binding chimeric protein made by fusion of an anti-GFP ISVD (top) and a circularly permuted variant of the human I L18 (bottom) via two peptide bonds or linkers that connect the ISVD to the scaffold.
• the peptide linkers to connect the ISVD to I L18 are underlined in dashed lines.
• the peptide linking the /V-terminus and the C-terminus of I L18 to make a circularly permuted variant is depicted in italics.
• Figure 36 An AlphaFold model of a 33 kD GFP-binding chimeric protein built from a circularly permuted variant of IL18 inserted into the first P-turn connecting P-strands A and B of an anti- GFP ISVD.
• A Model of an antigen-binding chimeric protein made by fusion of an anti-GFP ISVD (top) and a circularly permuted variant of the human I L18 (bottom) via two peptide bonds or linkers that connect the ISVD to the scaffold.
• Figure 37 An AlphaFold model of a 33 kD GFP-binding chimeric protein built from a circularly permuted variant of IL18 inserted into the first P-turn connecting P-strands A and B of an anti- GFP ISVD.
• A Model of an antigen-binding chimeric protein made by fusion of an anti-GFP ISVD (top) and a circularly permuted variant of the human I L18 (bottom) via two peptide bonds or linkers that connect the ISVD to the scaffold.
• the peptide linkers to connect the ISVD to I L18 are underlined in dashed lines.
• the peptide linking the /V-terminus and the C-terminus of I L18 to make a circularly permuted variant is depicted in italics
• FIG 38 mAb D044-3 binds IL18-ISVD Megabody proteins.
• FIG 39 IL18BP binds I L18-ISVD Megabody proteins.
• cells were pre-incubated with a fluorescent IL18-BP. A variation in the binding of IL18-BP to the different IL18JSVD207 Megabody proteins is seen.
• FIG. 40 GFP binds to I L18-ISVD Megabody proteins.
• a staining of the cells with GFP was performed.
• a dot plot representation was created of the relative fluorescence intensity of individual EBY100 yeast cells each transformed with a pCTCON2 derivative encoding either the control proteins or a different IL18JSVD207 Megabody protein. Gates were set using the negative and positive controls.
• Figure 41 Experimental set-up to harvest displayed proteins from the yeast cell wall.
• IL18[K70-E69]V5b and IL18JSVD207 Megabody proteins in fusion with the acyl carrier protein and a number of accessory peptides, displayed on the surface of the yeast cells can be labeled with biotin using Biotin-PEG3-CoenzymeA and SFP synthase and released from the cell wall by adding DTT.
• FIG. 42 Biolayer Interferometry (BLI) analysis of GFP binding to IL18 ISVD207 Megabody proteins.
• BLI Biolayer Interferometry
• FIG 43 Biolayer Interferometry (BLI) analysis of GFP binding to IL18 ISVD207 IL18[K70- E69] ISVD207 VI.
• BBI Biolayer Interferometry
• Figure 44 Plasma PK profile of IL-2 containing compounds after /'n vivo intravascular treatment of naive female C57BI/6N mice. The results are expressed as mean ⁇ SD.
• LOQ Limit Of Quantification of the plasma PK assay.
• Figure 45 Phosphorylation of STAT5 (pSTAT5) in different immune cells at two timepoints (24h and 48h) after in vivo intravascular treatment of naive female C57BI/6N mice with IL-2 containing compounds.
• CD3+CD4-CD8+ cells are shown in A
• CD3+CD4+CD8-CD25-Foxp3- cells are shown in B
• CD3+CD4+CD8-CD25+Foxp3+ cells are shown in C
• CD3-NK1.1+ are shown in D.
• the results are expressed as median (grey bars) and individual data (symbols).
• Figure 46 Ki67 expression in different immune cells at two timepoints (48h and 72h) after in vivo intravascular treatment of naive female C57BI/6N mice with IL-2 containing compounds.
• CD3+CD4-CD8+ cells are shown in A
• CD3+CD4+CD8-CD25-Foxp3- cells are shown in B
• CD3+CD4+CD8-CD25+Foxp3+ cells are shown in C
• CD3-NK1.1+ are shown in D.
• the results are expressed as median (grey bars) and individual data (symbols):
• Figure 47 Proliferation of different immune cells at 72h after in vivo intravascular treatment of naive female C57BI/6N mice with IL-2 containing compounds.
• CD3+CD4-CD8+ cells are shown in A
• CD3+CD4+CD8-CD25+Foxp3+ cells are shown in B
• CD3-NK1.1+ are shown in C.
• the results are expressed as median (grey bars) and individual data (symbols):
• Figure 48 Ratio between the different immune cell populations at 72h after in vivo intravascular treatment of naive female C57BI/6N mice with IL-2 containing compounds. The ratio between CD3+CD4-CD8+ cells and CD3+CD4+CD25+Foxp3+ cells is shown in A, and the ratio between CD3-NK1.1+ cells and CD3+CD4+CD25+Foxp3+ cells is shown in B. The results are expressed as median (grey bars) and individual data (symbols):
• Similar is interchangeable for alike, analogous, comparable, corresponding, and -like, and is meant to have the same or common characteristics, and/or in a quantifiable manner to show comparable results i.e. with a variation of maximum 20 %, 10 %, more preferably 5 %, or even more preferably 1 %, or less.
• sequence as used herein (for example in terms like “immunoglobulin sequence”, “antibody sequence”, “variable domain sequence”, “VHH sequence” or “protein sequence”), should generally be understood to include both the relevant amino acid sequence as well as nucleic acids or nucleotide sequences encoding the same, unless the context requires a more limited interpretation.
• Amino acid sequences are interpreted to mean a single amino acid or an unbranched sequence of two or more amino acids, depending on the context.
• Nucleotide sequences are interpreted to mean an unbranched sequence of 3 or more nucleotides.
• Nucleotide sequence refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double- and single-stranded DNA, and RNA. It also includes known types of modifications, for example, methylation, "caps" substitution of one or more of the naturally occurring nucleotides with an analogue.
• nucleic acid construct it is meant a nucleic acid sequence that has been constructed to comprise one or more functional units notfound together in nature. Examples include circular, linear, double-stranded, extrachromosomal DNA molecules (plasmids), cosmids (plasmids containing COS sequences from lambda phage), viral genomes comprising non-native nucleic acid sequences, and the like.
• plasmids extrachromosomal DNA molecules
• cosmids plasmids containing COS sequences from lambda phage
• viral genomes comprising non-native nucleic acid sequences, and the like.
• any reference to the amino acid sequences of the invention is meant to encompass post-translational modifications of these sequences occurring in mammalian cells such as CHO cells, including, but not limited to, /V-glycosylation, O-glycosylation, deamidation, Asp isomerization/fragmentation, pyro-glutamate formation, removal of C-terminal lysine, and Met/Trp oxidation.
• nucleotide sequence or amino acid sequence is said to "comprise” another nucleotide sequence or amino acid sequence, respectively, or to “essentially consist of” another nucleotide sequence or amino acid sequence, this may mean that the latter nucleotide sequence or amino acid sequence has been incorporated into the first mentioned nucleotide sequence or amino acid sequence, respectively, but more usually this generally means that the first mentioned nucleotide sequence or amino acid sequence comprises within its sequence a stretch of nucleotides or amino acid residues, respectively, that has the same nucleotide sequence or amino acid sequence, respectively, as the latter sequence, irrespective of how the first mentioned sequence has actually been generated or obtained (which may for example be by any suitable method described herein).
• Coding sequence is a nucleotide sequence, which is transcribed into mRNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus.
• a coding sequence can include, but is not limited to mRNA, cDNA, recombinant nucleotide sequences or genomic DNA, while introns may be present as well under certain circumstances.
• Promoter region of a gene refers to a functional DNA sequence unit that, when operably linked to a coding sequence and possibly placed in the appropriate inducing conditions, is sufficient to promote transcription of said coding sequence.
• "Operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
• a promoter sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the promoter sequence.
• Gene as used here includes both the promoter region of the gene as well as the coding sequence. It refers both to the genomic sequence (including possible introns) as well as to the cDNA derived from the spliced messenger, operably linked to a promoter sequence.
• the term “terminator” or “transcription termination signal” encompasses a control sequence which is a DNA sequence at the end of a transcriptional unit which signals 3' processing and polyadenylation of a primary transcript and termination of transcription.
• the terminator can be derived from the natural gene, from a variety of other plant genes, or from T-DNA.
• the terminatorto be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.
• a “genetic construct”, “chimeric gene”, “chimeric construct” or “chimeric gene construct” is meant a recombinant nucleic acid sequence in which a promoter or regulatory nucleic acid sequence is operatively linked to, or associated with, a nucleic acid sequence that codes for an mRNA, such that the regulatory nucleic acid sequence is able to regulate transcription or expression of the associated nucleic acid coding sequence.
• the regulatory nucleic acid sequence of the chimeric gene is not operatively linked to the associated nucleic acid sequence as found in nature.
• the term “genetic fusion construct” as used herein refers to the genetic construct encoding the mRNA that is translated to the fusion protein of the invention as disclosed herein.
• vector is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked, and includes any vector known to the skilled person, including any suitable type including, but not limited to, plasmid vectors, cosmid vectors, phage vectors, such as lambda phage, viral vectors, such as adenoviral, AAV or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or Pl artificial chromosomes (PAC).
• plasmid vectors such as lambda phage
• viral vectors such as adenoviral, AAV or baculoviral vectors
• artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or Pl artificial chromosomes (PAC).
• BAC bacterial artificial chromosomes
• YAC yeast artificial chromosomes
• Expression vectors comprise plasmids as well as viral vectors and generally contain a desired coding sequence and appropriate DNA sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in in vitro expression systems.
• Expression vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors having an origin of replication which functions in the host cell).
• Other vectors can be integrated into the genome of a host cell upon introduction into the host cell and are thereby replicated along with the host genome.
• Suitable vectors have regulatory sequences, such as promoters, enhancers, terminator sequences, and the like as desired and according to a particular host organism (e.g., bacterial cell, yeast cell).
• Cloning vectors are generally used to engineer and amplify a certain desired DNA fragment and may lack functional sequences needed for expression of the desired DNA fragments.
• the construction of expression vectors for use in transfecting prokaryotic cells is also well known in the art, and thus can be accomplished via standard techniques (see, for example, Sambrook, et al.
• “Host cells” can be either prokaryotic or eukaryotic.
• the cells can be transiently or stably transfected.
• Such transfection of expression vectors into prokaryotic and eukaryotic cells can be accomplished via any technique known in the art, including but not limited to standard bacterial transformations, calcium phosphate co-precipitation, electroporation, or liposome mediated-, DEAE dextran mediated-, polycationic mediated-, or viral mediated transfection.
• Recombinant host cells are those which have been genetically modified to contain an isolated DNA molecule, nucleic acid molecule or expression construct or vector of the invention.
• the DNA can be introduced by any means known to the art which are appropriate for the particular type of cell, including without limitation, transformation, lipofection, electroporation or viral mediated transduction.
• a DNA construct capable of enabling the expression of the chimeric protein of the invention can be easily prepared by the art-known techniques such as cloning, hybridization screening and Polymerase Chain Reaction (PCR). Standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and commonly employed by those skilled in the art. A number of standard techniques are described in Sambrook et al. (2012), Wu (ed.) (1993) and Ausubel et al. (2016). Representative host cells that may be used with the invention include, but are not limited to, bacterial cells, yeast cells, insect cells, plant cells and animal cells.
• Bacterial host cells suitable for use with the invention include Escherichia spp. cells, Bacillus spp. cells, Streptomycesspp. cells, Erwinia spp. cells, Klebsiella spp. cells, Serratia spp. cells, Pseudomonas spp. cells, and Salmonella spp. cells.
• Animal host cells suitable for use with the invention include insect cells and mammalian cells (most particularly derived from Chinese hamster (e.g., CHO), and human cell lines, such as HeLa.
• Yeast host cells suitable for use with the invention include species within Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia (e.g., Pichia pastoris), Hansenula (e.g., Hansenula polymorpha), Yarowia, Schwaniomyces, Schizosaccharomyces, Zygosaccharomyces and the like. Saccharomyces cerevisiae, S. carlsbergensis and K. iactis are the most commonly used yeast hosts and are convenient fungal hosts.
• the host cells may be provided in suspension or flask cultures, tissue cultures, organ cultures and the like. Alternatively, the host cells may also be transgenic animals.
• protein protein
• polypeptide peptide
• proteins proteins
• polypeptide amino acid polymers
• amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers, as described below.
• This term also includes posttranslational modifications of the polypeptide, such as glycosylation, phosphorylation and acetylation. Based on the amino acid sequence and the modifications, the atomic or molecular mass or weight of a polypeptide is expressed in (kilo)Dalton (kDa).
• recombinant polypeptide is meant a polypeptide made using recombinant techniques, i.e., through the expression of a recombinant or synthetic polynucleotide.
• recombinant polypeptide is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20 %, more preferably less than about 10 %, and most preferably less than about 5 % of the volume of the protein preparation.
• the amide bond in the primary structure of polypeptides is in the order that the amino acids are written, in which the amine end (/V-terminus) of a polypeptide is always on the left, while the acid end (C-terminus) is on the right.
• Any amino acid sequence that contains post- translationally modified amino acids may be described as the amino acid sequence that is initially translated using the symbols shown in Table 1 below with the modified positions, e.g., hydroxylations or glycosylations, but these modifications shall not be shown explicitly in the amino acid sequence.
• Any peptide or protein that can be expressed as a sequence modified linkages, cross links and end caps, non-peptidyl bonds, etc., is embraced by this definition.
• isolated is meant material that is substantially or essentially free from components that normally accompany it in its native state.
• an "isolated polypeptide” refers to a polypeptide which has been purified from the molecules which flank it in a naturally-occurring state, e.g., a fusion protein as disclosed herein which has been removed from the molecules present in the production host that are adjacent to said polypeptide.
• An isolated chimer can be generated by amino acid chemical synthesis or can be generated by recombinant production.
• heterologous protein may mean that the protein is not derived from the same species or strain that is used to display or express the protein.
• Homologue of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived.
• amino acids are organic compounds that contain aminofa] (- N H + s) and carboxylate (- CO ⁇ 2) functional groups, along with a side chain (R group) specific to each amino acid.
• amino acids include those L-amino acids commonly found in naturally occurring proteins.
• Amino acids in the context of the present invention, also include D-amino acids and nonnatural, unusual or unnatural amino acids, as described below. Amino acid residues will be indicated according to the standard three-letter or one-letter amino acid code. Reference is made to Table A-2 on page 48 of WO 08/020079. Examples of amino acids commonly found in proteins and represented in the genetic code are listed in Table 1 below. Other common amino acids (excluding those listed in Table 1 below) are described on the table on p.
• D-amino acids are also encompassed by the definition of "amino acid".
• the term "D-amino acid” refers to amino acids where the stereogenic carbon alpha to the amino group has the D-configuration.
• unusual, unnatural or non-natural amino acids are also encompassed by the definition of "amino acid".
• the term "unnatural amino acid” or “non-canonical amino acid” or “non-natural amino acid” or “novel amino acid” (or the like) refers to an amino acid that is not one of the twenty amino acids commonly found in peptides synthesized in nature, and known by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V.
• Exemplary unnatural amino acids are described in Young et al., "Beyond the canonical 20 amino acids: expanding the genetic lexicon," J. of Biological Chemistry, 285(15): 11039- 11044 (2010), the disclosure of which is incorporated herein by reference.
• the percentage of "sequence identity" between a first nucleotide sequence and a second nucleotide sequence may be calculated by dividing [the number of nucleotides in the first nucleotide sequence that are identical to the nucleotides at the corresponding positions in the second nucleotide sequence] by [the total number of nucleotides in the first nucleotide sequence] and multiplying by [100%], in which each deletion, insertion, substitution or addition of a nucleotide in the second nucleotide sequence - compared to the first nucleotide sequence - is considered as a difference at a single nucleotide (position).
• the degree of sequence identity between two or more nucleotide sequences may be calculated using a known computer algorithm for sequence alignment such as NCBI Blast v2.0, using standard settings.
• a known computer algorithm for sequence alignment such as NCBI Blast v2.0
• Some other techniques, computer algorithms and settings for determining the degree of sequence identity are for example described in WO 04/037999, EP 0967284, EP 1085089, WO 00/55318, WO 00/78972, WO 98/49185 and GB 2357768.
• nucleotide sequence with the greatest number of nucleotides will be taken as the "first" nucleotide sequence, and the other nucleotide sequence will be taken as the "second" nucleotide sequence.
• the percentage of "sequence identity" between a first amino acid sequence and a second amino acid sequence may be calculated by dividing [the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at the corresponding positions in the second amino acid sequence] by [the total number of amino acid residues in the first amino acid sequence] and multiplying by [100%], in which each deletion, insertion, substitution or addition of an amino acid residue in the second amino acid sequence - compared to the first amino acid sequence - is considered as a difference at a single amino acid residue (position), i.e., as an "amino acid difference" as defined herein.
• the degree of sequence identity between two amino acid sequences may be calculated using a known computer algorithm, such as those mentioned above for determining the degree of sequence identity for nucleotide sequences, again using standard settings.
• a known computer algorithm such as those mentioned above for determining the degree of sequence identity for nucleotide sequences, again using standard settings.
• the amino acid sequence with the greatest number of amino acid residues will be taken as the "first" amino acid sequence, and the other amino acid sequence will be taken as the "second" amino acid sequence.
• amino acid substitutions can generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue of similar chemical structure, and which has little or essentially no influence on the 3D structure, function, activity, or other biological properties of the polypeptide.
• Such conservative amino acid substitutions are well known in the art, for example from WO 04/037999, GB 335768, WO 98/49185, WO 00/46383, and WO 01/09300; and (preferred) types and/or combinations of such substitutions may be selected on the basis of the pertinent teachings from WO 04/037999 as well as WO 98/49185 and from the further references cited therein.
• Such conservative substitutions preferably are substitutions in which one amino acid within the following groups (a) - (e) is substituted by another amino acid residue within the same group: (a) small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gin; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, He, Vai and Cys; and (e) aromatic residues: Phe, Tyr and Trp.
• Particularly preferred conservative substitutions are as follows: Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into His; Asp into Glu; Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; He into Leu or into Vai; Leu into lie or into Vai; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into He; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and/or Phe into Vai, into He or into Leu.
• amino acid sequences and nucleic acid sequences are said to be “exactly the same” if they have 100% sequence identity (as defined herein) over their entire length.
• amino acid difference refers to an insertion, deletion or substitution of a single amino acid residue on a position of the first sequence, compared to the second sequence; it being understood that two amino acid sequences may contain one, two or more such amino acid differences.
• substitution results from the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively as compared to an amino acid sequence or nucleotide sequence of a parental protein or a fragment thereof. It is understood that a protein or a fragment thereof may have conservative amino acid substitutions which have substantially no effect on the protein's activity.
• wild-type refers to a gene or gene product isolated from a naturally occurring source.
• a wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designed the "normal” or “wild-type” form of the gene.
• modified refers to a gene or gene product that displays modifications in sequence, post-translational modifications and/or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.
• a variant may also include synthetic molecules, e.g., a chemokine ligand variant may be similar in structure and/or function to the natural chemokine, but may concern a small molecule, or a synthetic peptide or protein, which is man-made. Said variants with different functional properties may concerns super-agonists, super antagonists, among other functional differences, as known to the skilled person.
• a "protein domain” is a distinct functional and/or structural unit in a protein. Usually, a protein domain is responsible for a particular function or interaction, contributing to the overall role of a protein. Domains may exist in a variety of biological contexts, where similar domains can be found in proteins with different functions. Protein secondary structure elements (SSEs) typically spontaneously form as an intermediate before the protein folds into its three-dimensional tertiary structure. The two most common secondary structural elements of proteins are alpha (a) helices and beta (P) sheets, though s-turns and omega loops occur as well.
• SSEs Protein secondary structure elements
• a beta barrel is a beta-sheet composed of tandem repeats that twists and coils to form a closed toroidal structure in which the first strand is bonded to the last strand (hydrogen bond). Beta strands in many beta-barrels are arranged in an antiparallel fashion. Beta sheets consist of beta strands (also p-strand) connected laterally by at least two or three back-bone hydrogen bonds, forming a generally twisted, pleated sheet.
• a p-strand is a stretch of poly-peptide chain typically 3 to 10 amino acids long with backbone in an extended conformation.
• a "turn” is a type of non-regular secondary structure in proteins that causes a change in direction of the polypeptide chain.
• Beta turns P-turns, p-bends, tight turns or reverse turns
• the polypeptide chain makes a 180° change in direction in the beta turn.
• Loops are irregular structures which connect two secondary structure elements in proteins. They are generally located on the protein's surface in solvent exposed areas in the proteins (Choi Y. et al., "How long is a piece of loop?", PeerJ., 2013, l:el).
• loops are longer in amino acid number than turns, see, e.g., Milner-White and Poet, "Loops, bulges, turns and hairpins in proteins", Trends in Biochemical Sciences, 1987, 12:189-192.
• loops that have only 4 or 5 amino acid residues, when they have internal hydrogen bonds can also be referred to as turns.
• circular permutation of a protein refers to a molecule which in its linear form has the termini joined together, either directly or through a linker, to produce a circular molecule (as an intermediate), followed by opening or cleaving of the circular molecule at another location or position to produce a new molecule which in its linear form is a molecule with termini (XI and X2) different from the termini in the original molecule.
• the opening or cleaving of the circular molecule at another location may comprise the removal of one or more nucleotides/amino acids of the original sequence.
• Circularly permuted molecules include those molecules whose structure is equivalent to a molecule that has been circularized and then opened, and/or with regards to proteins include those wherein the amino and carboxy ends are joined together, directly or through a linker, and new amino and carboxy terminal ends are formed at a different location within the protein sequence.
• a circularly permuted molecule may also be synthesized de novo starting from a new linearform of the molecule (as compared to the original molecule) and never go through a circularization and opening step.
• Circularly permuted molecules provide for a rearrangement in the molecule as compared to the original wild type molecule, though without impact on activity or functionality, since the folding or appearance of the final (folded) molecule is similar or the same as the original molecule, with the only difference that the beginning and end point is at a different location.
• one or more nucleotides/amino acids of the original molecule are removed from the original molecule.
• So circularly permuted molecules which may be nucleic acid molecules, or proteins, have their normal termini fused, often with a linker, and contain new termini at another position. See Goldenberg, et al. J. Mol.
• Circular permutation is functionally equivalent to taking a straight-chain molecule, fusing the ends to form a circular molecule, and then cutting the circular molecule at a different location to form a new straight chain molecule with different termini. Circular permutation thus has the effect of essentially preserving the sequence and identity of the amino acids of a protein while generating new termini at different locations (also see Pastan et al. - EP 0754 192 Bl).
• Proteins for which a circular permutation is straightforward to design are those in which the termini of the original protein are in close proximity and favourably oriented, for instance, where the termini are naturally situated close together a direct fusion of the termini to each other or introduction of a short linker will have relatively little effect.
• the linker may be of any length, close proximity of the native termini is not an absolute requirement.
• the particular circular permutation of a molecule is designated by squared brackets containing the amino acid residues between which the peptide bond is eliminated.
• PRT[AAX2-AAXI] designates a circularly permuted protein "PRT" in which the opening site (position at which the peptide bond is eliminated) occurred between amino acid (AA) residues at positions X2 and XI of the unpermuted or unmodified protein.
• the terms “circular permutation”, “circularly permuted”, or “circularly permutated”, refer to the process of taking a protein, or its cognate nucleic acid sequence, and fusing the /V- and C-termini (directly or through a linker, e.g., using protein or recombinant DNA methodologies) to form a circular molecule, and then cutting (opening) the circular molecule at a different location to form a new protein, or cognate nucleic acid molecule, with termini different from the termini in the original molecule.
• Circular permutation thus preserves the overall sequence (besides the linkers, if introduced, and the one or more amino acids removed, if any), structure, and function of a protein, while generating new C- and /V-termini at different locations that results in an improved orientation forfusing a desired polypeptide fusion partner as compared to the original molecule.
• a circularly permuted molecule may be synthesized de novo as a linear molecule and nevergo through a circularization and opening step.
• the fusion of the N- and C-termini of the molecule may take place between the original N- and C-termini of the protein or may take place between the N- and C-termini created after deletion of one or more residues, such as one, two, three, four, five or more residues from the original /V-terminus, between the N- and C-termini created after deletion of one or more residues, such as one, two, three, four, five or more residues from the original C-terminus, or between the N- and C-termini created after deletion of one or more residues, such as one, two, three, four, five or more residues from the both the original N- and C-termini.
• the different possibilities for the fusion of the N- and C-termini of the linear molecule may lead to different versions of circularly permuted proteins designated as PRT[AAx2-AAxi]Vn.
• the designation IFNA2a[D77-W76]V2 and IFNA2a[D77-W76]V4 indicate both circularly permuted proteins IFNA2a in which the opening site (position at which the peptide bond is eliminated) occurred between amino acid residues at positions 77 and 76 of the unpermuted or unmodified protein but having differently fused original /V- and C-termini.
• the design of a circularly permuted protein by opening of the circular molecule may also not occur at two consecutive amino acid positions, resulting in the deletion of one or more amino acids from the protein.
• the designation I L-2[F42-M39] indicates a circularly permuted IL-2 cytokine in which the opening site (position at which the peptide bond is eliminated) occurred between residues at positions 42 and 39 of the unpermuted or unmodified IL-2. Residues 40 and 41 of the original protein have been deleted to generate the circularly permuted protein.
• the opening of the circular molecule is preferably performed at an accessible or exposed site (preferentially a R-turn or loop) of said protein, so that the folding (3D structure) of the circularly permuted protein is retained or similar as compared to the folding of the wild-type protein.
• the term "circular permutation of a protein” or “circularly permuted protein” refers to a protein which has a changed order of amino acids in its amino acid sequence, as compared to the wild-type protein sequence, with as a result a protein structure with different connectivity, but overall similar three-dimensional (3D) shape.
• a circular permutation of a protein is analogous to the mathematical notion of a cyclic permutation, in the sense that the sequence of the first portion of the wild-type protein (adjacent to the /V-terminus) is related to the sequence of the second portion of the resulting circularly permuted protein (near its C-terminus), as described for instance in Bliven and Prlic (2012) (Circular permutation in proteins. PLOS Comput. Biol. 8(3):el002445).
• a circular permutation of a protein as compared to its wild protein is obtained through genetic or artificial engineering of the protein sequence, whereby the N- and C- terminus of the wild-type protein are 'connected' (directly, by means of a linker and/or with one or more amino acids having been removed, as explained above) and the protein sequence is interrupted at another site (where one or more amino acids can be removed, as explained above), to create a novel N- and C-terminus of said protein.
• the circularly permuted proteins of the invention are the result of a connected N- and C-terminus of the wild-type cytokine sequence, and a cleavage or interrupted sequence at an accessible or exposed site (preferentially a p-turn or loop) of said cytokine, whereby the folding (3D structure) of the circularly permuted cytokine is retained or similar as compared to the folding of the wild-type protein.
• connection of the N- and C-terminus in said circularly permuted cytokine may be the result of a peptide bond linkage, or of introducing a peptide linker, or of a deletion of a peptide stretch near the original /V- and C-terminus in the wild-type protein, followed by a peptide bond or the remaining amino acids.
• the terms "circularly permuted” and “circular permutation” are well known in the art, see, e.g., "CPSARST: Circular Permutation Search Aided by Ramachandran Sequential Transformation"
• CPSARST an efficient circular permutation search tool applied to the detection of novel protein structural relationships. Genome Biol. 2008 Jan 18;9(1):R11, or "CPDB - the Circular Permutation Database" (http://10.life.nctu.edu.tw/cpdb/). Circular permutation is performed to obtain a circularly permuted protein.
• fused to refers, in particular, to "genetic fusion”, e.g., by recombinant DNA technology, as well as to "chemical and/or enzymatic conjugation” resulting in a stable covalent link.
• chimeric polypeptide refers to a protein that comprises at least two separate and distinct polypeptide components that may or may not originate from the same protein, e.g., a protein that comprises an immunoglobulin single variable domain (ISVD) fused with a cytokine.
• ISVD immunoglobulin single variable domain
• the term also refers to a non-naturally occurring molecule, which means that it is man-made.
• fusion of the two or more polypeptide components e.g., of an ISVD and a cytokine, as described herein, may be a direct fusion of the sequences or it may be an indirect fusion, e.g., with intervening amino acid sequences or linker sequences, or chemical linkers.
• the fusion of two polypeptides may also refer to a non-covalent fusion obtained by chemical linking.
• protein complex or “complex” refers to a group of two or more associated macromolecules, whereby at least one of the macromolecules is a protein.
• a protein complex typically refers to associations of macromolecules that can be formed under physiological conditions. Individual members of a protein complex are linked by non-covalent interactions.
• a protein complex can be a non-covalent interaction of only proteins, and is then referred to as a protein-protein complex; for instance, a non-covalent interaction of two proteins, of three proteins, of four proteins, etc. More specifically, a complex of the chimeric protein and the cytokine receptor, or a complex of the cytokine or chemokine-comprising ligand protein (such as a chimeric protein) and its specifically bound interactor, such as the cytokine receptor that is capable of binding to the cytokine ligand.
• a complex of the chimeric protein and the cytokine receptor or a complex of the cytokine or chemokine-comprising ligand protein (such as a chimeric protein) and its specifically bound interactor, such as the cytokine receptor that is capable of binding to the cytokine ligand.
• the protein complex of the chimeric protein comprising an ISVD fused with a cytokine, bound by its chemokine receptor-interacting region (its /V-terminus) to a chemokine receptor, for which it is known to bind to said chemokine ligand, to the chemokine receptor, will be a complex formed that is used herein.
• determining As used herein, the terms “determining”, “measuring”, “assessing” and “assaying” are used interchangeably and include both quantitative and qualitative determinations.
• suitable conditions refers to the environmental factors, such as temperature, movement, other components, and/or "buffer condition(s)” among others, wherein “buffer conditions” refers specifically to the composition of the solution in which the assay is performed.
• the said composition includes buffered solutions and/or solutes such as pH buffering substances, water, saline, physiological salt solutions, glycerol, preservatives, etc. for which a person skilled in the art is aware of the suitability to obtain optimal assay performance.
• binding specifically refers to the number of different target molecules, such as antigens, to which a particular binding unit can bind with sufficiently high affinity (see below).
• Specificity refers to the number of different target molecules, such as antigens, to which a particular binding unit can bind with sufficiently high affinity (see below).
• Specificity refers to the number of different target molecules, such as antigens, to which a particular binding unit can bind with sufficiently high affinity (see below).
• binding specifically are used interchangeably herein with “selectivity”, “binding selectively” or “selective binding”.
• binding units such as binding ISVDs or cytokines, specifically bind to their designated targets or receptors.
• the specificity /selectivity of a binding unit can be determined based on affinity.
• the affinity denotes the strength or stability of a molecular interaction.
• the affinity is commonly given by the KD, or dissociation constant, which has units of mol/litre (or M).
• the affinity can also be expressed as an association constant, KA, which equals 1/KD and has units of (mol/ litre) 1 (or M 1 ).
• the "affinity" is a measure for the binding strength between a moiety and a binding site on a target molecule: the lower the value of the KD, the stronger the binding strength between a target molecule and a targeting moiety.
• the off-rate k O ff has units s 1 (where s is the SI unit notation of second).
• the on-rate k on has units M -1 s -1 .
• the on-rate may vary between 10 2 M -1 s 1 to about 10 7 M -1 s -1 , approaching the diffusion-limited association rate constant for bimolecular interactions.
• the measured KD may correspond to the apparent KD if the measuring process somehow influences the intrinsic binding affinity of the implied molecules for example by artefacts related to the coating on the biosensor of one molecule. Also, an apparent KD may be measured if one molecule contains more than one recognition sites for the other molecule or molecules. In such situation the measured affinity may be affected by the avidity of the interaction by the two molecules.
• the dissociation constant (KD) may be the actual or apparent dissociation constant, as will be clear to the skilled person. Methods for determining the KD will be clear to the skilled person, and for example include the techniques mentioned below.
• the affinity of a molecular interaction between two molecules can be measured via different techniques known perse, such as the well-known surface plasmon resonance (SPR) biosensor technique (see for example Ober et al. 2001, Intern. Immunology 13: 1551-1559).
• SPR surface plasmon resonance
• the term "surface plasmon resonance” (SPR), as used herein, refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, where one molecule is immobilized on the biosensor chip and the other molecule is passed over the immobilized molecule under flow conditions yielding k on , k O ff measurements and hence KD (or KA) values.
• BIAcore® system (BIAcore International AB, a Cytiva lifesciences company, Uppsala, Sweden and Piscataway, NJ).
• BIAcore International AB a Cytiva lifesciences company, Uppsala, Sweden and Piscataway, NJ.
• Johnsson et al. (1995, J. Mol. Recognit. 8: 125-131
• Johnnson et al. (1991, Anal. Biochem. 198: 268-277).
• bio-layer interferometry refers to a label-free optical technique that analyzes the interference pattern of light reflected from two surfaces: an internal reference layer (reference beam) and a layer of immobilized protein on the biosensor tip (signal beam).
• reference beam an internal reference layer
• signal beam a layer of immobilized protein on the biosensor tip
• BLI can for example be performed using the well-known Octet® Systems (ForteBio, a division of Pall Life Sciences, Menlo Park, USA).
• affinities can be measured in Kinetic Exclusion Assay (KinExA) (see for example Drake et al., "Characterizing high-affinity antigen/antibody complexes by kinetic- and equilibrium-based methods", Anal. Biochem., 2004, 328: 35-43), using the KinExA® platform (Sapidyne Instruments Inc, Boise, USA).
• KinExA Kinetic Exclusion Assay
• Equilibrated solutions of a binding unit/target complex such as an antibody/antigen complex, are passed over a column with beads precoated with antigen (or antibody), allowing the free antibody (or antigen) to bind to the coated molecule. Detection of the antibody (or antigen) thus captured is accomplished with a fluorescently labeled protein binding the antibody (or antigen).
• the GYROLAB® immunoassay system provides a platform for automated bioanalysis and rapid sample turnaround (Fraley et al., "The GyrolabTM immunoassay system: a platform for automated bioanalysis and rapid sample turnaround", Bioanalysis 2013, 5: 1765-74).
• a temperature specified in °C with no decimal place shall have an error margin of ⁇ 1°C (e.g., a temperature value of about 50°C means 50°C ⁇ 1°C); a time indicated in hours shall have an error margin of 0.1 hours irrespective of the decimal places (e.g., a time value of about 1.0 hours means 1.0 hours ⁇ 0.1 hours; a time value of about 0.5 hours means 0.5 hours ⁇ 0.1 hours).
• Methods of determining the spatial conformation of amino acids and proteins are known in the art, and include, for example, X-ray crystallography and multi-dimensional nuclear magnetic resonance.
• conformation or “conformational state” of a protein refers generally to the range of structures that a protein may adopt at any instant in time.
• determinants of conformation or conformational state include a protein's primary structure as reflected in a protein's amino acid sequence (including modified amino acids) and the environment surrounding the protein.
• the conformation or conformational state of a protein also relates to structural features such as protein secondary structures (e.g., a-helix, p-sheet, p-barrel, among others), tertiary structure (e.g., the three- dimensional folding of a polypeptide chain), and quaternary structure (e.g., interactions of a polypeptide chain with other protein subunits).
• protein secondary structures e.g., a-helix, p-sheet, p-barrel, among others
• tertiary structure e.g., the three- dimensional folding of a polypeptide chain
• quaternary structure e.g., interactions of a polypeptide chain with other protein subunits.
• Post-translational and other modifications to a polypeptide chain such as ligand binding, phosphorylation, sulfation, glycosylation, or attachments of hydrophobic groups, among others, can influence the conformation of a protein.
• conformational state of a protein may be determined by either functional assay for activity or binding to another molecule or by means of physical methods such as X-ray crystallography, NMR, or spin labelling, among other methods.
• protein solubility is a thermodynamic parameter defined as the concentration of protein in a saturated solution that is in equilibrium with a solid phase, either crystalline or amorphous, under a given set of conditions (see, e.g., Kramer RM. et al., "Toward a molecular understanding of protein solubility: increased negative surface charge correlates with increased solubility", Biophys J., 2012, 102(8):1907-15).
• the term “functional chimeric protein”, “functional fusion protein” or “conformation- selective fusion protein” in the context of the present invention refers to a fusion protein that is functional in binding to its cytokine and/or to the ISVD target, optionally in a conformation- selective manner, and/or is functional in activation/inactivation of the cytokine receptor and/or ISVD target (depending on the known features of the ligand: agonist, antagonist, inverse agonist).
• a binding domain that selectively binds to a particular conformation of a target protein refers to a binding domain that binds with a higher affinity to a target in a subset of conformations than to other conformations that the target may assume.
• binding domains that selectively bind to a particular conformation of a target will stabilize or retain the target in this particular conformation.
• an active state conformation-selective binding domain will preferentially bind to a target in an active conformational state and will not or to a lesser degree bind.
• the chimeric protein of the present invention is a chimeric protein of the present invention.
• the present invention provides a chimeric protein which comprises an immunoglobulin single variable domain (ISVD) fused with a cytokine.
• ISVD immunoglobulin single variable domain
• the chimeric protein of the present invention can also be referred to as a "fusion protein” or "chimera”.
• immunoglobulin single variable domain defines immunoglobulin molecules wherein the antigen binding site is present on, and formed by, a single immunoglobulin domain. This sets ISVDs apart from “conventional” immunoglobulins (e.g., monoclonal antibodies) or their fragments (such as Fab, Fab', F(ab')2, scFv, di-scFv), wherein two immunoglobulin domains, in particular two variable domains, interact to form an antigen binding site.
• a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form an antigen binding site.
• the complementarity determining regions (CDRs) of both VH and VL will contribute to the antigen binding site, i.e., a total of 6 CDRs will be involved in antigen binding site formation.
• the antigen-binding domain of a conventional 4-chain antibody such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art
• a conventional 4-chain antibody such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art
• a Fab fragment, a F(a b')2 fragment, an Fv fragment such as a disulphide linked Fv or a scFv fragment, or a diabody (all known in the art) derived from such conventional 4-chain antibody would normally not be regarded as an ISVD as, in these cases, binding to the respective epitope of an antigen would normally not occur by one single immunoglobulin domain but by a pair of associating immunoglobulin domains such as light and heavy chain variable domains, i.e., by a VH-VL pair of immunoglobulin domains, which jointly bind to an epitope of the respective antigen.
• ISVDs are capable of specifically binding to an epitope of the antigen without pairing with an additional immunoglobulin variable domain.
• the binding site of an ISVD is formed by a single VH, a single VHH or single VL domain.
• the ISVD may be a light chain variable domain sequence (e.g., a V sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a Vn-sequence or VHH sequence) or a suitable fragment thereof.
• An ISVD which may preferably be comprised in the chimeric protein of the present invention can for example be a heavy chain ISVD, such as a VH, VHH, including a camelized VH or humanized VHH.
• Heavy chain ISVDs can be derived from a conventional four-chain antibody or from a heavy chain antibody.
• the ISVD may be a single domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody), a "dAb” or dAb (or an amino acid sequence that is suitable for use as a dAb) or a Nanobody® ISVD (as defined herein and including but not limited to a VHH); other single variable domains, or any suitable fragment of any one thereof.
• the ISVD is a VH, a humanized VH, a human VH, a VHH, a humanized VHH or a camelized VH. More preferably, the ISVD is a Nanobody® ISVD (such as a VHH, including a humanized VHH or camelized VH) or a suitable fragment thereof.
• Nanobody® is a registered trademark from Ablynx N.V.
• VHH domains also known as VHHS, VHH antibody fragments, and VHH antibodies
• VHH domains have originally been described as the antigen binding immunoglobulin variable domain of "heavy chain antibodies”; i.e., of “antibodies devoid of light chains”, see Hamers-Casterman et al., Nature, 363: 446-448, 1993.
• the term “VHH domain” has been chosen in order to distinguish these variable domains from the heavy chain variable domains that are present in conventional 4-chain antibodies, which are referred to herein as "VH domains”, and from the light chain variable domains that are present in conventional 4-chain antibodies, which are referred to herein as "VL domains".
• VHH domains can be obtained from heavy chain-only antibodies (HCAbs) that are circulating in Camelidae, see e.g., Muyldermans S., "A guide to: generation and design of nanobodies", FEBS J., 2021, 288(7):2084-2102.
• HCAbs heavy chain-only antibodies
• immunoglobulins typically involve the immunization of experimental animals, fusion of immunoglobulin producing cells to create hybridomas and screening for the desired specificities.
• immunoglobulins can be generated by screening of naive, immune, or synthetic libraries, e.g., by phage display.
• VHHS immunoglobulin sequences
• WO 94/04678 Hamers-Casterman et al. 1993 ("Naturally occurring antibodies devoid of light chains", Nature, 363: 446-448, 1993) and Muyldermans et al. 2001 (“Single domain camel antibodies: current status", J Biotechnol., 2001, 74: 277- 302) can be exemplified.
• camelids are immunized with the target antigen in order to induce an immune response against said target antigen.
• the repertoire of VHHS obtained from said immunization is further screened for VHHS that bind (or not) a target antigen.
• immunoglobulin sequences of different origin may be used, comprising mouse, rat, rabbit, donkey, human and camelid immunoglobulin sequences.
• fully human, humanized or chimeric sequences are also included.
• a “humanized VHH” comprises an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VHH domain, but that has been “humanized” , i.e., by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring VHH sequence (and in particular in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a VH domain from a conventional 4-chain antibody from a human being (e.g., indicated above).
• This can be performed in a manner known perse, which will be clear to the skilled person, for example on the basis of the further description herein and the prior art (e.g., WO 2008/020079).
• VHH humanized VHH can be obtained in any suitable manner known per se and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring VHH domain as a starting material.
• the building block of the present invention is a VHH
• the VHH is a humanized VHH.
• a “camelized VH” comprises an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VH domain, but that has been “camelized”, i.e., by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain from a conventional 4-chain antibody by one or more of the amino acid residues that occur at the corresponding position(s) in a VHH domain of a heavy chain antibody.
• This can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the further description herein and the prior art (e.g., WO 2008/020079).
• the VH sequence that is used as a starting material or starting point for generating or designing the camelized VH is a VH sequence from a mammal, or the VH sequence of a human being, such as a VH3 sequence.
• camelized VH can be obtained in any suitable manner known per se and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring VH domain as a starting material.
• the structure of an ISVD sequence can be considered to be comprised of four framework regions ("FRs”), which are referred to in the art and herein as “Framework region 1" ("FR1”); as “Framework region 2” (“FR2”); as “Framework region 3” ("FR3”); and as “Framework region 4" ("FR4"), respectively; which framework regions are interrupted by three complementary determining regions (“CDRs"), which are referred to in the art and herein as “Complementarity Determining Region 1" (“CDR1”); as “Complementarity Determining Region 2" (“CDR2”); and as “Complementarity Determining Region 3" (“CDR3”), respectively.
• CDRs complementary determining regions
• the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering. That is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering. This means that, generally, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence.
• the total number of amino acid residues in a VH domain and a VHH domain will usually be in the range of from 110 to 120, often between 112 and 115. It should however be noted that smaller and longer sequences may also be suitable for the purposes described herein.
• CDR sequences may also be described according to Kabat numbering with AbM CDR annotation, as described in Kontermann and Dubel (Eds. 2010, Antibody Engineering, vol 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 33-51).
• FR1 comprises the amino acid residues at positions 1-25
• CDR1 comprises the amino acid residues at positions 26-35
• FR2 comprises the amino acids at positions 36-49
• CDR2 comprises the amino acid residues at positions 50-58
• FR3 comprises the amino acid residues at positions 59-94
• CDR3 comprises the amino acid residues at positions 95-102
• FR4 comprises the amino acid residues at positions 103-113.
• CDR regions may also be done according to different methods.
• FR1 of an ISVD comprises the amino acid residues at positions 1-30
• CDR1 of an ISVD comprises the amino acid residues at positions 31-35
• FR2 of an ISVD comprises the amino acids at positions 36-49
• CDR2 of an ISVD comprises the amino acid residues at positions 50-65
• FR3 of an ISVD comprises the amino acid residues at positions 66-94
• CDR3 of an ISVD comprises the amino acid residues at positions 95-102
• FR4 of an ISVD comprises the amino acid residues at positions 103-113.
• the framework sequences may be any suitable framework sequences, and examples of suitable framework sequences will be clear to the skilled person, for example on the basis the standard handbooks and the further disclosure and prior art mentioned herein.
• the framework sequences are a suitable combination of immunoglobulin framework sequences or framework sequences that have been derived from immunoglobulin framework sequences, for example by humanization or camelization.
• the framework sequences may be framework sequences derived from a light chain variable domain (e.g., a V sequence) and/or from a heavy chain variable domain (e.g., a Vn-sequence or VHH sequence).
• the framework sequences are either framework sequences that have been derived from a VHH-sequence in which said framework sequences may optionally have been partially or fully humanized or are conventional VH sequences that have been camelized (as defined herein).
• the framework sequences present in the ISVD sequences referred to in the present invention may contain one or more of Hallmark residues (as defined herein), such that the ISVD sequence is a Nanobody® ISVD, such as, e.g., a VHH, including a humanized VHH or camelized VH.
• Hallmark residues as defined herein
• the origin of the ISVD sequence or the origin of the nucleotide sequence used to express it is not limited, nor as to the way that the ISVD sequence or nucleotide sequence is or has been generated or obtained.
• the ISVD sequences may be naturally occurring sequences (from any suitable species) or synthetic or semi-synthetic sequences.
• the ISVD sequence is a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence, including but not limited to "humanized” (as defined herein) immunoglobulin sequences (such as partially or fully humanized mouse or rabbit immunoglobulin sequences, and in particular partially or fully humanized VHH sequences), "camelized” (as defined herein) immunoglobulin sequences, as well as immunoglobulin sequences that have been obtained by techniques such as affinity dematuration (for example, starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to the skilled person; or any suitable combination of any of the foregoing.
• immunoglobulin sequences such as partially or fully humanized mouse or rabbit immunoglobulin sequences, and in particular partially or fully humanized VHH sequences
• camelized as defined herein immunoglobul
• nucleotide sequences may be naturally occurring nucleotide sequences or synthetic or semi-synthetic sequences, and may for example be sequences that are isolated by PCR from a suitable naturally occurring template, e.g., DNA or RNA isolated from a cell, nucleotide sequences that have been isolated from a library (and in particular, an expression library), nucleotide sequences that have been prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique known per se, such as mismatch PCR), nucleotide sequence that have been prepared by PCR using overlapping primers, or nucleotide sequences that have been prepared using techniques for DNA synthesis known per se.
• a suitable naturally occurring template e.g., DNA or RNA isolated from a cell
• nucleotide sequences that have been isolated from a library and in particular, an expression library
• nucleotide sequences that have been prepared by introducing mutations into a naturally occurring nucleotide sequence using any suitable
• Nanobody® ISVDs For a general description of Nanobody® ISVDs, reference is made to the present description, as well as to the prior art cited herein. In this respect, it should however be noted that this description and the prior art mainly described Nanobody® ISVDs of the so-called "VH3 class", i.e., Nanobody® ISVDs with a high degree of sequence homology to human germline sequences of the VH3 class such as DP-47, DP-51 or DP-29.
• VH3 class i.e., Nanobody® ISVDs with a high degree of sequence homology to human germline sequences of the VH3 class such as DP-47, DP-51 or DP-29.
• Nanobody® ISVD any type of Nanobody® ISVD, and for example also uses the Nanobody® ISVDs belonging to the so-called "VH4 class", i.e., Nanobody® ISVDs with a high degree of sequence homology to human germline sequences of the VH4 class such as DP-78, as for example described in WO 2007/118670.
• VH4 class i.e., Nanobody® ISVDs with a high degree of sequence homology to human germline sequences of the VH4 class such as DP-78, as for example described in WO 2007/118670.
• the ISVD comprised in the chimeric molecule of the present invention is derived from a Nanobody® ISVD belonging to the so-called "VH3 class", i.e., a Nanobody® ISVDs with a high degree of sequence homology to human germline sequences of the VH3 class such as DP-47, DP-51 or DP-29.
• VH3 class i.e., a Nanobody® ISVDs with a high degree of sequence homology to human germline sequences of the VH3 class such as DP-47, DP-51 or DP-29.
• Nanobody® ISVDs in particular VHH sequences, including (partially) humanized VHH sequences and camelized VH sequences
• VHH sequences including (partially) humanized VHH sequences and camelized VH sequences
• a Nanobody® ISVD can be defined as an immunoglobulin sequence with the (general) structure:
• FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which one or more of the Hallmark residues are as further defined herein.
• Nanobody® ISVD can be an immunoglobulin sequence with the (general) structure
• FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which the framework sequences are as further defined herein.
• Nanobody® ISVD can be an immunoglobulin sequence with the (general) structure
• FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which: one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table 2 below.
• Nanobody® ISVD can be defined as an amino acid sequence with the (general) structure
• FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table 2.
• the ISVD comprised in the chimeric protein of the present invention derives from an ISVD, such as from a heavy-chain ISVD, preferably from a Nanobody® ISVD, which has been further engineered/modified to include mutations which prevent/remove binding of pre-existing antibodies/factors. Examples of such mutations are described, e.g., in WO 2012/175741 and WO 2015/173325.
• the amino acid at position 11 may be Vai or Leu, preferably Vai; and/or the amino acid at position 89 (according to Kabat) may be preferably Vai, Thr or Leu, preferably Leu; and/or the amino acid at position 110 (according to Kabat) may be preferably Thr, Lys or Gin, preferably Thr; and/or the amino acid at position 112 (according to Kabat) may be Ser, Lys or Gin, preferably Ser; and/or the ISVD- based building block may contain a C-terminal extension of 1-5 amino acids chosen from any naturally occurring amino acid.
• the ISVD comprised in the chimeric protein of the present invention specifically binds its target (antigen), indicating that such an interaction of the ISVD with its antigen is characterized by a high specificity and/or high affinity, as defined herein. .
• the ISVD comprised in the chimeric protein of the present invention does not specifically bind its target (antigen).
• the ISVD comprised in the chimeric protein shows any interaction with its original target (antigen), or with any other protein, such interaction is characterized by low specificity and/or low affinity, as defined herein.
• the ISVD comprised in the chimeric protein of the present invention may derive from an ISVD (the "ISVD precursor").
• the "ISVD precursor" is an ISVD which is modified (e.g., by point mutations and/or by addition/deletion of amino acids to its sequence) to generate the ISVD comprised in the chimeric protein of the present invention, in this particular embodiment.
• the "ISVD precursor” is modified so that it no longer specifically binds any molecule to which the ISVD precursor specifically binds (the ISVD precursor target (antigen)).
• Cytokines are a class of small proteins (5-20 kDa) that act as cell signalling molecules at picomolar or nanomolar concentrations to regulate inflammation and modulate cellular activities such as migration, growth, survival, and differentiation. Cytokines are an exceptionally large and diverse group of pro- or anti-inflammatory factors that are grouped into families based upon their structural homology or that of their receptors. Cytokines may include chemokines, interferons, interleukins, lymphokines, tumor necrosis factors, hormones, or growth factors. Interleukins (ILs) form a group of cytokines with complex immunomodulatory functions including cell proliferation, maturation, migration and adhesion, playing an important role in immune cell differentiation and activation.
• ILs Interleukins
• ILs can also have pro- and anti-inflammatory effects and are under constant pressure to evolve due to continual competition between the host's immune system and infecting organisms; as such, ILs have undergone significant evolution, which has resulted in little amino acid conservation between orthologous proteins, complicating the gene family organisation.
• the fusion between the ISVD and cytokine is made at an internal fusion site of the ISVD.
• an "internal fusion site” is defined herein to refer to a position between two amino acids anywhere present in a polypeptide sequence, more specifically in the ISVD and/or cytokine as used herein, more specifically with internal referring to the fusion site not being the /V- or C- terminus of the protein.
• the internal fusion site may alternatively be defined as a position between two amino acids anywhere present in a protein variant of said ISVD or cytokine wherein a few amino acids are deleted or added at said fusion site as compared to the original protein sequence.
• Said internal fusion site is the position that needs to be cleaved, so that another protein sequence can be inserted by creating peptide bonds between the cleaved protein sequence and the inserted protein sequence.
• a chimierc protein of said structure can also be obtained by designing a genetic fusion.
• a chimeric protein of the present invention is thus obtained by translation of a genetic fusion of said chimeric protein, corresponding to the protein sequence which starts with the /V-terminal portion of an ISVD which ends at the internal fusion site (and/or a variant with few amino acids added or deleted at said fusion site, such as from 1 to 10 amino acids added or deleted at said fusion site, such as from 1 to 7, or from 1 to 5, such as 1, 2, 3, 4 or 5 amino acids added or deleted at said fusion site); connected to the /V-terminus of the insertion protein, which is a cytokine or circularly permuted variant of a cytokine in the present invention, of which the C-terminus is then connected to the remaining part of the ISVD.
• an 'internal fusion site' is meant a position in a polypeptide sequence wherein the original peptide bond between two amino acids present in the ISVD or cytokine sequence in particular, is interrupted, as to provide for a point where two novel peptidic bonds are made, specifically one peptidic bond connecting the amino acid sequence /V-terminally located of the internal fusion site with the /V-terminus of the inserted protein sequence, and one peptidic bond connecting the C-terminus of the inserted protein sequence with the sequence C- terminally located of the internal fusion site.
• an "internal fusion site” is a location within the sequence of the ISVD (and/or cytokine, if circularly permuted) in which the link (fusion) to the cytokine (or ISVD) is established.
• the internal fusion site may thus be used as a reference point in the amino acid sequence dividing its original protein sequence in a sequence located /V-terminally of the internal fusion site, and a sequence located C-terminally of the internal fusion site of said protein.
• the internal fusion site of the ISVD is preferably located at a loop or turn, preferably a beta turn, in the folded protein, more preferably between two beta strands, even more preferably between beta strands A and B, see below for more details.
• the internal fusion site of the circularly permuted cytokine comprised in the chimeric protein of the present invention is located in a turn or loop between two secondary elements of the cytokine, e.g., in a turn or loop between two
• the internal fusion site of the cytokine is located at the position in the protein that will result in an altered cytokine-receptor binding and/or in an altered cytokine-receptor downstream activity and/or in an altered receptor/receptor's subunit oligomerization upon cytokine binding and/or in an altered cytokine-receptor/receptor's subunit-binding functionality for the chimeric molecule made by fusing an ISVD at said cytokine internal fusion site.
• the term "accessible site(s)", or “exposed site”, are used interchangeably herein and all referto amino acid sites of the protein sequence that are structurally accessible, preferably positions at the surface of the protein, or exposed to the surface.
• the 'internal fusion site' as referred to herein is an amino acid site of the protein that is preferably also an accessible site or exposed site.
• the exposed or accessible sites are located preferably in a turn or loop between two secondary elements (e.g., two
• two secondary elements e.g., two
• the ISVD is linked to said circularly permuted cytokine at an internal fusion site of the ISVD, wherein the circularly permuted cytokine protein sequence is inserted as described above, wherein the /V- and C-terminus of said circularly permuted cytokine protein sequence is provided by cleavage of the protein sequence at an internal fusion site as defined herein, to provide an /V-terminus and C-terminus for formation of the peptidic bonds to create the chimeric protein.
• the amino acids at the internal fusion site of the ISVD are linked to an amino acids at the internal fusion site of the cytokine.
• the internal fusion site is located in a loop or turn between two secondary structure elements in both the ISVD and in the circularly permuted cytokine.
• the internal fusion site of the ISVD may be located in a loop or turn between two secondary structure elements, such as in a beta turn.
• the internal fusion site of the circularly permuted cytokine may be located in a loop or turn and thus provides the position for fusion to the ISVD protein sequence, also at an internal fusion site position of said ISVD.
• the internal fusion site of the ISVD is not located in any of the CDRs of the ISVD.
• the loop or turn (such as beta turn) where the internal fusion site of the ISVD is located is not a CDR.
• the internal fusion site of the ISVD is thus the position where the amino acids positioned N- terminally from the site are connected at the C-terminal ending to the /V-terminus of the cytokine (or circularly permuted cytokine) protein, and wherein the amino acids positioned C- terminally of the internal fusion site are connected with the C-terminus of the cytokine (or circularly permuted cytokine) protein (see also Figure 32 as an example for clarification).
• the cytokine is a circularly permuted cytokine, as previously described.
• the cytokine is circularly permuted
• the original N- and C- termini of the cytokine protein sequence are linked to each other (because it is a circularly permuted cytokine).
• the /V- and C- termini of the cytokine may be linked to each other directly or by means of a linker, as described herein.
• from 0 to 10 amino acids from the (original) N- and/or C-terminal part of the cytokine are removed before linking the N- and C-termini of the cytokine to each other.
• from 0 to 7 amino acids from the N- and/or C-terminal part of the cytokine are removed before linking the N- and C-termini of the cytokine to each other, even more preferably from 0 to 5 amino acids, such as 0, 1, 2, 3 or 4 amino acids from the N- and/or C-terminal part of the cytokine are removed before linking the N- and C-termini of the cytokine to each other (directly or by means of a linker, as described herein).
• an internal chimeric fusion in the ISVD is obtained using a cytokine which is not circularly permuted.
• the cytokine keeps its original /V-and C-termini, i.e., no new N- and C-termini are created somewhere else in the sequence of the cytokine.
• the cytokine is fused to the amino acids of the ISVD that are located at the internal fusion site of the ISVD through the (original) N- and C-termini of the cytokine.
• the cytokine is not linked to the amino acids of the internal fusion site of the ISVD through amino acids located at an internal fusion site of the cytokine.
• from 0 to 7 amino acids from the original N- and/or C-termini of the cytokine may be removed before fusing the cytokine to the amino acid(s) located at the internal fusion site of the ISVD.
• from O to 7 amino acids from the N- and/or C-termini of the cytokine are removed before fusing the cytokine to the amino acid(s) of the internal fusion site of the ISVD, even more preferably from 0 to 5 amino acids, such as 0, 1, 2, 3 or 4 amino acids from the N- and/or C-termini of the cytokine are removed before fusing the cytokine to the amino acid(s) located at the internal fusion site of the ISVD.
• a peptide linker as described herein may be added to the N- and/or C-termini of the cytokine before fusing the cytokine to the amino acid(s) at the internal fusion site of the ISVD.
• the chimeric protein of the present invention is a continuous amino acid sequence.
• the /V-terminal sequence of the ISVD, located /V-te rm inally from the internal fusion site is linked through a peptide linker to the original C-terminal portion of the cytokine, corresponding to the sequence which is C-terminally located to the internal fusion site of the cytokine, and the /V-terminal portion of the original cytokine, corresponding to the sequence located /V-terminally from the internal fusion site of the cytokine, is linked through a peptide linker and/or peptidic bond, to the C-terminal sequence of the ISVD, located C-terminally of the internal fusion site of the ISVD, to form the continuous amino acid sequence. See Figures 2 and 32 for further details.
• the tertiary structure of the ISVD and of the cytokine in the chimeric protein is maintained except for the structure of amino acids at the internal fusion sites which link the ISVD and the cytokine, if applicable.
• the tertiary structure of the ISVD and of the cytokine in the chimeric protein is maintained as compared with the tertiary structure of the ISVD and cytokine when they are not part of the chimeric protein, except for the structure of the amino acids at the internal fusion sites which link the ISVD and the cytokine, if applicable.
• the tertiary structure can be partially maintained, such as at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 99% of the tertiary structure the ISVD and/or of the cytokine is maintained as compared with the tertiary structure of the ISVD and cytokine when they are not part of the chimeric protein, except for the structure of the amino acids at internal fusion sites which link the ISVD and the cytokine, if applicable (i.e., if the cytokine is circularly permuted and thus linked to the amino acids at the internal fusion site of the ISVD and at the internal fusion site of the cytokine, as described in detail above).
• a protein tertiary structure is the three-dimensional shape of a protein.
• the tertiary structure is primarily due to interactions between the side chain groups of the amino acids that make up the protein.
• Side-chain group interactions that contribute to tertiary structure include non- covalent interactions such as hydrogen bonding, ionic bonding, dipole-dipole interactions, and London dispersion forces.
• hydrophobic interactions in which amino acids with non-polar, hydrophobic side chain groups cluster together on the inside of the protein, leaving hydrophilic amino acids on the outside to interact with surrounding water molecules.
• disulphide bonds covalent linkages between the sulphur-containing side chains of cysteines
• X-ray crystallography nuclear magnetic resonance spectroscopy (NMR), cryogenic electron microscopy or dual polarisation interferometry are tools that can be used to ascertain the tertiary structure of a given protein.
• NMR Nuclear magnetic resonance spectroscopy
• cryogenic electron microscopy or dual polarisation interferometry are tools that can be used to ascertain the tertiary structure of a given protein.
• the way the ISVD and cytokine are fused in the chimeric protein of the present invention provides for a chimera with more rigid non-flexible connections.
• a classical junction of polypeptide components while typically joined in their native state, is performed by joining their respective /V- and C-termini directly or through a peptide linkage to form a single continuous polypeptide. These fusions are often made via flexible linkers, or at least connected in a flexible manner, which means that the fusion partners are not in a stable position or conformation with respect to each other.
• a rigid chimeric/fusion protein as presented herein with one or more fusion points or connections within the primary topology of two or more proteins, possesses at least one non-flexible fusion point (see Figure 1).
• the chimeric protein of the present invention originates through generation of fusions between the ISVD and the cytokine, as explained above, wherein the cytokine (preferably circularly permuted) interrupts the topology of the ISVD.
• the chimeric protein of the present invention is a continuous amino acid sequence, preferably obtained by a genetic fusion.
• An embodiment provides a chimeric protein wherein the ISVD is fused with the cytokine (preferably circularly permuted) in such a manner that the cytokine (preferably circularly permuted) is "interrupting" the ISVD's topology.
• the "topology” of a protein refers to the orientation of regular secondary structures with respect to each other in three- dimensional space. Protein folds are defined mostly by the polypeptide chain topology (Orengo, C., Jones, D. & Thornton, J., "Protein superfamilies and domain superfolds", Nature, 1994, 372:631-634).
• the 'primary topology' is defined as the sequence of secondary structure elements (SSEs), which is responsible for protein fold recognition motifs, and hence secondary and tertiary protein /domain folding.
• SSEs secondary structure elements
• the true or primary topology is the sequence of SSEs, i.e., if one imagines of being able to hold the /V- and C-terminal ends of a protein chain, and pull it out straight, the topology does not change whatever the protein fold.
• the protein fold is then described as the tertiary topology, in analogy with the primary and tertiary structure of a protein (also see Martin AC., "The ups and downs of protein topology; rapid comparison of protein structure", Protein Eng. 2000, 13(12):829-37).
• the ISVD comprised in the chimeric protein of the invention is hence interrupted in its primary topology, by introducing the cytokine (which is preferably circularly permuted).
• novel chimeric proteins are fused in a unique manner to avoid that the junction is a flexible, loose, weak link / region within the chimeric protein structure.
• a convenient means for linking or fusing two polypeptides is by expressing them as a fusion protein from a recombinant nucleic acid molecule, which comprises a first polynucleotide encoding a first polypeptide operably linked to a second polynucleotide encoding the second polypeptide, in the classical known manner.
• the interruption of the topology of the ISVD by the cytokine is also reflected in the design of the genetiefusion from which said chimeric protein is expressed.
• the chimeric protein is encoded by a chimeric gene formed by recombining parts of a gene encoding for an ISVD, and parts of a gene encoding the cytokine, wherein said cytokine interrupts the primary topology of the encoded ISVD at one or more internal fusion sites of said ISVD via at least two or more direct fusions or fusions made directly or by encoded peptide linkers.
• the polynucleotides encoding the polypeptides to be fused are fragmented and recombined in such a way to provide the chimeric protein that provides a rigid non-flexible link, connection or fusion between said proteins.
• the novel chimera proteins are made by fusing the cytokine with the ISVD in such a manner that the primary topology of the ISVD is interrupted, meaning that the amino acid sequence of the antigen-binding domain is interrupted at an internal fusion site, and joined to the amino acid(s) in the cytokine.
• the amino acid sequence of the antigen-binding domain is interrupted at an internal fusion site, and joined to the amino acid(s) at the internal fusion site of the cytokine, which sequence is therefore also interrupted.
• the internal fusion site is located in a loop or a turn between two secondary structure elements, as described above.
• the amino acid of the ISVD /V-terminally positioned of the internal fusion site of the ISVD is linked at its C-terminus to the /V-terminus of the cytokine, and the C-terminus of the cytokine is linked to the amino acid present at the C-terminal end of the internal fusion site of the ISVD, to form the continuous amino acid sequence.
• the /V-terminus of the chimeric protein contains the ISVD amino acid sequence /V-terminally positioned from the internal fusion site of the ISVD, followed by the (genetically fused) amino acid sequence of the circularly permuted cytokine sequence, followed by the ISVD amino acid sequence C-terminally positioned of the internal fusion site of the ISVD, wherein the circularly permuted cytokine sequence is provided as described herein, particularly wherein the circularly permuted cytokine is fused to the ISVD, by linking the /V-terminus of the amino acid C-terminally located at the (cleaved) internal fusion site of the circularly permuted cytokine to the C-terminus of the ammino acid /V-terminally located at the
• the /V- and C-terminal sequences preceding or following the internal fusion sites of the ISVD, respectively, and/or the /V- and C-terminal sequences preceding or following the internal fusion site of the circularly permuted cytokine correspond to at least a part of the sequence of the loop or turn between two secondary structure elements of the respective internal fusion sites (in the original sequences of the ISVD and/or cytokine, if applicable).
• the /V- and C-terminal sequences preceding or following the internal fusion site of the ISVD correspond to at least a part of the sequence of the loop or turn (e.g., a R-turn) between two secondary structure elements (e.g., two R-strands) of the internal fusion site of the ISVD.
• the /V- and C-terminal sequences preceding or following of the internal fusion site of the cytokine correspond to at least a part of the sequence of the loop or turn between two secondary structure elements (e.g., two R-strands, or two a-helices, or one R-strand and one a-helix) of the internal fusion site of the cytokine.
• the correspondence may be "to at least a part of the sequence of the loop or turn". This is because from O to 10, preferably from 0 to 5, more preferably from 0 to 4, even more preferably from 0 to 3, such as 0, 1, 2 or 3 (preferably continuous) amino acid residues of the loop or turns of one or both proteins (i.e., ISVD and/or cytokine, if applicable) may be missing in the chimeric protein.
• the ISVD and the cytokine (preferably circularly permuted) may be fused through a peptide linker, as described above.
• the amino acid sequence positioned /V- and C- terminally of the internal fusion sites of the ISVD and/or of the cytokine (if applicable) exactly correspond to the sequence of the loop or turn between two secondary structure elements of the respective internal fusion sites in the original sequences of the ISVD and/or of the cytokine (i.e., no amino acid residues are missing in the loop or turn of one or both proteins in the chimeric protein).
• the N- and C- termini of the chimeric protein correspond to the N- and C- termini of the ISVD, respectively.
• the amino acid sequence of the chimeric protein of the present invention (which is preferably a continuous amino acid sequence, as described above) comprises:
• amino acid sequence of the chimeric protein of the present invention (which is preferably a continuous amino acid sequence, as described above) comprises:
• the amino acid sequence of the chimeric protein first comprises the /V-terminal amino acids of the ISVD (e.g., R-strand A in Figure 1), followed by the C-terminus of the amino acid at the internal fusion site of the ISVD, which is linked to the /V-terminus of the circularly permuted cytokine (which is made by cleavage of the sequence at an internal fusion site located in a turn or loop, to form a novel /V- and C-terminus of the circularly permuted cytokine as compared to the original cytokine).
• the ISVD e.g., R-strand A in Figure 1
• the C-terminus of the amino acid at the internal fusion site of the ISVD which is linked to the /V-terminus of the circularly permuted cytokine (which is made by cleavage of the sequence at an internal fusion site located in a turn or loop, to form a novel /V- and C-terminus of
• the amino acid sequence of the chimeric protein continues with the (rest of the) sequence of the circularly permuted cytokine, ending in its C-terminus (which corresponds to the ammino acid that was /V-terminally located at the internal fusion site of the cytokine to design the circularly permuted cytokine), and finally linked to the /V-terminus of the C-terminally located amino acid at the internal fusion site of the ISVD (located in a turn or loop, in this case in a beta turn, represented by a black line in Figure 1) and the rest of the sequence of the ISVD (the C-terminal part of the ISVD).
• the primary amino acid sequence of the circularly permuted cytokine is interjected in the primary sequence of the ISVD (or the amino acid sequence of the circularly permuted cytokine interrupts the primary sequence (the amino acid sequence) of the ISVD).
• the fusions can be direct fusions, or fusions made by a linker peptide, said fusion sites being designed to result in a rigid, non-flexible fusion protein.
• the length and type of the linker peptide contributes to the rigidity of the resulting chimeric protein.
• polypeptides constituting the chimeric protein are fused to each other directly, by connection via a peptide bond, or indirectly, whereby indirect coupling assembles two polypeptides through connection via a short peptide linker.
• Preferred "linker molecules”, “linkers”, or “short polypeptide linkers” are peptides with a length of maximum ten amino acids, more likely four amino acids, typically is only three or four amino acids in length but is preferably only two or even more preferred only a single amino acid to provide the desired rigidity to the junction of fusion at the accessible sites.
• Nonlimiting examples of suitable linker sequences are described in Table A-l and in the Example section, which can be randomized, and wherein linkers have been successfully selected to keep a fixed distance between the structural domains, and/or as to maintain the fusion partners their independent functions (e.g., antigen-binding and/or cytokine receptor binding), if this is desired for the chimeric protein of the present invention.
• Non-limiting examples of such linkers are GSGG (SEQ. ID NO.: 120), GGSG (SEQ ID NO.: 121) or GSG (SEQ ID NO.: 5).
• rigid linkers In the embodiment relating to the use of rigid linkers, these are generally known to exhibit a unique conformation by adopting a-helical structures or by containing multiple proline residues. Under many circumstances, they separate the functional domains more efficiently than flexible linkers, which may as well be suitable, preferably in a short length of only 1-4 amino acids.
• the ISVD and the cytokine are fused through at least one, preferably two peptide linkers as defined above.
• the ISVD sequence /V-terminally located of the internal fusion site of the ISVD is linked through a peptide linker to the circularly permuted cytokine sequence, and/or the /V-terminal part of the original cytokine is linked through a peptide linkerto the sequence C-terminally located of the internal fusion site of the ISVD.
• the fusion between the ISVD and the cytokine may take place by first removing some amino acids from the internal fusion site(s) of the ISVD and/or the cytokine, if it is circularly permuted (or from the /V- and/or C-terminal of the cytokine, if not circularly permuted).
• the sequence /V-terminally located from the internal fusion site of the ISVD and the sequence of the (circularly permuted) cytokine and the sequence C- terminally located from the internal fusion site of the ISVD are linked to each other (directly or by means of a linker, as defined above) by first removing from O to 10, preferably from O to 5, more preferably from O to 3, such as 0, 1, 2 or 3 (continuous) amino acids from the sequence at the N-terminus of the (circularly permuted) cytokine, and linking the sequence /V-terminally of the internal fusion site of the ISVD to the adapted /V-terminus of the (circularly permuted) cytokine to the sequence C-terminally located of the internal fusion site of the ISVD, optionally through a peptide linker, as described above.
• sequence /V-terminally located of the internal fusion site of the ISVD and the sequence of the (circularly permuted) cytokine and/orthe sequence C-terminally located of the internal fusion site of the ISVD are linked to each other (directly or by means of a linker, as defined above) by first removing from 0 to 10, preferably from 0 to 5, more preferably from 0 to 3, such as 0, 1, 2 or 3 (continuous) amino acids from the sequence at the internal fusion site of the ISVD, and linking the sequence /V-terminally located of the internal fusion site of the ISVD to the (circularly permuted) cytokine to the sequence C-terminally positioned at the internal fusion site of the ISVD, optionally though a peptide linker, as described above.
• the internal fusion site(s) of the ISVD are located in an exposed region of the domain fold. Said exposed regions are identified as less fixed amino acid stretches, that are mostly located at the surface of the protein, and on the edges of a structure.
• the internal fusion site is located in an exposed loop between two p-strands in the ISVD, e.g., in an exposed turn such as a R-turn as defined by IMGT, see below. More preferably, the internal fusion site(s) of the ISVD is located at a p-turn, such as an exposed
• the internal fusion site(s) of the ISVD is(are) comprised in a loop or turn as defined by IMGT (Lefranc MP, "Immunoglobulin and T Cell Receptor Genes: IMGT(®) and the birth and Rise of Immunoinformatics", Front Immunol., 2014, 5:22), preferably in a beta turn.
• IMGT Lefranc MP, "Immunoglobulin and T Cell Receptor Genes: IMGT(®) and the birth and Rise of Immunoinformatics", Front Immunol., 2014, 5:22
• the cytokine preferably circularly permuted
• the internal fusion site is located in: a. the first R-turn that connects beta-strands A and B of the ISVD; or b.
• the R-turn that connects beta-strands C and C' of the ISVD or c. the R-turn that connects beta-strands C" and D of the ISVD; or d. the R-turn that connects beta-strands D and E of the ISVD; or e. the R-turn that connects beta strands E and F of the ISVD.
• the internal fusion site(s) in the ISVD are located in the exposed region of the AB R-turn, which connects the A and B R-strands of the ISVD.
• the internal fusion site(s) in the ISVD are positioned in an exposed region defined by the CC' R- turn, connecting the C and C'R-strands of the ISVD.
• Another embodiment comprises internal fusion site(s) in the C"D R-turn, or the EF R-turn. In fact, those are the surface loops connecting the R-strands A and B, C and C', C" and D, or E and F, respectively, constituting the R-strands of the typical sandwich to provide the immunoglobulin fold.
• the CDRs concern exposed regions (loops or turns) between two secondary elements (see, e.g., Figure 1).
• the interruption of those sites for fusing the ISVD to the cytokine may lead to loss of antigen-binding capacity.
• the ISVD comprised in the chimeric protein of the present invention may no longer have the capacity of specifically binding its antigen, as defined above. If it is desired to retain the antigen-binding capacity, the CDRs would not be the most suitable internal fusion sites of the ISVD.
• the internal fusion site(s) are in an exposed region, loop or turn, so that the CDRs of the ISVD retain their ability to bind the epitope of the target protein.
• the ISVD comprised in the chimeric protein of the present invention is a functional ISVD, i.e., an ISVD which specifically binds its antigen.
• the internal fusion site(s) of the cytokine comprised in the chimeric protein of the present invention when circularly permuted, is(are) located in a turn or loop between two secondary elements of the cytokine, e.g., in a p-turn, or in a loop between two p-strands, or between two a-helices, or between one p-strand and one a-helix.
• the internal fusion site of the cytokine is located at a position in the protein that will result in an altered cytokinereceptor binding or in an altered cytokine-receptor downstream activity and/or in an altered receptor/receptor's subunit oligomerization upon cytokine binding and/or in an altered cytokine-receptor/receptor's subunit-binding functionality for the chimeric molecule made by fusing an ISVD at said cytokine internal fusion site.
• the internal fusion site(s) of the cytokine is located in an exposed region of the domain fold.
• the internal fusion site is an exposed loop or turn between two p-strands or between two a-helices in the cytokine. More preferably, the internal fusion site(s) of the cytokine is a p-turn, such as an exposed p- turn. In another embodiment, the internal fusion site(s) of the cytokine is located in a loop or turn located between one p-strand and one a-helix.
• the chimeric protein (with two peptide bonds or two short linkers) is obtained by connecting the ISVD to the cytokine, via interruption of the ISVD's primary topology at an internal fusion site in its sequence located in the AB beta turn, through fusion with a circularly permuted cytokine at its internal fusion site located in an exposed region of its sequence (turn or loop, as defined above) (wherein said exposed or accessible site is not the original /V- or C-terminal of the cytokine, as explained above).
• the chimeric protein (with two peptide bonds or two short linkers) is obtained by connecting the ISVD to the cytokine, via interruption of the ISVD's primary topology at an internal fusion site in its sequence located in the AB beta turn, through fusion with a non-circularly permuted cytokine, i.e., the fusion site of the ISVD is fused to the cytokine through the (original) /V- and C- termini of the cytokine (though peptide linkers and/or with one or more amino acids deleted from the N- and/or C-termini of the cytokine, as explained above).
• the ISVD and the cytokine comprised in the chimeric protein are further connected via a disulphide bond.
• the disulphide bond may be formed by cysteine residues located within the ISVD, preferably near the turn or loop, preferably near the AB beta turn, at the end of R-strand A, and/or at the end of the R-strand G.
• the ISVD and the cytokine are further connected via a disulphide bond to improve rigidity of the chimeric protein.
• the cytokine present in the chimeric protein of the present invention is preferably a circularly permuted cytokine.
• the /V- and C-termini of the cytokine i.e., the original /V- and C-termini, before the circular permutation
• the circularly permuted cytokine comprised in the chimeric protein of the present invention is generated by linking the (original) /V- and C-termini of the cytokine to each other by first removing 0 to 10, preferably 0 to 5, more preferably 0 to 3, such as 0, 1, 2 or 3 (continuous) amino acids from the /V- and/or C-termini of the cytokine, and then linking the /V- and C-termini (i.e., the original /V- and C-termini of the cytokine before the circular permutation is performed) directly or through a peptide linker, as defined above and in Table A-l.
• the cytokine present in the chimeric protein of the present invention is not circularly permuted as described herein.
• the /V- and C-termini of the cytokine i.e., the original /V- and C-termini, although one or more amino acids from the N- and/or C-termini may be removed, as explained above
• the /V- and C-termini of the cytokine are not linked to each other, and are used to fuse (directly or by means of a linker, as explained above) the cytokine to the amino acids at the internal fusion site of the ISVD.
• the cytokine comprised in the chimeric protein of the present invention is any kind of cytokine, for instance an interleukin, a chemokine, an interferon, a colony stimulating factor (CSF), a transforming growth factor or a tumor necrosis factor.
• the cytokines comprise very diverse superfamilies of ligands, such as cytokine superfamilies with a R-stra nd-based or R-strand-containing conserved core domain or motif, revealing internal fusion sites at their exposed regions present in R-turns or loops that interconnect these R- strands.
• the cytokine comprised in the chimeric protein of the present invention may be selected from:
• Interleukins - subfamilies o IL-1 family o IL-2 family o IL-6 family o IL-10 family o IL-12 family o IL-17 family
• CSF Colony-stimulating factors
• TGF Transforming growth factors
• TNF Tumor necrosis factors
• the cytokine is selected from an interleukin or an interferon. More preferably, the cytokine is IL-2, IFNA2a or I L18.
• Interleukin-2 (e.g., Gene ID: 3558) is a member of a cytokine family ("IL-2 family"), each member of which has a four alpha helix bundle; the family also includes IL-4, IL-7, IL-9, IL-15 and IL-21.
• I L-2Rs IL-2 receptors
• monomeric, dimeric, and trimeric I L-2Rs see, e.g., Figures 12-15 and Figure 1 of Arenas-Ramirez, N., et al., "Interleukin-2: biology, design and application", 2015, Trends in Immunology, 36(12):763-777.
• Monomeric IL-2Rs, comprising IL-2a (CD25) are usually cell membrane associated but also exist in soluble form and bind IL-2 with a low Kd of ⁇ 10 -8 M.
• Dimeric I L-2Rs comprise IL-2RP (CD122) and IL-2Ry [better known as common y-chain (y c ) or CD132], whereas trimeric I L-2Rs comprise CD25, CD122, and y c .
• dimeric I L-2Rs can be referred to as low-affinity (Kd ⁇ 10 -9 M) and trimeric I L-2Rs as high-affinity (Kd ⁇ 10 11 M) I L-2Rs.
• IL-2 On a molecular level, a single trimeric IL- 2R binds IL-2 with roughly 10- 100-fold higher affinity than a single dimeric IL-2R (from Arenas- Ramirez, N., et al., "Interleukin-2: biology, design and application", 2015, Trends in Immunology, 36(12):763-777).
• IFNA2a Human interferon alpha-2
• the mature protein is made of 165 amino acids.
• the secondary structure of IFNA2a consists of five a-helices: A to E, from the /V-terminal to the C-terminal end. Helices A, B, C and E are organized as a bundle with a long loop between the helices A and B (the A-B loop) and two disulphide bonds which connect helix E to the A-B loop and helix C to the Interminal end.
• the type I IFN receptor is composed of two subunits, IFNAR 1 and IFNAR 2, see, e.g., Figures 24-27.
• Interleukin-18 (e.g., Gene ID: 3606) belongs to the IL-1 superfamily and it is folded as all-beta pleated sheet molecule, see, e.g., Figure 30.
• the 'IL-1 receptor-type interleukin' superfamily or 'IL-1 family' interleukins comprises for instance the interleukins IL-la, IL-ip, IL-IRa, IL-18, IL-33, IL-36a, IL-36p, IL-36y, IL-36Ra, IL-37, IL-38.
• These cytokines are related to each other by origin, receptor structure, and signal transduction pathways.
• the receptors for IL-1 superfamily interleukins share a similar architecture, comprised of three Ig-like domains in their ectodomains, and an intracellular Toll/IL-1 R (TIR) domain that is also found among Toll-like receptors.
• TIR Toll/IL-1 R
• the initiation of cytokine signalling requires two receptors, a primary specific receptor and an accessory receptor that can be shared in some cases.
• the primary receptor is responsible for specific cytokine binding, while the accessory receptor by itself does not bind the cytokine but associates with the preassembled binary complexes from the cytokine and the primary receptor.
• the binding of the cytokines to their respective receptors results in a signalling ternary complex, leading to the dimerization of the TIR domains of the two receptors.
• MARK mitogen-activated protein kinases
• NF-kB nuclear factor kappa-light-chain- enhancer of activated B cells
• the IL-18 binds first to the IL-18a receptor and forms a lower affinity complex. Upon binding with I L-18P receptor, a hetero-trimeric complex with higher affinity is formed that initiates the signal transduction process.
• the three-dimensional structures of several interleukin cytokines of the IL-1 superfamily have been determined, and demonstrate that despite having limited sequence similarity, these cytokines adopt a conserved signature p-trefoil fold comprised of 12 anti-parallel
• the p-barrel core motif is packed by various amounts of helices in each cytokine structure.
• Superimposition of the Ca atoms of each of the human cytokines reveals a conserved hydrophobic core, with significant flexibility in the loop regions.
• IL-18 shares 65% sequence identity to murine IL-18 while sharing only 15% and 18% identity to human IL-1 a and human IL-ip, respectively. Nevertheless, IL-18 shows striking similarity to other IL-1 cytokines in its three-dimensional structure.
• this I L-l-like receptor interleukins provide for an example of a superfamily within the cytokines with a s-strand-based conserved structural core domain that is interconnected by flexible
• the cytokine comprised in the chimeric protein of the present invention is not erythropoietin (EPO), such as human EPO (hEPO).
• the cytokine is not granulocyte colony-stimulating factor, such as human granulocyte colony-stimulating factor (hGCSF).
• the cytokine is neither hEPO nor hGCSF.
• the cytokine comprised therein may be functional (in that it retains its receptor-binding functionality in a similar manner as compared to the cytokine not fused to the ISVD) or non-functional (in that it does not retain its receptor-binding functionality in a similar manner as compared to the cytokine not fused to the ISVD, as described above in the context of the ISVD).
• the receptor-binding functionality of the cytokine comprised in the chimeric protein of the present invention may be modulated by its fusion to the ISVD, as described in detail below.
• the oligomerization of the cytokine receptor/receptor's subunits may be affected/altered upon binding of the cytokine comprised in the chimeric protein as compared with the oligomerization of the cytokine receptor/receptor's subunits upon binding if the cytokine is not comprised in the chimeric protein (i.e., if the cytokine is not fused via 2 peptide bonds to the ISVD).
• the cytokine signalling may be modulated with the cytokine comprised in the chimeric protein of the present invention.
• the cytokine comprised in the chimeric protein of the present invention may thus not retain its receptor-binding functionality in a similar manner as compared to the cytokine not fused to the ISVD.
• the cytokine comprised in the chimeric protein of the present invention may bind its receptor with better specificity and/or higher affinity as compared to the cytokine not fused to the ISVD.
• the cytokine comprised in the chimeric protein of the present invention may bind its receptor with lower specificity and/or lower affinity as compared to the cytokine not fused to the ISVD.
• downstream signalling of the cytokine receptor upon binding of the cytokine present in the chimeric protein of the present invention may be different as compared with the downstream signalling of the cytokine receptor upon binding of the cytokine not fused to the ISVD.
• the receptor/receptor's subunit oligomerization upon binding of the cytokine present in the chimeric protein of the present invention may be affected (e.g., may be different as compared to the receptor/receptor's subunit oligomerization upon binding of the cytokine not fused to the ISVD).
• the receptor-binding functionality of the cytokine comprised in the chimeric protein of the present invention may be modulated (e.g., improved or worsened, or simply altered), as described in detail below.
• the internal fusion site of the cytokine may be a R-turn of the interleukin R-barrel core motif, as described above.
• the chimeric protein comprises an anti-GFP ISVD, preferably ISVD comprising a sequence as defined in SEQ ID NO.: 1, or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 1.
• the chimeric protein comprises an ISVD acting as a half-life extension moiety or with half-life extension properties.
• half-life as used here can generally be defined as described in paragraph o) on page 57 of WO 2008/020079 and as mentioned therein refers to the time taken for the serum concentration of the compound or polypeptide to be reduced by 50%, in vivo, for example due to degradation of the sequence or compound and/or clearance or sequestration of the sequence or compound by natural mechanisms.
• the in vivo half-life of the protein-based carrier building block and/or molecule of the invention can be determined in any manner known per se, such as by pharmacokinetic analysis. Suitable techniques will be clear to the person skilled in the art and may for example generally be as described in paragraph o) on page 57 of WO 2008/020079.
• the half-life can be expressed using parameters such as the ti/2-alpha, ti/2- beta and the area under the curve (AUG).
• the term "half-life” as used herein in particular refers to the ti/2-beta or terminal half-life (in which the ti/2-alpha and/or the AUC or both may be kept out of considerations).
• (In vivo) half-life can be extended by an increase in the hydrodynamic radius (size) or by a decrease in the molecule's clearance.
• (In vivo) half-life extending moieties such as binding units that can bind to, e.g., serum albumin, increase the half-life of the molecules to which they are attached by binding, e.g., to serum albumin.
• Albumin is the most abundant plasma protein, is highly soluble, very stable and has an extraordinarily long circulatory half-life as a direct result of its size and interaction with the FcRn mediated recycling pathway, see, e.g., Sleep D. et al., "Albumin as a versatile platform for drug half-life extension", Biochim Biophys Acta, 2013, 1830(12):5526-34.
• WO 2004/041865 describes ISVDs binding to serum albumin (and in particular against HSA) that can be used to increase the half-life of the chimeric protein or polypeptide of the present invention.
• ISVDs against (human) serum albumin.
• ISVDs include the ISVDs called Alb-1 (SEQ ID NO: 52 in WO 2006/122787) and humanized variants thereof, such as Alb-8 (SEQ ID NO: 62 in WO 2006/122787). Again, these can be used to extend the half-life of therapeutic proteins and polypeptides, and other entities or moieties, such as the chimeric protein or polypeptide of the present invention.
• the chimeric protein or polypeptide of the present invention comprises a serum albumin binding moiety selected from Alb-1, Alb-3, Alb-4, Alb-5, Alb-6, Alb-7, Alb-8, Alb-9, Alb-10 (described in WO 2006/122787) and Alb-23.
• the serum albumin binding moiety is Alb-8 or Alb-23 or its variants, as shown on pages 7-9 of WO 2012/175400.
• the serum albumin binding moiety is selected from the albumin binders described in WO 2012/175741, WO 2015/173325, WO 2017/080850, WO 2017/085172, WO 2018/104444, WO 2018/134235, and WO 2018/134234, the content of which is herein incorporated by reference.
• Some serum albumin binders are also shown in Table 3 below.
• the chimeric protein or polypeptide of the present invention comprises the serum albumin binding moiety Alb23 (SEQ ID NO.: 123) as defined in Table 3 below.
• the molecule of the present invention comprises the serum albumin binding moiety Alb23002 (SEQ ID NO.: 55) as defined in Table 3 below.
• the molecule of the present invention comprises the serum albumin binding moiety Alb23002(ElD) (SEQ ID NO.: 137) as defined in Table 3 below.
• the molecule of the present invention comprises a HLE moiety as described in the following item A:
• An ISVD that binds to HSA and comprises i. a CDR1 that is the amino acid sequence of SEQ ID NO: 138 or an amino acid sequence with 2 or 1 amino acid difference with SEQ ID NO: 138; ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 139 or an amino acid sequence with 2 or 1 amino acid difference with SEQ ID NO: 139; and iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 140 or an amino acid sequence with 2 or 1 amino acid difference with SEQ ID NO: 140.
• the ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO: 138, a CDR2 that is the amino acid sequence of SEQ ID NO: 139 and a CDR3 that is the amino acid sequence of SEQ ID NO: 140.
• Examples of such an ISVD that binds to HSA have one or more, or all, framework regions as indicated for construct ALB23002 (SEQ I D NO.: 55) in Tables 4 and 5 (in addition to the CDRs as defined in the preceding item A). In one embodiment, it is an ISVD comprising or consisting of the full amino acid sequence of construct ALB23002 (SEQ ID NO: 55).
• Table 4 Sequences for CDRs according to AbM CDR and framework annotation ("ID" refers to the given SEQ ID NO)
• Item A' can be also described using the Kabat CDR definition as:
• An ISVD that binds to HSA and comprises i. a CDR1 that is the amino acid sequence of SEQ ID NO: 146 or an amino acid sequence with 2 or 1 amino acid difference with SEQ ID NO: 146; ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 148 or an amino acid sequence with 2 or 1 amino acid difference with SEQ ID NO: 148; and iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 140 or an amino acid sequence with 2 or 1 amino acid difference with SEQ ID NO: 140.
• the ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO: 146, a CDR2 that is the amino acid sequence of SEQ ID NO: 148 and a CDR3 that is the amino acid sequence of SEQ ID NO: 140.
• Examples of such an ISVD that binds to HSA have one or more, or all, framework regions as indicated for construct ALB23002 in Table 5 (in addition to the CDRs as defined in the preceding item A'). In one embodiment, it is an ISVD comprising or consisting of the full amino acid sequence of construct ALB23002 (SEQ ID NO: 55, see also Table 5).
• the amino acid sequence of an ISVD binding to HSA may have a sequence identity of more than 90%, such as more than 95% or more than 99%, with SEQ ID NO: 55, wherein the CDRs are as defined in the preceding item A or A'.
• the ISVD binding to HSA comprises or consists of the amino acid sequence of SEQ ID NO: 55.
• the ISVD has at least half the binding affinity, or at least the same binding affinity, to HSA compared to construct ALB23002 (SEQ ID NO: 55), wherein the binding affinity is measured using the same method, such as SPR.
• such an ISVD binding to HSA when such an ISVD binding to HSA has a C-terminal position, it exhibits a C-terminal extension, such as a C-terminal alanine, cysteine, or glycine extension.
• a C-terminal extension such as a C-terminal alanine, cysteine, or glycine extension.
• such an ISVD is selected from SEQ ID Nos: 124, 125, 127, 129, 130, 131, 132, 133, 134, and 55 (see Table 3 above).
• the ISVD binding to HSA has another position than the C-terminal position (i.e., is not the C-terminal ISVD of the molecule of the present invention).
• such an ISVD is selected from SEQ ID Nos: 55, 122, 123, 136, 128 and 137 (see Table 3 above).
• said one or more other groups, residues, moieties or binding units that provide the molecule with increased half-life is a peptide that can bind to HSA.
• the "serum-albumin binding polypeptide or binding domain” may be any suitable serum-albumin binding peptide capable of increasing the half-life (preferably T1/2R, as defined above) of the molecule (compared to the same molecule without the serum-albumin binding peptide or binding domain).
• the polypeptide sequence suitable for extending serum half-life is a polypeptide sequence capable of binding to a serum protein with a long serum half-life, such as serum albumin, transferrin, IgG, etc, in particular human serum albumin (HSA).
• serum albumin transferrin, IgG, etc
• HSA human serum albumin
• Polypeptide sequences capable of binding to serum albumin have previously been described and may in particular be serum albumin binding peptides as described in WO 2008/068280 (and in particular WO 2009/127691 and WO 2011/095545), the content of which is herewith incorporated by reference.
• the chimeric protein of the present invention comprises an ISVD acting as a half-life extension moiety or with half-life extension properties such as an anti-HSA ISVD, as described above, preferably an ISVD comprising a sequence as defined in SEQ ID NO.: 55, or in SEQ ID NO: 154, or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 55 or with SEQ ID NO: 154.
• an ISVD comprising a sequence as defined in SEQ ID NO.: 55, or in SEQ ID NO: 154, or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 55 or with SEQ ID NO: 154.
• the chimeric protein of the present invention comprises an ISVD comprising the CDR1, 2, and 3 sequences of SEQ ID NO: 154, as defined in SEQ ID NO: 151 for CDR1, SEQ ID NO: 152 for CDR2 and SEQ ID NO: 153 for CDR3 (disclosed as HSA binders in WO 2019/016237 Al).
• the chimeric protein comprises a cytokine which is IL-2, preferably a cytokine comprising a sequence as defined in SEQ ID NO.: 2 (IL-2), or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 2.
• the chimeric protein comprises a cytokine comprising a sequence as defined in SEQ ID NO.: 3 (I L-2(K35E,C125S)), or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 3.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 4 (circularly permutated IL- 2(K35E,C125S), referred to as IL-2(K35E,C125S)[S75-Q74]), or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 4.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 172 (circularly permutated IL-2(K35E,C125S), referred to as IL-2(K35E,C125S)[L17-L14]), or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 172.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 173 (circularly permutated IL-2(K35E,C125S), referred to as IL-2(K35E,C125S)[P34-Y31]), or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 173.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 174 (circularly permutated IL-2(K35E,C125S), referred to as IL-2(K35E,C125S)[F42-M39]), or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 174.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 175 (circularly permutated IL-2(K35E,C125S), referred to as IL-2(K35E,C125S)[M46-F42]), or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 175.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 176 (circularly permutated IL-2(K35E,C125S), referred to as I L-2(K35 E,C125S) [E62-L59]), or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 176.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 177 (circularly permutated IL-2(K35E,C125S), referred to as IL-2(K35E,C125S)[S75-N71]), or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 177.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 178 (circularly permutated IL-2(K35E,C125S), referred to as IL-2(K35E,C125S)[N77-S75]) or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 178.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 179 (circularly permutated IL-2(K35E,C125S), referred to as IL-2(K35E,C125S)[F78-Q74]) or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 179.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 180 (circularly permutated IL-2(K35E,C125S), referred to as I L-2(K35E,C125S)[L85-P82]) or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 180.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 181 (circularly permutated IL-2(K35E,C125S), referred to as IL-2(K35E,C125S)[T101-G98]) or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 181.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 182 (circularly permutated IL-2(K35E,C125S), referred to as IL-2(K35E,C125S)[T102-E100]) or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 182.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 183 (circularly permutated IL-2(K35E,C125S), referred to as IL-2(K35E,C125S)[F103-S99]) or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 183.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 184 (circularly permutated IL-2(K35E,C125S), referred to as I L-2(K35E,C125S) [L132-1129]) or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 184.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 185 (I L-2(K35E,C125S) without the first four amino acids) or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 185.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 186 (circularly permutated IL-2(K35E,C125S), referred to as IL-2(K35E,C125S)[K35-K32]) or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 186.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 187 (circularly permutated IL-2(K35E,C125S), referred to as IL-2(K35E,C125S)[I92-I89]) or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 187.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 188 (circularly permutated IL-2(K35E,C125S), referred to as IL-2(K35E,C125S)[L96-V93]) or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 188.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 189 (circularly permutated IL-2(K35E,C125S), referred to as IL-2(K35E,C125S)[S4-T133]) or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 189.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 262 (circularly permutated IL-2, referred to as I L-2[L132-I129], or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 262.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 264 (circularly permutated IL-2, referred to as IL-2[F42-M39], or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 264.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 268 (circularly permutated IL-2, referred to as I L-2[S75-N71], or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 268.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 270 (circularly permutated IL-2, referred to as IL-2[T102-E100], or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 270.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 272 (circularly permutated IL-2, referred to as I L-2[F103-S99], or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 272.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 274 (circularly permutated IL-2, referred to as I L-2[L85-P82], or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 274.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 190 (IL-2 in TP072), or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 190.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 191 (IL-2 in TP075), or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 191.
• the chimeric protein comprises a cytokine which is IFNA2a, preferably a cytokine comprising a sequence as defined in SEQ ID NO.: 56, or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 56.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 58 (circular permuted IFNA2a, referred to as IFNA2a[D77-W76]V2), or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 58.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 59 (circular permuted IFNA2a, referred to as IFNA2a[D77-W76]V4), or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 59.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 192 (IFNA2a in TP093), or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 192.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 193 (IFNA2a in TP095), or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 193.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 194 (IFNA2a in TP098), or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 194.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 195 (IFNA2a in TP109), or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 195.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 197 (IFNA2a in TP089), or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 197.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 199 (IFNA2a in TP090), or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 199.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 201 (IFNA2a in TP091), or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 201.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 203 (IFNA2a in TP092), or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 203.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 205 (IFNA2a in TP095), or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 205.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 207 (IFNA2a in TP096), or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 207.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 209 (IFNA2a in TP097), or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 209.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 211 (IFNA2a in TP099), or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 211.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 213 (IFNA2a in TP100), or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 213.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 215 (IFNA2a in TP101), or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 215.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 217 (IFNA2a in TP102), or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 217.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 220 (IFNA2a in TP104), or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 220.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 222 (IFNA2a in TP105), or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 222.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 225 (IFNA2a in TP107), or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 225.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 227 (IFNA2a in TP108), or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 227.
• the chimeric protein comprises a cytokine which is IL-18, preferably a cytokine comprising a sequence as defined in SEQ ID NO.: 64, or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 64.
• the chimeric protein comprises a cytokine which is IL-18, preferably a cytokine comprising a sequence as defined in SEQ ID NO.: 66, or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 66.
• the chimeric protein comprises a cytokine which is IL-18, preferably a cytokine comprising a sequence as defined in SEQ ID NO.: 68, or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 68.
• the chimeric protein comprises a cytokine which is IL-18, preferably a cytokine comprising a sequence as defined in SEQ ID NO.: 70, or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 70.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 244 (circular permuted IL-18, referred to as IL18[K79-N78], or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 244.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 245 (circular permuted IL-18, referred to as IL18[Q56-S55], or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 245.
• the circularly permuted cytokine comprised in the chimeric protein of the present invention comprises a sequence as defined in SEQ ID NO.: 246 (circular permuted IL-18, referred to as IL18[P57-Q56], or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 246.
• the chimeric protein of the present invention is selected from a protein comprising or consisting of a sequence as defined in SEQ ID NO.: 7-25, 36-54, 60-63, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218-219, 221, 223-224, 226, 230-237, 261, 263, 265-267, 269, 271 or 273, or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 7-25, 36- 54, 60-63, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218-219, 221, 223-224, 226, 230-237, 261, 263, 265-267, 269, 271 or 273.
• the chimeric protein of the present invention comprises an ISVD fused to a cytokine (preferably circularly permuted), wherein the cytokine comprises or consists of a sequence as defined in SEQ ID NO.: 4, 58, 59, 66, 68, 70, 172-195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 220, 222, 225, 227, 244-246, 262, 264, 268, 270, 272 or 274, or a sequence with at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97% identity with SEQ ID NO.: 4, 58, 59, 66, 68, 70, 172-195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 220, 222, 225, 227, 244-246, 262, 264, 268, 270, 27
• polypeptide of the present invention is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoe of the present invention
• the present invention further provides a polypeptide comprising the chimeric protein of the present invention.
• the polypeptide of the present invention may comprise, besides the chimeric protein, further groups, residues, moieties or binding units.
• such further groups, residues, moieties or binding units may be one or more additional immunoglobulins, so as to form a (fusion) protein or (fusion) polypeptide (the polypeptide of the present invention).
• the one or more other groups, residues, moieties or binding units are ISVDs.
• the one or more other groups, residues, moieties or binding units are chosen from the group consisting of domain antibodies, ISVDs that are suitable for use as a domain antibody, single domain antibodies, ISVDs that are suitable for use as a single domain antibody, "dAb"'s, ISVDs that are suitable for use as a dAb, VHHS, humanized VHHS, camelized VHS, or Nanobody® VHHS.
• groups, residues, moieties or binding units may for example be chemical groups, residues, moieties, which may or may not by themselves be biologically and/or pharmacologically active.
• such groups may be linked to the one or more domains in the polypeptides of the invention so as to provide a "derivative" of the polypeptide of the invention, as further described herein.
• a polypeptide of the invention may also include additional groups with certain functionalities, such as a label, a toxin, one or more linkers, a binding sequence, etc. These additional functionalities include both amino acid-based and non-amino acid-based groups.
• the polypeptide of the present invention may further comprise (besides the chimeric protein of the present invention), one or more ISVDs.
• the ISVD may be a HLE ISVD, a targeting ISVD or a therapeutic ISVD.
• the one or more ISVDs which may be further comprised (besides the one comprised in the chimeric protein) in the polypeptide of the present invention may thus form a "multivalent" or "multispecific" polypeptide or construct.
• polypeptides that comprise of two or more ISVDs will be referred to herein as “multivalent polypeptides” or as “multivalent constructs”, and these may provide certain advantages compared to the corresponding monovalent polypeptide.
• proteins or polypeptides that comprise a single ISVD (such as the chimeric protein of the invention) will be referred to herein as “monovalent” proteins or polypeptides or as “monovalent constructs”.
• multivalent polypeptides of the invention may for example, without limitation, be multispecific (such as bispecific or trispecific) or multiparatopic (such as biparatopic) constructs (or be both multiparatopic and multispecific), and may for example be constructs that comprise at least two binding domains or binding units that are each directed towards a different epitope on the same subunit, constructs that comprise at least two binding domains or binding units that each have a different biological function (for example one binding domain that can block or inhibit receptor-ligand interaction, and one binding domain that does not block or inhibit receptor-ligand interaction), or constructs that comprise at least two binding domains or binding units that are each directed towards a different target.
• multispecific such as bispecific or trispecific
• multiparatopic constructs or be both multiparatopic and multispecific
• constructs that comprise at least two binding domains or binding units that are each directed towards a different epitope on the same subunit constructs that comprise at least two binding domains or binding units that each have a different biological function (for example one binding domain that can block or
• polypeptide construct and “polypeptide” can be used interchangeably herein (unless the context clearly dictates otherwise).
• polypeptides of the invention can generally be prepared by a method which comprises at least one step of suitably linking the chimeric protein of the present invention to one or more further groups, residues, moieties or binding units, either directly or via one or more suitable linkers, as described herein.
• Polypeptides of the invention can also be prepared by a method which generally comprises at least the steps of providing a nucleic acid that encodes a polypeptide of the invention, expressing said nucleic acid in a suitable manner, and recovering the expressed polypeptide of the invention. Such methods can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the methods and techniques further described herein.
• the order of the chimeric protein and further groups, residues, moieties or binding units, if present, in the polypeptides of the invention such as, e.g., a first domain (e.g., the chimeric protein of the present invention), a second binding domain (e.g., a HSA-binding ISVD), a third binding domain (e.g., a domain binding to a therapeutically relevant target), etc., in the polypeptide (i.e., the orientation or configuration of the chimeric protein and further groups, residues, moieties or binding units, if present) can be chosen according to the needs of the person skilled in the art, as well as the relative affinities which may depend on the location of the chimeric protein and further groups, residues, moieties or binding units, if present, in the polypeptide.
• a first domain e.g., the chimeric protein of the present invention
• a second binding domain e.g., a HSA-binding ISVD
• polypeptide comprises one or more linkers to interconnect the chimeric protein and optionally further groups, residues, moieties or binding units is a matter of design choice. However, some orientations, with or without linkers, may provide preferred binding characteristics in comparison to other orientations. All different possible orientations are encompassed by the invention.
• sequence of the polypeptide of the present invention may comprise one or more ISVDs, linked together directly or by means of a linker, as defined herein, followed by the chimeric protein of the present invention, directly linked to the one or more ISVDs or linked by means of a linker, as defined herein.
• the one or more ISVDs would be located at the /V-terminal region of the polypeptide, whereas the chimeric protein would be located at the C-terminal region of the polypeptide.
• sequence of the polypeptide of the present invention may comprise the chimeric protein of the present invention followed by one or more ISVDs, linked together directly or by means of a linker, directly linked to the chimeric protein of the invention, or linked by means of a linker, as defined herein.
• the one or more ISVDs would be located at the C-terminal region of the polypeptide, whereas the chimeric protein would be located at the /V-terminal region of the polypeptide.
• sequence of the polypeptide of the present invention may comprise one or more ISVDs, linked together directly or by means of a linker, directly linked to the chimeric protein of the invention, or linked by means of a linker, followed by a further one or more ISVDs, linked together directly or by means of a linker.
• the chimeric protein of the present invention would be flanked by one or more ISVDs at the /V- and C-terminal regions of the polypeptide.
• linkers to connect two or more (poly)peptides is well known in the art.
• One frequently used class of peptide linkers are known as the "Gly-Ser” or “GS” linkers. These are linkers that essentially consist of glycine (G) and serine (S) residues, and usually comprise one or more repeats of a peptide motif such as the GGGGS (SEQ ID NO: 155) motif (for example, exhibiting the formula (Gly-Gly-Gly-Gly-Ser)n in which n may be 1, 2, 3, 4, 5, 6, 7 or more).
• 9GS linkers e.g., GGGGSGGGS, SEQ I D NO: 156
• the linker is chosen from the group consisting of linkers of GSG, GSGG, GGSG, 3A, 3GS, 5GS, 7GS, 9GS, 10GS, 15GS, 18GS, 20GS, 25GS, 30GS and 35GS, see Table A-l.
• the polypeptide of the present invention may additionally comprise a group, residue, moiety or binding unit which provides the chimeric protein/polypeptide of the present invention with increased (in vivo) half-life compared to the corresponding chimeric protein/polypeptide without said one or more other groups, residues, moieties or binding units ("(in vivo) half-life extending moiety", or "half-life extending (HLE) moiety").
• the (multivalent and multispecific) polypeptide of the present invention may comprise at least two HLE moieties: the ISVD comprised in the chimeric molecule and at least one further HLE moiety.
• the HLE moieties comprised in the polypeptide of the present invention may be the same or different.
• the term "half-life" as used here has been defined in the context of the chimeric protein of the present invention, and is equally applicable to the present embodiment.
• the further binding unit comprised in the polypeptide of the present invention is an ISVD, for instance an HSA-binding ISVD.
• HSA-binding ISVDs have already been defined in the context of the chimeric protein of the present invention and are equally applicable to the present embodiment.
• HLE moieties that may be comprised in the polypeptide of the present invention are HLE moieties such as polyethylene glycol or ELNN polypeptides, which increase the size of the molecules to which they are attached, therefore bypassing renal clearance, and thus increasing the half-life of those molecules.
• HLE groups, residues, moieties or binding units is not generally restricted and may for example be chosen from the group consisting of a polyethylene glycol (PEG) molecule, ELNN polypeptides or fragments thereof, as described above, serum proteins or fragments thereof, binding units that can bind to serum proteins, such as HSA-binding ISVDs, as described above, an Fc portion, and small proteins or peptides that can bind to serum proteins.
• PEG polyethylene glycol
• the polypeptide of the present invention may additionally comprise (besides the chimeric protein) one or more targeting moieties.
• a "targeting moiety”, as defined herein, is any group, residue, moiety, or binding unit which is capable of being directed through its binding to a target.
• amino acid sequence such as an ISVD, an antibody, antigen-binding domains or fragments such as VHH domains or VH/VL domains, or generally an antigen binding protein or polypeptide or a fragment thereof
• an amino acid sequence that "(specifically) binds", that "can (specifically) bind to”, that "has affinity for” and/or that "has specificity for” a specific antigenic determinant, epitope, antigen or protein, or for a specific non-protein molecule, such as nucleic acids (such as DNA or RNA) or glycans (or for at least one part, fragment or epitope thereof) is said to be "against” or “directed against” said antigenic determinant, epitope, antigen, protein or nonprotein molecule.
• Specific binding of an antigen-binding protein to an antigen or antigenic determinant can be determined in any suitable manner known perse, including, for example, Scatchard analysis and/or competitive binding assays, such as radio-immunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known perse in the art; as well as the other techniques mentioned herein.
• Scatchard analysis and/or competitive binding assays such as radio-immunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known perse in the art; as well as the other techniques mentioned herein.
• polypeptide of the present invention may additionally comprise one or more therapeutic moieties.
• a "therapeutic moiety", as defined herein, is any group, residue, moiety, or binding unit which is capable of exerting a therapeutic activity in the animal and/or human body.
• the therapeutic moiety may also be in the form of a precursor, which then gets activated to exert its therapeutic activity.
• Non-limiting examples of therapeutic moieties which may be present in the polypeptide of the present invention are Programmed deathligand 1 (PD-Ll)binding molecules.
• the nucleic acid of the present invention is a nucleic acid of the present invention.
• the present invention further provides a nucleic acid molecule encoding the chimeric protein of the present invention and/or the polypeptide of the present invention.
• a nucleic acid may be used to transform/transfect a host cell or host organism, e.g., for expression and/or production of a polypeptide.
• Suitable (non-human) hosts or host cells for production purposes will be clear to the skilled person, and may for example be any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism.
• a host or host cell comprising a nucleic acid encoding the chimeric protein and/or polypeptide of the present invention is also encompassed by the present invention.
• a nucleic acid may be for example DNA, RNA, or a hybrid thereof, and may also comprise (e.g., chemically) modified nucleotides, like PNA. It can be single- or double-stranded. In one embodiment, it is in the form of double-stranded DNA.
• the nucleotide sequences of the present invention may be genomic DNA, cDNA.
• nucleic acids of the present invention can be prepared or obtained in a manner known perse, and/or can be isolated from a suitable natural source.
• Nucleotide sequences encoding naturally occurring (poly)peptides can for example be subjected to site-directed mutagenesis, so as to provide a nucleic acid molecule encoding polypeptide with sequence variation.
• site-directed mutagenesis so as to provide a nucleic acid molecule encoding polypeptide with sequence variation.
• nucleic acid also several nucleotide sequences, such as at least one nucleotide sequence encoding a targeting moiety and for example nucleic acids encoding one or more linkers can be linked together in a suitable manner.
• nucleic acids may for instance include, but are not limited to, automated DNA synthesis; site-directed mutagenesis; combining two or more naturally occurring and/or synthetic sequences (or two or more parts thereof), introduction of mutations that lead to the expression of a truncated expression product; introduction of one or more restriction sites (e.g., to create cassettes and/or regions that may easily be digested and/or ligated using suitable restriction enzymes), and/or the introduction of mutations by means of a PCR reaction using one or more "mismatched" primers.
• a chimeric gene is described with at least a promoter, said nucleic acid molecule encoding the chimeric protein, and a 3' end region containing a transcription termination signal.
• Another embodiment relates to an expression cassette encoding the chimeric protein of the present invention or comprising the nucleic acid molecule or the chimeric gene encoding the chimeric protein.
• Said expression cassettes are in certain embodiments applied in a generic format as an immune library, containing a large set of ISVD to select for the most suitable binders of the target (if ISVD binding to a target is desired).
• the vector of the present invention is a vector of the present invention.
• the present invention further provides a vector comprising the nucleic acid molecule of the present invention.
• a vector as used herein is a vehicle suitable for carrying genetic material into a cell.
• a vector includes naked nucleic acids, such as plasmids or mRNAs, or nucleic acids embedded into a bigger structure, such as liposomes or viral vectors.
• vectors comprise at least one nucleic acid that is optionally linked to one or more regulatory elements, such as for example one or more suitable promoter(s), enhancer(s), terminator(s), etc.).
• the vector is an expression vector, i.e., a vector suitable for expressing an encoded polypeptide or construct under suitable conditions, e.g., when the vector is introduced into a (e.g., human) cell.
• DNA-based vectors include the presence of elements for transcription (e.g., a promoter and a polyA signal) and translation (e.g., Kozak sequence).
• vectors comprising said expression cassette or nucleic acid molecule encoding the chimeric protein and/or polypeptide of the invention.
• vectors for expression in E. coli or 5. cerevisiae allow to produce the chimeric proteins and/or polypeptides and purify them.
• said at least one nucleic acid and said regulatory elements are "operably linked" to each other, by which is generally meant that they are in a functional relationship with each other.
• a promoter is considered “operably linked” to a coding sequence if said promoter is able to initiate or otherwise control/regulate the transcription and/or the expression of a coding sequence (in which said coding sequence should be understood as being “under the control of” said promotor).
• two nucleotide sequences when operably linked, they will be in the same orientation and usually also in the same reading frame. They will usually also be essentially contiguous, although this may also not be required.
• any regulatory elements of the vector are such that they are capable of providing their intended biological function in the intended host cell or host organism.
• a promoter, enhancer or terminator should be "operable" in the intended host cell or host organism, by which is meant that for example said promoter should be capable of initiating or otherwise controlling/regulating the transcription and/or the expression of a nucleotide sequence - e.g., a coding sequence - to which it is operably linked.
• the present invention providing said vectors further encompasses the option for high throughput cloning in a generic fusion vector.
• Said generic vectors are preferably specifically suitable for surface display in yeast, phages, bacteria or viruses.
• said vectors find applications in selection and screening of immune libraries comprising such generic vectors or expression cassettes with a large set of different ISVDs, wherein the same /V-terminal end of the conserved ISVD, and the cytokine, are fused with the remaining ISVD sequences provided by the library. So, the differential sequence in said libraries constructed for the screening of novel chimeric proteins for specific targets is provided by the difference in the ISVD sequence, and more particularly in the CDR regions of said ISVD library.
• the host cell of the present invention is a cell of the present invention.
• Alternative embodiments relate to host cells comprising the chimeric protein and/or polypeptide of the invention, or the nucleic acid molecule or expression cassette or vector encoding the chimeric protein of the invention.
• Suitable host cells or host organisms are clear to the skilled person, and are for example any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism. Specific examples include HEK293 cells, CHO cells, 5. cerevisiae, Escherichia coli or Komagataella phaffii (Pichia pastoris, see Bernauer L., et al. ("Komagataella phaffii as emerging model organism in fundamental research", Front. Microbiol., 2021, 11:1- 16)). In one embodiment, the host is Komagataella phaffii (Pichia pastoris).
• the host is Escherichia coli. In a preferred embodiment, the host is 5. cerevisiae.
• cell free systems may also be employed to produce the protein-based carrier building block and/or the molecule of the present invention, as reviewed, for instance, in Gregorio NE, Levine MZ, Oza JP, "A user's guide to cell-free protein synthesis", Methods Protoc., 2019, 2(1):24.
• Another embodiment discloses the use of said host cells, or a membrane preparation isolated thereof, or proteins isolated therefrom, for ligand screening, drug screening, protein capturing and purification, or biophysical studies.
• a method for producing a chimeric protein and/or polypeptide of the present invention is to produce a chimeric protein and/or polypeptide of the present invention.
• Another embodiment of the invention relates to a method for producing the chimeric protein and/or the polypeptide of the present invention, wherein the method comprises the steps of:
• the method of the present invention comprises the step (iv) of recovering the obtained chimeric protein and/or polypeptide, and optionally purifying it.
• the above method may further comprise, after step (i), the step of selecting one or more further groups, residues, moieties or binding units which may be comprised in the polypeptide, besides the chimeric protein of the present invention. If this is the case, step (ii) should further comprise the design of the genetic construct which comprises, besides the chimeric protein, the one or more further groups, residues, moieties or binding units.
• the host cell or host organism or cell free system may generally be kept, maintained and/or cultured under conditions such that the (desired) chimeric protein and/or polypeptide of the invention is optimally expressed/produced. Suitable conditions will be clear to the skilled person and will usually depend upon the host cell/host organism or cell free system used, as well as on the regulatory elements that control the expression of the chimeric protein and/or polypeptide of the invention.
• Suitable host cells or host organisms for production purposes will be clear to the skilled person, and may for example be any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism.
• Specific examples include HEK293 cells, CHO cells, 5. cerevisiae, Escherichia coli or Komagataella phaffii (Pichia pastoris).
• the host is Komagataella phaffii (Pichia pastoris).
• the host is Escherichia coli.
• the host is 5. cerevisiae.
• the method of the present invention further comprises a step (v) of screening for chimeric proteins which bind to at least one of the cytokine receptors or receptor subunits with increased or decreased affinity, as compared to the binding of the wild-type cytokine.
• the method of the present invention further comprises a step (v) of screening for chimeric proteins wherein cytokine comprised therein shows modified cytokine signaling as compared with the cytokine not fused to an ISVD, or for chimeric proteins which affect receptor or receptor's subunit oligomerization upon binding of the cytokine comprised therein to at least one of its receptors or receptor's subunits.
• the present invention further provides a method for modulating the activity of a cytokine by fusing (directly or by means of a linker, as described above) the cytokine to an ISVD to form a fusion protein.
• the cytokine or circularly permuted variant of the cytokine is inserted in the ISVD as described herein (e.g., as "Mega body” -type fusion, as described herein).
• the inventors have surprisingly found that, by fusing a cytokine to an ISVD (directly or by means of a linker, as described herein), as an internal fusion, results in altered or modified cytokine binding to its receptor or receptor subunits the activity (or altering downstream consequences of the binding of the cytokine to at least one of its receptors or receptor subunits, such as modified cytokine signaling and/or modified or affected receptor or receptor's subunit oligomerization upon binding of the cytokine, as compared with cytokines not fused to an ISVD).
• the inventors have surprisingly found that, by fusing a cytokine to an ISVD (directly or by means of a linker, as described herein) the receptorbinding functionality of the cytokine may be altered as compared with the receptor-binding functionality of the cytokine not fused to the ISVD.
• the binding affinity of a cytokine to at least one of its receptors or receptor subunits can be modulated (increased or decreased) by fusing the cytokine to an ISVD in a fusion protein.
• the downstream signalling generated by the interaction of the cytokine to at least one of its receptors or receptor subunits can also be modulated by fusing the cytokine to an ISVD.
• the efficacy of a cytokine/receptor interaction can also be modulated (increased or decreased) in this way, as well as the potency of the cytokine bound to the ISVD.
• receptor or receptor's subunit oligomerization can also be affected (modified, modulated) upon binding of the cytokine fused to the ISVD as compared with the receptor or receptor's subunit oligomerization upon binding of the cytokine not fused to the ISVD.
• the present invention provides a method for modulating the efficacy of the cytokine receptor, wherein the method comprises the step of fusing a cytokine to an ISVD (directly or by means of a linker, as described herein), or a method for modulating the downstream signalling generated by the interaction of the cytokine to at least one of its receptors or receptor subunits, or a method for modulating (modifying, affecting) receptor or receptor's subunit oligomerization upon binding of the cytokine fused to the ISVD.
• the method for modulating the activity of a cytokine of the present invention comprises the step of fusing a cytokine to an ISVD to form a chimeric protein, as described in this specification.
• the activity or downstream consequences/receptor or receptor's subunit oligomerization of the binding of the cytokine to at least one of its receptors or receptor subunits, as described above
• the cytokine can be modulated.
• the chimeric protein of the present invention may comprise a circularly permuted cytokine variant, such as a circularly permuted cytokine mutant (e.g., as exemplified herein using circularly permuted I L-2(K35E)) with different properties, which may also generate a modulation of the cytokine receptor efficacy.
• a circularly permuted cytokine variant such as a circularly permuted cytokine mutant (e.g., as exemplified herein using circularly permuted I L-2(K35E)) with different properties, which may also generate a modulation of the cytokine receptor efficacy.
• the method of the present invention may further comprise a step of screening for fusion or chimeric proteins which cytokine comprised therein shows a receptor-binding functionality different as compared with the receptor-binding functionality of the cytokine not fused to the ISVD, as described above.
• the screening step may comprise screening for fusion or chimeric proteins which cytokine comprised therein shows a different (increased or decreased) binding affinity to at least one of its receptors or receptor subunits, as compared with the binding affinity of the cytokine not fused to the ISVD.
• the screening step may comprise screening for fusion or chimeric proteins which cytokine comprised therein shows a different (increased or decreased) efficacy of the cytokine/receptor interaction, as compared with the efficacy of the cytokine not fused to the ISVD.
• the screening step may comprise screening for fusion or chimeric proteins which cytokine comprised therein generates a different (increased or decreased) downstream signalling when interacting with one of its receptors or receptor subunits, as compared with the downstream signalling generated by the interaction of the cytokine not fused to the ISVD with the same receptor or receptor subunit.
• the screening step may comprise screening for fusion or chimeric proteins which cytokine comprised therein generates a different receptor or receptor's subunit oligomerization upon binding of the cytokine fused to the ISVD as compared with the receptor or receptor's subunit oligomerization upon binding of the cytokine not fused to the ISVD.
• fusion of a cytokine to an ISVD may modify (e.g., by sterically hindering and/or conformational changes to the cytokine ligand induced by fusing it to the ISVD) the binding between the cytokine present in the chimeric protein and its receptor.
• a different downstream effect (or signalling) triggered by the binding of the cytokine to at least one of its receptors or receptor subunits may be obtained, as compared to the binding of a wild-type (not fused to the ISVD, e.g., not part of the fusion or chimeric protein) cytokine to at least one of its receptors or receptor subunits.
• the present invention provides a method for modulating the functionality of the cytokine receptor upon interaction with a cytokine fused (directly or by means of a linker, as described herein) to an ISVD (a fusion protein), as described herein, or upon interaction with the circularly permuted cytokine comprised in the chimeric protein of the present invention.
• the method comprises contacting the cytokine (fused to the ISVD, as described herein) with at least one of the cytokine's receptors or receptor subunits, to modulate the functionality of the cytokine receptor (e.g., to modulate the downstream signalling and/or receptor/receptor's subunit oligomerization generated by the binding of the cytokine with at least one of its receptors or receptor subunits).
• the present invention further comprises a method for modulating cytokine signaling comprising the steps of: providing the chimeric protein of the present invention; and screening for a chimeric protein or polypeptide wherein the cytokine comprised therein shows modified cytokine signaling as compared with the cytokine not fused to an ISVD.
• the present invention thus provides the use of a cytokine fused to an ISVD, or of the chimeric protein, or of the polypeptide of the present invention for modulating the activity of the cytokine comprised in the chimeric protein.
• the present invention provides the use of a cytokine fused to an ISVD, or of the chimeric protein, or of the polypeptide of the present invention for modulating the binding affinity of a cytokine to at least one of its receptors or receptor subunits.
• a cytokine fused to an ISVD, the chimeric protein, or the polypeptide of the present invention can be used to modulate the downstream signalling generated by the interaction of the cytokine to at least one of its receptors or receptor subunits.
• the present invention provides the use of a cytokine fused to an ISVD, of the chimeric protein, or of the polypeptide of the present invention for modulating the efficacy of a cytokine/receptor interaction, as well as for modulating the potency of the cytokine present in the chimeric protein.
• the present invention provides the use of a cytokine fused to an ISVD, of the chimeric protein, or of the polypeptide of the present invention for modulating the efficacy and/or the functionality of the cytokine receptor upon interaction with the chimeric protein of the present invention.
• the present invention also provides the use of a cytokine fused to an ISVD, or of the chimeric protein or polypeptide of the present invention for modulating the binding affinity of the cytokine to its receptor and/or for altering or modifying the cytokine signaling and/or for affecting, altering or modifying receptor oligomerization upon binding of the cytokine to at least one of its receptors or receptor subunits.
• the present invention further provides the chimeric protein or the polypeptide of the present invention for use in medicine.
• the chimeric protein or the polypeptide of the present invention may be used in the treatment of cancer and/or in the treatment of inflammatory diseases.
• the present invention thus provides the chimeric protein or the polypeptide of the present invention for use used in the treatment of cancer and/or in the treatment of inflammatory diseases.
• the cancer may be a solid and/or a liquid tumour.
• antigen-binding chimeric proteins also called herein Megabody® proteins, Megabody® protein constructs or Megabody® constructs
• these antigen-binding chimeric proteins can serve different applications.
• IL-2 Megabody proteins are built from ISVDs grafted onto interleukin-2 (IL-2).
• IL-2 interleukin-2
• the topology of the IL-2 molecule was investigated, and different sites were chosen to graft an ISVD.
• Example 1 Design and generation of a 29 kDa antigen-binding chimeric protein built from a circularly permuted variant of IL-2 inserted into the first p-turn connecting p-strands A and B of an anti-GFP ISVD.
• the ISVD used is an anti-GFP ISVD as depicted in SEQ ID NO: 1.
• the scaffold protein is a variant of IL-2 (SEQ ID NO: 3). All parts were connected to each other from the amino to the carboxy terminus in the next given order by peptide bonds: p-strand A of the anti-GFP ISVD (residues 1-12 of SEQ ID NO: 1), a short peptide linker (SEQ ID NO: 5 or SEQ ID NO: 120), a C-terminal part of IL-2(K35E,C125S) (SEQ ID NO: 3 from amino acid position X2 until amino acid position 133), a peptide linker to (SEQ ID NO: 6) connecting the C-terminus to an /V-terminal part of IL- 2(K35E,C125S) to produce a circularly permuted variant of the scaffold protein, an /V-terminal part of IL-2(K35E,C125S) starting from residue 4
• IL-2(K35E,C125S)_ISVD207 Megabody proteins displayed them on the surface of yeast (Boder, E. T., and Wittrup, K. D., "Yeast surface display for screening combinatorial polypeptide libraries", Nat Biotechnol, 1997, 15:553-557) and examined the specific binding of the cognate antigen (GFP) to yeast cells displaying this Megabody protein by flow cytometry.
• Each construct was introduced into the yeast strain EBY100 (5. cerevisiae), and EBY100 clones of each construct bearing a corresponding plasmid were grown and induced overnight by changing growing conditions from glucose-rich to galactose-rich media to trigger the expression and secretion of the IL-2(K35E,C125S)_ISVD207-Aga2p-ACP fusion protein.
• IL-2(K35E,C125S)_ISVD207-Aga2p-ACP fusion protein For the orthogonal staining of ACP, cells were incubated for 1 h in the presence a fluorescently labelled CoA analogue (coA-647, 2 pM) and catalytic amounts of the SFP synthase (1 pM).
• GFP does not bind to EBY100 yeast cells that have been stained in the same way but do not express the Megabody protein, neither to EBY100 yeast cells that express only the circularly permuted IL-2(K35E,C125S)[S75-Q74] (SEQ ID NO: 4).
• SEQ ID NO: 34 As a positive control cYgjk_ISVD207 Megabody protein in fusion to a number of accessory peptides and proteins, (SEQ ID NO: 34) was expressed and displayed on the surface of yeast cells.
• This Megabody protein is a chimeric polypeptide concatenated from parts of the anti- GFP ISVD and parts of YgjK, a 86 kDa periplasmic protein of E. coli (PDB 3W7S), according to Figure 1 to form a 100 kDa Megabody protein that was shown to bind GFP ( Figure 9).
• yeast cells expressing these IL- 2(K35E,C125S)_ISVD207 Megabody proteins were incubated lh in the presence of monoclonal antibody mAb5111-humanFc at a final concentration of 2 pg/ml.
• the epitope of mAb5111 on IL-2 is known (PDB 5UTZ), in some constructs the epitope of mAb5111-humanFc is not present and/or not accessible in the Megabody protein due to fact that ISVD207 is inserted in or nearthe epitope. Indeed the mAb5111-humanFc antibody could not bind to any of the IL-2(K35E,C125S)_ISVD207 Megabody protein constructs where ISVD207 was inserted near the epitope (AA16-31; AA70-AA86).
• Example 3 Binding of CD25 or CD122/CD132 to IL-2(K35E,C125S)_ISVD207 Megabody proteins. As binding of monoclonal antibody mAb5111-humanFc to IL-2(K35E,C125S)_ISVD207 Megabody proteins was confirmed for defined clones, we checked whether we could confirm binding of CD25 or CD122/CD132 to IL-2(K35E,C125S)_ISVD207 Megabody proteins displayed on the surface of yeast cells.
• Yeast cells expressing and displaying the different IL- 2(K35E,C125S)_ISVD207 Megabody proteins were incubated lh in the presence of CD25 protein with His tag (Human IL-2 R alpha Acrobiosystems #ILA-H52H9) at a final concentration of 4 pg/ml.
• yeast cells from the same batch expressing and displaying the different IL-2(K35E,C125S)_ISVD207 Megabody proteins were incubated 1 h in the presence of CD122/CD132 protein with His-tag (Human IL-2 R beta&IL-2 R gamma heterodimer protein, His tag & Twin strep tag, Acrobiosystems #ILG-H5283) at a final concentration of 4 pg/ml. All cells were washed 3 times and cells were incubated lh in the presence of a mouse Anti-His-PE antibody (miltenyibiotec / #130-120-718; 1/50 diluted), washed 3 times, and were analysed using flow cytometry.
• His-tag Human IL-2 R beta&IL-2 R gamma heterodimer protein, His tag & Twin strep tag, Acrobiosystems #ILG-H5283
• CD122/CD132 could hardly bind the IL-2(K35E,C125S)[L132- I129]_ISVD2O7 Megabody protein while CD122/CD132 binding was seen at IL- 2(K35E,C125S)[N77-S75]_ISVD207 Megabody protein.
• IL-2 Megabody proteins were designed with an anti-HSA ISVD or an anti-PD-Ll ISVD instead of the anti-GFP ISVD and were expressed in Pichia pastoris or CHO EBNALT85 and purified according to standard protocols.
• FLAG3HIS6 tag (SEQ ID NO: 35) was fused to the Mega body proteins at the C-terminus.
• HSA Human Serum Albumin
• GFP green fluorescent protein
• SPR Surface Plasmon Resonance
• mAb anti-FLAG M2 monoclonal antibody
• Binding to human PD-L1 was probed by Surface Plasmon Resonance (SPR) (Cytiva, Biacore 8K+, #2743662). Briefly, an anti-humanFc binder was immobilized on a CM5 sensor (Cytiva, BR100399) using standard amine coupling chemistry. Human PD-Ll-hFc was injected at 1 pg/mL at a flow rate of 10 pL/min for 180 seconds.
• SPR Surface Plasmon Resonance
• HSA human serum albumin
• ISVD ALB23002
• GFP ISVD207, table 6b
• PD- L1 ISVD10F11; table 6c.
• fold difference is calculated for the different compounds compared to the respective references.
• Table 6a Affinity of the IL-2(K35E, C125S)_ALB23002 Megabody proteins for HSA
• the data show that the ISVD moiety maintains its binding to the target when formatted in a Megabody construct.
• the maximal fold difference is 3 compared to the reference ISVD.
• the maximal fold difference is 2 and this is even lower for the ISVD-PD-L1 interaction further stressing that the ISVD is fully functional upon formatting into a Megabody construct.
• the IL-2 Megabody proteins were characterized for induction of STAT5 phosphorylation in different subsets of T cells to demonstrate their differential signaling profile depending on interaction properties with IL-2 receptors (I L-2Ra, I L-2R , I L-2Ry).
• STAT5 phosphorylation in CD8+ T cells, CD25-CD4+ T cells and CD25+FoxP3+CD4+ Treg cells was determined via flow cytometry.
• PBMCs isolated from healthy donors within 4 hours after blood drawing were retrieved from cryogenic storage and thawed in culture medium (RPMI 1640, Glutamax, 25mM Hepes, Gibco 72400-021 supplemented with 10% heat-inactivated FBS, Sigma F7524, 1 mM sodium pyruvate, Gibco 11360-039, MEM Non-Essential Amino Acids, Gibco 11140-035 and lx penicillin/streptomycin, Life Technologies 15140).
• PBMCs were rested overnight at 5,000,000 cells/mL in culture medium in a 5% CO2 atmosphere at 37°C.
• PBMCs were washed once with D-PBS (Gibco 14190) prior to staining with ZombieNIR fixable live/dead stain (Biolegend, 423105) for 15 min at RT in the dark. After a wash step with culture medium, 300,000 PBMCs were seeded per well of a 96-well U-bottom deepwell plate (Thermo Scientific 260251) in 250 pL culture medium and incubated for at least 30 min at 37°C in a 5% CO2 atmosphere. An equal volume of test compound with or without the addition of human serum albumin (HSA, final concentration 30 pM, CSL Behring 2160-679) was then added and cells were incubated for 15 min at 37°C.
• HSA human serum albumin
• Table 7a EC50 (M) of IL-2(K35E,C125S) Megabody proteins with HSA-binding ISVD in PBMC - pSTAT5 assay (Donor ABX-00168), in absence and presence of HSA
• fold difference is calculated between EC50(M) on IL-2R0y-expressing cells (average of CD8+ and CD4+CD25- T cells) versus IL-2Ra0y-expressing cells (CD4+CD25+ Treg cells).
• STAT5 STAT5
• TP027 and TP028 there is a fold difference of at least 100 between the potency on I L-2RPy- (CD8+ and CD4+CD25-) versus IL-2RaPy- (CD4+CD25+) expressing cells. This difference is higher for wildtype IL-2 (TP027), suggesting K35E and/or C125S impact the interaction with IL-2 receptors, mainly IL-2Ra.
• Table 8a EC50 (M) of IL-2 Megabody proteins with HSA-binding ISVD in PBMC - pSTAT5 assay (Donor ABL-0312-03), in absence and presence of
• Table 8b Fold difference in EC50 (M) between absence and presence of HSA of IL-2 Megabody proteins with HSA-binding ISVD in PBMC - pSTAT5 assay (Donor ABL-0312-03)
• IL-2 Megabody proteins with HSA-targeting ISVD induce phosphorylation of STAT5 in primary T cell subsets, with different profiles. Differences in EC50 values and the calculated fold difference versus previous experiment can be attributed to a difference between PBMC donors, overall trends and ranking are the same.
• TP027 there is a 61-fold difference between the potency on I L-2R(3y- versus IL-2RaPy-expressing cells.
• the ratio is below 3 indicating that IL-2Ra is not engaged for downstream signaling.
• reference compound 2 mainly interaction with cells that express ILRPy but not IL-2Ra is affected, as expected based on literature.
• Binding of the ISVD to its target, HSA can reduce potency, though in a compound-dependent manner. The biggest impact is observed forTP075, which shows a 40-fold reduced potency on both I L-2R V- and IL-2RaPy-expressing cells. Apart from the Megabody proteins, also the /V- terminal to C-terminal fused ISVD-cytokine construct (TP121) shows lower potency in presence HSA. Potency of the reference compounds, that do not contain an HSA-binding ISVD, was not affected by the presence of HSA in the assay, as expected.
• IL-2(K35E,C125S) Megabody proteins were formatted with a GFP binding ISVD (ISVD207) and compared with the HSA- binding ISVD - IL-2(K35E,C125S) Megabody proteins. Data is shown in Table 9.
• Table 9 EC50 (M) of IL-2(K35E,C125S) Megabody proteins with GFP- and HSA-binding ISVD in PBMC - pSTAT5 assay (Donor ABX-00168)
• IL-2(K35E,C125S) Megabody proteins with HSA-targeting ISVD Similar to the IL-2(K35E,C125S) Megabody proteins with HSA-targeting ISVD, also IL- 2(K35E,C125S) Megabody proteins with GFP-targeting ISVD (ISVD207) induce phosphorylation of STAT5 in primary T cell subsets, with different profiles.
• IL-2RPy- versus IL-2RaPy- expressing cells This difference is higher for wild-type IL-2, suggesting K35E and/or C125S impact the interaction with IL-2 receptors, mainly IL-2Ra.
• the ratio is below 3.
• TP031 and TP019 show lower fold difference between IL-2RPy- and IL-2RaPy-expressing cells compared to TP028, while TP030 and TP018 show higher fold difference between IL-2RPy- and IL-2RaPy-expressing cells compared to TP028.
• Example 6 Activity of the IL-2 Megabody proteins in a PBMC proliferation assay
• the IL-2 Megabody proteins were characterized for stimulation of proliferation of CD4+ and CD8+ T cells in a PBMC proliferation assay with Ki67 read-out. Additionally, CD25 was added to discriminate between IL-2Ra-positive and -negative populations. Ki67 is a nuclear protein that is associated with cell proliferation.
• PBMCs isolated from healthy donors were retrieved from cryogenic storage and thawed in thawing medium (RPMI 1640 Medium, GlutaMAXTM Supplement, HEPES (Life Technologies - Gibco, 72400-021), supplemented with 10% heat- inactivated FBS (Sigma F9665), and 1% penicillin/streptomycin (Life Technologies, 15140)).
• PBMCs were seeded at 300,000 cells/well in 100 pL culture medium (CTSTM OpTmizerTM T Cell Expansion SFM and OpTmizerTM T-Cell Expansion Supplement (Life Technologies - Gibco, A10221-01 - A10484-02), 2 mM L-Glutamine (Life Technologies - Gibco, A2916801), 5% CTSTM Immune Cell SR (Life Technologies - Gibco, A25961-01), and 1% penicillin/streptomycin (Life Technologies, 15140)) in a 96-well U-bottom plate (Costar, 3799).
• CTSTM OpTmizerTM T Cell Expansion SFM and OpTmizerTM T-Cell Expansion Supplement (Life Technologies - Gibco, A10221-01 - A10484-02)
• 2 mM L-Glutamine Life Technologies - Gibco, A2916801
• CTSTM Immune Cell SR Life Technologies - Gibco, A25961-01
• test compound An equal volume of test compound, with or without the addition of human serum albumin (HSA, final concentration 30 pM, CSL Behring 2160-679) was then added and cells were incubated for 6 days at 37°C in a 5% CO2 atmosphere. After this incubation, Ki67 expression in CD4+ and CD8+ T cells was determined by flow cytometry with CD25 as additional marker. Cells were transferred to a V- bottom plate (Greiner, 651180) and washed with D-PBS (Gibco, 14190) prior to staining with ZombieAqua fixable live/dead stain (Biolegend, 423102) for 15 min at RT.
• HSA human serum albumin
• Table 10a EC50 (M) of IL-2(K35E,C125S) Megabody proteins with HSA-binding ISVD in PBMC proliferation assay (Donor D1622), in absence and presence of HSA
• Table 10b Fold difference in EC50 (M) between absence and presence of HSA of IL-2(K35E,C125S) Megabody proteins with HSA-binding ISVD in
• IL-2(K35E,C125S) Megabody proteins with HSA-targeting ISVD are capable of driving proliferation of different immune cell populations (CD8+CD25-, CD4+CD25- and CD4+CD25+).
• TP027 there is a 160-fold difference between the potency on I L-2RPy- (CD8+CD25- and CD4+CD25-) versus IL-2RaPy- (CD4+CD25+) expressing cells.
• the ratio is below 3. No full dose response curve was obtained for reference compound 2 on CD8+CD25- and CD4+CD25- T cells while this compound maintained its potency on CD4+CD25+ T cells compared to wild-type human IL-2 (TP027).
• Table 11b Fold difference in EC50 (M) between absence and presence of HSA of IL-2 Megabody proteins with HSA-binding ISVD in PBMC proliferation assay (Donor 1622)
• IL-2 Megabody proteins with HSA-targeting ISVD are capable of driving proliferation of different immune cell populations (CD8+CD25-, CD4+CD25- and CD4+CD25+).
• IL-2RPy- CD8+CD25- and CD4+CD25-
• IL-2RaPy- CD4+CD25+
• An AlphaFold model for TP118 ( Figure 14 (SA17678)) shows that the ISVD is positioned towards the IL-2Ry protein thereby interfering with the IL-2-IL-2Ry interaction while retaining binding to IL-2Ra.
• An AlphaFold model for TP116 ( Figure 15 (SA17659) shows that the ISVD is positioned towards the I L-2R protein thereby interfering with the IL-2-IL-2RP interaction.
• IL-2 Megabody proteins different functional profiles are detected indicating that the insertion position of the ISVD into the cytokine can modulate the interaction of the cytokine with its receptors.
• Example 7 Binding of the IL-2 Megabody proteins to IL-2 receptors
• Binding to human IL-2Ra was probed by Surface Plasmon Resonance (SPR) (Cytiva, Biacore 8K+, #2743662). Briefly, an anti-humanFc binder was immobilized on a CM5 sensor (Cytiva, BR100399) using standard amine coupling chemistry. Human IL-2Ra was injected at 0.75, 1 or 10 pg/mL at a flow rate of 10 pL/min for 180 seconds.
• SPR Surface Plasmon Resonance
• test compounds were injected as a 7-point dilution series starting at 500 or 100 nM (dilution factor of 2.5) at a flow rate of 30 pL/min for 2 minutes to allow for binding to the target, followed by 10 minutes running buffer (Cytiva, BR100669, 10 mM HEPES, 150 mM NaCI, 3 mM EDTA, 0.005%(v/v) Surfactant P20, pH 7.4) allowing dissociation from the target.
• the chip was regenerated using 2 pulses of 30 seconds of 0.85% H3PO4 at 30 pL/min.
• the binding data were collected at 25 °C and analyzed according to a 1:1 binding fit model using the Insight Evaluation Software Version 3.0.12 supplied by the manufacturer (Cytiva).
• Binding to human IL-2RP was probed by Surface Plasmon Resonance (SPR) (Cytiva, Biacore 8K+, #2743662). Briefly, an anti-humanFc binder was immobilized on a CM5 sensor (Cytiva, BR100399) using standard amine coupling chemistry. Human I L-2R was injected at 5, 10 or 20 pg/mL at a flow rate of 10 pL/min for 180 seconds.
• SPR Surface Plasmon Resonance
• test compounds were injected as a 7-point dilution series starting at 500 or 250 nM (dilution factor of 2.5) at a flow rate of 30 pL/min for 2 minutes to allow for binding to the target, followed by 10 minutes running buffer (Cytiva, BR100669, 10 mM HEPES, 150 mM NaCI, 3 mM EDTA, 0.005%(v/v) Surfactant P20, pH 7.4) allowing dissociation from the target.
• the chip was regenerated using 2 pulses of 30 seconds of 0.85% H3PO4 at 30 pL/min.
• the binding data were collected at 25°C and analyzed according to a 1:1 binding fit model using the Insight Evaluation Software Version 3.0.12 supplied by the manufacturer (Cytiva).
• IL-2R6/y binding Binding human IL-2RP/y (human IL-2RB(ECD)(T191C)-THR-zipper, in-house produced was probed by Surface Plasmon Resonance (SPR) (Cytiva, Biacore 8K+, #2626160). Briefly, Strep- Tactin®XT (Iba Life Sciences, Twin-Strep-tag Capture kit, 2-4370-000) was immobilized on a CM5 sensor (Cytiva, BR100399) as described in the kit protocol. Human I L-2RP/y was injected at 0.75 pg/mL at a flow rate of 10 pL/min for 180 seconds.
• SPR Surface Plasmon Resonance
• test compounds were injected as a 7-point dilution series starting at 250 or 150 nM (dilution factor of 2.5) at a flow rate of 30 pL/min for 2 minutes to allow for binding to the target, followed by 10 minutes running buffer (Cytiva, BR100669, 10 mM HEPES, 150 mM NaCI, 3 mM EDTA, 0.005%(v/v) Surfactant P20, pH 7.4) allowing dissociation from the target.
• the chip was regenerated using 3 pulses of 70 seconds of 3M GuHCI at 30 pL/min.
• the binding data were collected at 25 °C and analyzed according to a 1:1 binding fit model using the Insight Evaluation Software Version 3.0.12 supplied by the manufacturer (Cytiva).
• Table 12a Kinetic parameters for interaction of IL-2 Megabody proteins with IL-2 receptor alpha. In addition, fold difference is calculated compared to reference TP027
• Table 12b Kinetic parameters for interaction of IL-2 Megabody proteins with IL-2 receptor beta. In addition, fold difference is calculated compared to reference TP027
• Table 12c Kinetic parameters for interaction of IL-2 Megabody proteins with IL-2 receptors beta/gamma. In addition, fold difference is calculated compared to reference TP027
• IL-2 Megabody proteins show differential binding profiles towards the cytokine receptors.
• Compounds TP115, TP116 and TP119 show significant reduced affinity towards IL-2Ra.
• Compound TP116 also has reduced affinity for I L-2R , though can still bind on I L-2RP/y heterodimer.
• a similar trend is observed for compound TP118.
• the differential binding towards the IL-2RP/y heterodimer is less pronounced in the current assay.
• these different binding profiles indicate that the insertion position of the ISVD into the cytokine can modulate the interaction of the cytokine with its receptors.
• Example 8 Activity of the IL-2 Megabody proteins in a Tetanus toxoid (TT) recall assay
• the multivalent constructs i.e., Megabody proteins fused to an ISVD, were tested for primary T cell activation (monitored via IFNy production) in an autologous Tetanus Toxoid recall assay.
• PBMCs isolated from healthy donors were retrieved from cryogenic storage and thawed in culture medium (RPMI 1640 Medium, GlutaMAXTM Supplement, 25mM HEPES (Life Technologies - Gibco, 72400-021) supplemented with 10% heat-inactivated FBS (Sigma F9665) and 1% penicillin/streptomycin (Life Technologies, 15140).
• Monocytes were isolated by negative magnetic separation according to the instructions of the supplier of the EasySep human monocyte isolation kit (Stemcell Technologies, 19359) and cultured for 7 days in human Mo-DC differentiation medium (Miltenyi Biotec, 130-094-812) with intermediate addition of an equal volume of fresh medium on day 3. After 7 days, the monocytes had differentiated to immature dendritic cells (i DC). The iDCs were harvested and frozen in liquid nitrogen for later use.
• PBMCs were thawed in assay medium (RPMI 1640 Medium, GlutaMAXTM Supplement, 25 mM HEPES (Life Technologies-Gibco, 72400-021) supplemented with 10% heat-inactivated human AB serum (BiolVT, SM-612-HSI) and 1% penicillin/streptomycin) and cultured for 7 days in assay medium supplemented with 0.5 pg/mL Tetanus Toxoid (TT) (Calbiochem, 582231) at 37°C in 5% CO2 atmosphere. After 7 days, the TT-specific T cells were enriched. The cells were harvested and frozen in liquid nitrogen for later use.
• assay medium RPMI 1640 Medium, GlutaMAXTM Supplement, 25 mM HEPES (Life Technologies-Gibco, 72400-021) supplemented with 10% heat-inactivated human AB serum (BiolVT, SM-612-HSI) and 1% penicillin/streptomycin
• assay medium
• iDCs were thawed in assay medium, seeded in 96-well U-bottom plates (Corning, 3799) at 5000 cells/well in assay medium supplemented with 0.5 pg/mL TT and incubated for 4 hours in a 5% CO2 atmosphere at 37°C.
• Autologous TT-enriched T cells were thawed in assay medium, 100,000 cells/well were added to the iDCs, and a dilution series of the multivalent constructs was added. After 3 days of co- culture, cell supernatant was harvested and IFNy concentration was determined via ELISA.
• Example 9 Design of circularly permuted variant of interferon alpha-2a (IFNA2a).
• IFNA2a_NbALB23 Megabody proteins To be able to design IFNA2a_NbALB23 Megabody proteins), a well-folded circularly permuted version of the interferon alpha-2a (IFNA2a) is required.
• IFNA2a interferon alpha-2a
• PBD 1ITF, 3S9D
• peptide linkers were introduced to connect the C- terminus of IFNA2a to its /V-terminus.
• 2 constructs were made and cloned into a yeast display vector with all the accessory proteins as described before to be able to display the circularly permuted variants on the surface of EBY100 yeast cells.
• IFNA2a[D77-W76]V2 SEQ ID NO: 58.
• Example 10 Binding of a specific anti-IFNA2a monoclonal to interferon alpha-2a and its circularly permuted variants.
• the circularly permuted versions of IFNA2a were expressed and displayed on the surface of EBY100 yeast cells.
• yeast cells expressing the different constructs were incubated lh in the presence of monoclonal antibody mAb93452 (Human IFN-alpha 2/IFNA2a Antibody, R&D systems: MAB93452) at a final concentration of 2.5 pg/ml.
• mAb93452 Human IFN-alpha 2/IFNA2a Antibody, R&D systems: MAB93452
• EBY1OO yeast cells not expressing any construct were also incubated lh in the presence of monoclonal antibody mAb93452 (Human IFN-alpha 2/IFNA2a Antibody, R&D systems: MAB93452) and serve as the negative control. After 3 washes, all cells were incubated lh in the presence of 2 pg/ml Anti-mouse IgG Fc (Phycoerythrin-conjugated AffiniPure Goat Anti-mouse IgG Fc gamma Specific, Jackson Immuno Research), they were washed 3 times, and cells were analysed using flow cytometry.
• monoclonal antibody mAb93452 Human IFN-alpha 2/IFNA2a Antibody, R&D systems: MAB93452
• MAB93452 Human IFN-alpha 2/IFNA2a Antibody, R&D systems: MAB93452
• Example 11 Binding of IFNAR2 to interferon alpha-2a and its circularly permuted variants.
• IFNA2a The circularly permuted versions of IFNA2a were expressed and displayed on the surface of EBY100 yeast cells.
• yeast cells expressing the different constructs were incubated lh in the presence of a his tagged IFNAR2 (Human IFN-alpha/beta R2 protein, His tag, Acrobiosystems) at a final concentration of 4 pg/ml.
• IFNAR2 Human IFN-alpha/beta R2 protein, His tag, Acrobiosystems
• EBY100 yeast cells not expressing any construct were also incubated lh in the presence IFNAR2 (Human IFN-alpha/beta R2 protein, His tag, Acrobiosystems) and serve as the negative control. After 3 washes, all cells were incubated 1 h in the presence of mouse Anti-His antibody-PE (miltenyibiotec / #130-120-718; 1/50 diluted), they were washed 3 times, and cells were analysed using flow cytometry.
• IFNAR2 Human IFN-alpha/beta R2 protein, His tag, Acrobiosystems
• yeast cells displaying either IFNA2a (SEQ ID NO: 56), IFNA2a[D77-W76]V2 (SEQ ID NO: 58) or IFNA2[D77- W76]V4 (SEQ ID NO: 59) was seen after incubation with IFNAR2 ( Figure 19), while a smaller shift in fluorescence was seen to the EBY1OO yeast cells not expressing any construct.
• Example 12 Design of a 31 kDa antigen-binding chimeric protein built from a circularly permuted variant of interferon alpha-2a inserted into the first p-turn connecting p-strands A and B of an anti-HSA ISVD.
• ISVD207 Megabody proteins and knowing that we can make a circularly permuted variant of IFNA2a, we also designed ISVD molecules that were grafted onto interferon alpha-2a (IFNA2a).
• interferon alpha-2a (IFNA2a PDB 1ITF, 3S9D, Figure 17) was examined and different sites were chosen to graft ISVD ALB23002 on. Twenty-three different versions of IFNA2_ALB23 Megabody proteins were created.
• the 31 kDa Megabody proteins described here are chimeric polypeptides concatenated from parts of a single-domain immunoglobulin and parts of a scaffold protein connected according to Figure 1.
• the immunoglobulin domain used is an anti-HSA ISVD as depicted in SEQ ID NO: 55.
• the scaffold protein is IFNA2a (SEQ ID NO: 56).
• Example 13 Binding of the IFNA2a Megabody proteins to IFNa receptor IFNAR2 and ISVD target human serum albumin (HSA)
• Binding to human IFNAR2 (Sino Biological, 10359-H02H) was probed by Surface Plasmon Resonance (SPR) (Cytiva, Biacore 8K+, #2626160). Briefly, an anti-humanFc binder was immobilized on a CM5 sensor (Cytiva, BR100399) using standard amine coupling chemistry. Human IFNAR2 was injected at 1 or 20 pg/mL at a flow rate of 10 pL/min for 180 seconds.
• SPR Surface Plasmon Resonance
• test compounds were injected as a 7-point dilution series starting at 500, 100, 50 or 25 nM (dilution factor of 2.5) at a flow rate of 30 pL/min for 2 minutes to allow for binding to the target, followed by 10 minutes running buffer (Cytiva, BR100669, 10 mM HEPES, 150 mM NaCI, 3 mM EDTA, 0.005%(v/v) Surfactant P20, pH 7.4) allowing dissociation from the target.
• the chip was regenerated using 2 pulses of 30 seconds of 0.85% H3PO4 at 30 pL/min.
• the binding data were collected at 25°C and analyzed according to a 1:1 binding fit model using the Insight Evaluation Software Version 3.0.12 supplied by the manufacturer (Cytiva).
• HSA human serum albumin
• SPR Surface Plasmon Resonance
• test compounds were injected as a 9-point dilution series starting at 2500 nM (dilution factor of 2.5) at a flow rate of 30 pL/min for 2 minutes to allow for binding to the target, followed by 10 minutes running buffer (Cytiva, BR100669, 10 mM HEPES, 150 mM NaCI, 3 mM EDTA, 0.005%(v/v) Surfactant P20, pH 7.4) allowing dissociation from the target.
• the chip was regenerated using 2 pulses of 30 seconds of 10 mM Glycine pH 1.5 at 30 pL/min.
• the binding data were collected at 25 °C and analyzed according to a 1:1 binding fit model using the Insight Evaluation Software Version 3.0.12 supplied by the manufacturer (Cytiva).
• the aim of the Megabody construct designs was to modulate the interaction of IFNA2a with its high affinity receptor IFNAR2, while maintaining interaction of the ISVD with its target. Scanning through the IFNA2a cytokine results in a spectrum of affinities (KD) towards IFNAR2, with affinities that are higher (Cluster C) or lower (Cluster B) compared to reference compound TP088. Compounds with an affinity similar to TP088 are grouped in Cluster A.
• a KD of 1-10 nM is anticipated. Binding of the ISVD with its target, HSA, is not hampered by formatting it into an IFNA2a Megabody protein. For all Megabody proteins, binding affinity towards HSA is within a 3.5-fold difference compared to reference compound TP113.
• Example 14 Activity of the IFNA2a Megabody proteins in a STAT1 phosphorylation assay
• A549 cells Human Lung Carcinoma, ATCC CCL-185
• STAT1 phosphorylation was determined via flow cytometry.
• A549 cells were grown in culture medium (Ham's F-12K (Kaighn's) medium supplemented with 10% heat-inactivated FBS; Life Technologies - Gibco 21127 and Sigma F9665 respectively) in a 5% CO2 atmosphere at 37°C.
• A549 cells were harvested, washed with D-PBS (Gibco 14190) prior to staining with ZombieNIR fixable live/dead stain (Biolegend, 423105) for 15 min at RT in the dark.
• D-PBS Gibco 14190
• ZombieNIR fixable live/dead stain Biolegend, 423105
• 150,000 cells were seeded per well of a 96-well U-bottom plate (Costar 3799) in 75 pl culture medium and incubated for at least 30 min at 37°C in a 5% CO2 atmosphere.
• HSA human serum albumin
• Example 15 Anti-proliferative activity of IFNA2a Megabody proteins on RPMI 8226 and NCI- H929 cells
• the Megabody proteins were characterized for their anti-proliferative effect on RPMI 8226 (Human B lymphocyte cell line from Plasmacytoma, ATCC CCL-155) and NCI-H929 cells (Human myeloma cell line, DSMZ ACC 163).
• RPMI 1640 Glutamax, 25 mM Hepes, Gibco 72400-021 supplemented with 10% heat- inactivated FBS, Sigma F9665, 1 mM sodium pyruvate, Gibco 11360-039, and 1% penicillin/streptomycin, Gibco 15140-122) and NCI-H929 cells (RPMI 1640, Glutamax, 25 mM Hepes, Gibco 72400-021 supplemented with 10% heat-inactivated FBS, Sigma F9665, 1 mM sodium pyruvate, Gibco 11360-039, 50 pM beta-mercaptoethanol, Gibco 21985-023, and 1% penicillin/streptomycin, Gibco 15140-122).
• Example 16 Design of circularly permuted variants of interleukin 18.
• I L18 interleukinl8
• PBD 1JOS, 3WO4, 3F62
• peptide linkers were introduced to connect the C- terminus of I L18 to its /V-terminus.
• 3 constructs were made and cloned into a yeast display vector with all the accessory proteins as described before to be able to display the circularly permuted I L18 variants on the surface of the yeast cell.
• the first 3 amino acids of I L18 were deleted and an 11 amino acid peptide linker (SEQ ID NO: 65) was introduced to connect the C-terminus of IL18 to its truncated /V-terminus to produce a circularly permuted variant of IL18 called IL18 [K70-E69]Vlb (SEQ ID NO: 66).
• IL18 [K70-E69]Vlb SEQ ID NO: 66
• the C-terminus of I L18 was connected to the /V-terminus by a 12 amino acid peptide linker (SEQ ID NO: 67) to connect the C-terminus of I L18 to its /V-terminus, this variant was called IL18[K70-E69]V5b (SEQ ID NO: 68).
• the circularly permuted versions of IL18 were expressed and displayed on the surface of EBY1OO yeast cells.
• yeast cells expressing the different constructs were incubated 1 h in the presence of monoclonal antibody D044-3 (mAbD044-3; Human IL18/IL-1F4 Antibody clone #125-2H, PDB 2VXT, R&D systems: D044-3) at a final concentration of 2 pg/ml in FACS buffer (PBS containing 1 % BSA).
• EBY100 yeast cells expressing ISVD207 were also incubated 1 h in the presence of monoclonal antibody D044-3 (Human I L18/I L-1F4 Antibody clone #125- 2H, R&D systems: D044-3) and serve as the negative control for the mAbD044-3 binding.
• monoclonal antibody D044-3 Human I L18/I L-1F4 Antibody clone #125- 2H, R&D systems: D044-3
• Example 18 Binding of IL18-binding protein to IL18 and to circularly permuted variants.
• IL18[K70-E69]Vlb SEQ ID NO: 66
• IL18[K70-E69]V5b SEQ ID NO: 68
• IL18[K70-E69]V7 SEQ ID NO: 70
• Yeast cells were incubated 1 h in the presence of Recombinant Human IL-18-binding protein (IL18-BP; Recombinant Human IL-18 BPa Fc Chimera Protein, R&D systems: 119-BP) at a final concentration of 1 pg/ml in FACS buffer (PBS containing 1% BSA).
• IL18-BP Recombinant Human IL-18 BPa Fc Chimera Protein, R&D systems: 119-BP
• ISVD207 SEQ ID NO:1
• Example 19 Design of a 33 kD antigen-binding chimeric protein built from a circularly permuted variant of interleukin 18 inserted into the first p-turn connecting p-strands A and B of an anti-GFP ISVD.
• Mb proteins SEQ ID NO: 230-237) where an ISVD molecule was grafted onto circularly permuted IL18.
• linker SEQ ID NO: 67
• interleukin 18 I L18 PDB 3WO4, 3F62 ( Figure 30), 1J0S
• the topology of interleukin 18 was examined and different sites were chosen to graft ISVD207 on.
• Eight different versions of IL18JSVD207 Megabody proteins were created (SEQ ID NO: 230-237).
• the 33 kD Megabody proteins described here are chimeric polypeptides concatenated from parts of a single-domain immunoglobulin and parts of a scaffold protein connected according to Figure 1.
• the immunoglobulin domain used is an anti-GFP ISVD as depicted in SEQ ID NO: 1.
• the scaffold protein is I L18 (SEQ ID NO: 64).
• the parts of the 33 kD Megabody proteins were connected to each other without short peptide linkers between the anti-GFP ISVD and the I L18 scaffold: the parts were connected to each other from the amino to the carboxy terminus in the next given order by peptide bonds: p-strand A of the anti-GFP ISVD (residues 1-12 of SEQ ID NO: 1), a C-terminal part of IL18 (SEQ ID NO: 64 from amino acid position X2 until amino acid position 157) a peptide linker (SEQ ID NO: 67) to connect the C-terminus of I L18 to its /V-terminus (SEQ ID NO: 64 amino acid 1) to produce a circularly permuted variant of the IL18 scaffold protein, an /V- terminal part of I L18 (SEQ ID NO: 64 from amino acid position 1 until amino acid position XI), followed by the P-strands B to G of the anti-GFP ISVD (residues 17-126
• Example 20 Binding of an anti-IL18 monoclonal to the IL18JSVD207 Megabody proteins
• yeast cells expressing these IL18JSVD207 Megabody proteins were incubated 1 h in the presence of monoclonal antibody D044-3 (mAbD044-3; Human IL18/IL-1F4 Antibody clone W125-2H, R&D systems: D044-3) at a final concentration of 2 pg/ml.
• EBY100 yeast cells not expressing any construct were also incubated 1 h in the presence of monoclonal antibody D044-3 and will serve as negative (ISVD207 & empty vector) and positive (IL18 & IL18[K70-E69]V5b) controls.
• IL18JSVD207 Megabody proteins As binding of monoclonal antibody D044-3 to IL18JSVD207 Megabody proteins was confirmed for defined clones, we checked whether we could confirm binding of the IL18-BP to IL18JSVD207 Megabody proteins displayed on the surface of yeast cells. Yeast cells expressing and displaying the different IL18JSVD207 Megabody proteins were incubated 1 h in the presence of recombinant IL18-BP (Recombinant Human IL-18 BPa Fc Chimera Protein, R&D systems: 119-BP) at a final concentration of 1 pg/ml.
• IL18-BP Recombinant Human IL-18 BPa Fc Chimera Protein, R&D systems: 119-BP
• EBY100 yeast cells not expressing any construct or expressing ISVD207 were also incubated 1 h in the presence recombinant IL18-BP (Recombinant Human IL-18 BPa Fc Chimera Protein, R&D systems: 119-BP) and serve as the negative control.
• EBY100 yeast cells expressing IL18 (SEQ ID NO: 64) or expressing IL18[K70-E69]V5b (SEQ ID NO: 68) were run in parallel and will serve as positive controls.
• the cells were washed 3 times and cells were incubated 1 h in the presence of Anti-human IgG Fc-PE (Phycoerythrin-conjugated AffiniPure F(ab)2 Fragment Goat Anti-Human IgG Fey Fragment Specific, Jackson Immuno Research: 109-116-170), the cells were washed 3 times and were analysed using flow cytometry. We observed detectable amounts of fluorescence bound to yeast cells displaying IL18 (SEQ ID NO: 64) or IL18[K70- E69]V5b (SEQ ID NO: 68) while no fluorescence was noticed in the negative controls.
• IL18 SEQ ID NO: 64
• IL18[K70- E69]V5b SEQ ID NO: 68
• IL18-BP could hardly bind the IL18[Q56-S55]_ISVD207 Megabody protein while the monoclonal antibody D044-3 (mAbD044-3; Human I L18/I L-1F4 Antibody clone W125-2H, R&D systems: D044-3) could clearly recognize the IL18 part within this construct.
• the IL18[K70-E69]_ISVD207 Megabody protein the binding of the IL18-BP was confirmed as well as binding to the monoclonal antibody D044-3 showing that not all IL18_ ISVD207 Megabody proteins behave the same.
• yeast cells each displaying a particular IL18JSVD207 Megabody protein were analysed by flow cytometry: yeast cells were incubated 1 h in the presence of 100 nM GFP (Scholz et al., 2000). After washing these cells with FACS buffer, we observed detectable amounts of GFP bound to different displayed IL18JSVD207 Megabody proteins.
• GFP does not bind to EBY100 yeast cells that were transformed with an empty vector and that have been stained in the same way but do not express the Megabody protein, neither to EBY100 yeast cells that express only I L18 (SEQ ID NO:64) or the circularly permuted IL18[K70-E69]V5b (SEQ ID NO:68).
• the HopQ_ISVD207 Megabody protein SEQ ID NO: 239) also in fusion to a number of accessory peptides and proteins, was expressed and displayed on the surface of yeast cells.
• This Megabody protein is a chimeric polypeptide concatenated from parts of the anti-GFP ISVD and parts of HopQ, a 43 kD circularly permutated variant of the Adhesin domain of HopQ of H. pylori (PDB 5LP2), according to Figure 1 to form a 58 kD Mb that was shown to bind GFP (Figure 40).
• PDB 5LP2 Adhesin domain of HopQ of H. pylori
• the acyl carrier protein in the IL18JSVD207 Megabody protein fusion was biotinylated as follows: 2xlOE9 yeast cells of each construct were collected and washed 3 times with PBS containing 1% BSA; next the cells were collected and incubated with 10 p.M Biotin-PEG3-CoenzymeA and 1 p.M SFP synthase in 50 mM HEPES pH 7.4, 10 mM MgCl2 and 0,1% BSA (10% stock) for lh at room temperature.
• yeast cell pellet of each construct were recovered and were resuspended in 0.5 ml 20 mM HEPES, pH 7.5, 150 mM NaCI, 2 mM DTT and incubated lh with head-over-head turning at 4°C. After spinning the tubes, the supernatant of each construct was collected and filtered through a 0.22 pM filter to remove residual cells.
• binding measurements were performed via Biolayer Interferometry (BLI) using an Octet R8 and Flat Bottom 96-well plates (Greiner, cat. no. 655076). During the experiment plates were kept at 25°C and shaken at 1000 rpm. Before use, streptavidin biosensors (Octet SA Biosensors, Sartorius 18-5019) were hydrated in PBS pH 7.4, 0.01 % Tween 20, 0.01 % BSA for 15 minutes.
• the SA biosensors were equilibrated in 200 pl of the same buffer, next the SA sensors were plunched in separate wells each filled with a 200 pl solution containing a particular biotinylated IL18JSVD207 Megabody protein and sensors were loaded till 1, 1-1,2 nm is reached. Subsequently the SA sensors loaded with the different biotinylated IL18JSVD207 Megabody proteins were incubated in wells containing 200 pl with 2 pg/ml of GFP. Binding to GFP was recorded.
• the cHopQ_ISVD207 Megabody (SEQ ID NO: 239) also in fusion with the acyl carrier protein and a number of accessory peptides, was used to confirm the binding to GFP.
• GFP does not bind to IL18[K70-E69]V5b (SEQ ID NO: 68) that was expressed and biotinylated in the same way as the IL18JSVD207 Megabody proteins (SEQ ID NO: 230-233; SEQ ID NO: 236-237) where binding of the IL18JSVD207 Megabody proteins to GFP was confirmed (Figure 42).
• Example 24 Binding of IL18[K70-E69]_ISVD207_V2 Megabody protein to GFP
• an affinity determination was done on the IL18[K70-E69]_ISVD207_V2 Megabody protein (SEQ ID NO: 233) using BLI on the Octet R8 and Flat Bottom 96-well plates as described before. Briefly, the biotinylated IL18[K70-E69]_ISVD207_V2 Megabody protein was loaded on 8 SA sensors till l,lnm was reached.
• the IL18[K70-E69]_ISVD207_V2 loaded SA sensors were incubated in different concentrations of GFP: 33.3, 11.1, 3.7, 1.23, 0.41, 0.137 and 0.46 nM in PBS pH 7.4, 0.01 % Tween 20, 0.01 % BSA allowing GFP to bind to IL18[K70- E69]_ISVD207_V2. After 15 minutes, dissociation started by moving the sensors to wells only containing PBS pH 7.4, 0.01 % Tween 20, 0.01 % BSA.
• Example 25 In vivo pharmacokinetics and pharmacodynamics profiles of IL-2 Megabody proteins in naive female C57BI/6N mouse
• IL-2 Megabody proteins with HSA-targeting ISVD having the same Megabody construction as of TP118 (IL-2[L132-I129]_ALB23OO2 Megabody protein), TP119 (IL-2[F42-M39]_ALB23002 Megabody protein) and TP121 (IL-2-35GS-ABL23002) have been produced in the production host Komagataella phaffii (De Groeve et al., "Optimizing expression of Nanobody® molecules in Pichia pastoris through co-expression of auxiliary proteins under methanol and methanol- free conditions", Microbial Cell Factories, 2023, 22:135), a well-known for production for ISVD(-based) molecules (Matsuzaki et al., "Production of Single-Domain Antibodies in Pichia pastoris", Methods in Molecular Biology (Clifton, N.J.), 2022, 2446:181-203).
• TP206 SEQ ID NO.: 265
• TP207 SEQ ID NO.: 266
• TP208 SEQ ID NO.: 260
• TP206 and TP207 have been tested in vivo after a single intravascular bolus dose in naive female C57BI/6N mice.
• Compound TP208 was used as a comparator.
• the pharmacodynamic (RD) and pharmacokinetic (PK) profiling of the different compounds were performed in C57BI/6N mice (Charles River Laboratories Germany GmbH, Freiburg, Germany). Animal welfare policies were implemented as by GV-SOLAS guidelines and by animal licenses in place with local veterinary authorities (Reg michsprasidium Freiburg, Germany). All animals, between 10 to 14 weeks old at the start of the experiment, were weighted before grouping and treatment. The body weight was used as numeric parameter to randomize selected animals into specified groups, with 12 animals per group.
• test item stock solutions and vehicles were thawed at +25°C using a water bath and swirled gently for 5-10 minutes.
• Stock solutions were diluted in the appropriate volume of commercial and sterile D-PBS under laminar flow.
• the compounds were administered to the animals via a single intravenous (bolus) injection to the tail vein, without sedation.
• Blood was collected by retro-bulbar sinus puncture under isoflurane anesthesia at three different timepoints (3 animals at 24 h, 3 animals at 48h, and 6 animals at 72h). Blood was collected in lithium Heparin coated tubes.
• whole blood was processed to plasma by a first centrifugation, at 300 g for 5 min at 2-8°C. After centrifugation the resulting plasma and the cell fraction were collected separately.
• the blood cell fraction was resuspended immediately and gently in PBS (same volume as the plasma volume removed), put on ice and processed immediately with fixation and flow cytometry protocol.
• the plasma fraction was submitted to a second centrifugation, at 2000 g for 5 min at 2-8°C, and transferred to a labeled polypropylene tubes, snap frozen in liquid nitrogen and stored frozen at -80°C.
• An assay was set up to measure the IL-2 Megabody proteins in plasma fractions.
• a streptavidin-coated MSD GOLD 96-well SMALLSPOT® plate (Meso Scale Discovery L45SA) was blocked with SuperBlockTM blocking buffer (ThermoFisher Scientific 37515) for 1 hour at room temperature (RT).
• the plate was then washed with PBS/0.05%Tw20 and incubated for 2 hours at RT and 600 rpm with 3.0 pg/mL biotinylated rabbit anti-human IL-2 polyclonal Ab (pAb, Bioscience 13-7028-85), directed against the IL-2 building block of the constructs.
• Calibrators and Quality Controls were prepared in pooled mouse plasma. After washing, calibrators, QCs and study samples were applied at a minimum required dilution (MRD) of 100 in PBS/0.1% casein (Biorad 161-0783) and incubated for 2 hours at RT and at 600 rpm. After washing, the plate was incubated for 1 hour at RT and at 600 rpm with 2.0 pg/mL sulfo- labelled ABH0085 mAb (Sanofi proprietary antibody), a mAb directed against the ALB23002 ISVD component of the IL-2 Megabody proteins.
• MRD minimum required dilution
• MSD Read buffer A (Meso Scale Discovery R92TG) was added and ECL values were measured with a Sector Imager Quickplex SQ 120 (Meso scale Discovery).
• Calibration curve responses were processed using a 5PL - 1/Y 2 weighted fit (with log(X) transformation) of electrochemiluminescence (ECL) responses versus concentrations. The concentrations of calibrators, QC and study samples (reported values) were calculated by interpolation based on the fit of the calibration curve.
• the pharmacodynamics readouts are pSTAT5 (receptor occupancy and early signaling), cell proliferation followed by expression of the molecular marker of proliferation (Ki67), and cell counts read by cytometry.
• the blood cell samples were treated with 1 mL of pre-warmed Lyse/Fix Buffer (BD Bioscience 558049) and incubated for 15 min at RT. After 5 wash steps with 1 mL FACS buffer (D-PBS, Gibco 14190 supplemented with 2% of heat-inactivated FBS, PAA A21-102, and 0,05% sodium azide) at 4°C, cells were transferred to a 96-well conical bottom plate and stored at 4°C until staining was performed.
• FACS buffer D-PBS, Gibco 14190 supplemented with 2% of heat-inactivated FBS, PAA A21-102, and 0,05% sodium azide
• the cells were resuspended in 50 pL/well of a 10 pg/mL solution of purified Rat Anti-Mouse CD16/CD32 (BD Bioscience 553142), and stained with a twofold concentrated mixture of anti-mouse FOXP3-Alexa Fluor® 488 (Invitrogen 53-5773-82), anti-mouse Ki67 Brilliant Violet 510TM (BD Biosciences 563462), anti-mouse phosphoSTAT5 (pY694)-PE (BD Bioscience 562077) diluted in FACS buffer for 50 min at RT under slow shaking (300 g).
• Flow cytometry data were analyzed with the FlowJo Data Analysis Software using relevant unstained and FMO samples to detect and exclude unspecific background signals and to set gates. Doublet exclusion was performed according to forward scatter height versus forward scatter area to include only single cells, followed by forward/sideward scatter to exclude debris and, finally live/dead discrimination. Controls (unstained and Fluorescence Minus One) were performed on a pool of cells. Further gating was performed to assess the different immune cell populations. The data for the phosphorylated STAT5 ( Figure 45) and for Ki67 ( Figure 46) were quantified by % positive in the different cell populations.
• Graphs were done using GraphPad Prism software (Version 10.1.2 (324)). At 72 h, for each marker (Ki67, % cells and ratio) and each subset of cells, a one-way analysis of variance (ANOVA) with group as fixed factor was performed on values after a log transformation. The statistical analysis compared each treated groups versus vehicle group, and TP208 control group versus TP207 and TP206. For each objective, it was followed by a contrast analysis with Bonferroni-Holm adjustment for multiplicity by objective. The ratio and associated 95% confidence interval (Cl) were estimated from the statistical model after back-transformation. The analysis was performed using SAS® version 9.4. Values of P ⁇ 0.05 were considered significant.
• TP206 and TP207 IL-2 Megabody proteins when combined with HSA-targeting ISVD, exhibited comparable plasma PK profiles to control IL-2 fused HSA-targeting ISVD (TP208) ( Figure 44). These PK profiles aligned with the anticipated extension of blood half-life achieved through albumin binding in mouse species (Hoefman et al., "Pre-clinical intravenous serum pharmacokinetics of albumin binding and non-half-life extended nanobodies®", Antibodies, 2015,4(3):141-156). The pSTAT5 induction in blood CD8+ T cells has been described to return to baseline after only 2 h in mice dosed with recombinant human IL-2 (Ptacin et al.
• TP207 showed higher pSTAT5 induction in CD8+ T cells (CD3+CD4-CD8+), naive CD4+ T cells (CD3+CD4+CD8-CD25-Foxp3-) and NK cells (CD3-NK1.1+), and lower pSTAT5 induction in Treg cells (CD3+CD4+CD8-CD25+Foxp3+) compared to the control TP208 ( Figure 45).
• Ki67 activation is significantly higher for TP207 at 72 h in CD8+ T cells, naive CD4+ T cells and NK cells, and significantly lower in Treg cells compared to the control TP208.
• the present invention provides the following items:
• a chimeric protein which comprises an immunoglobulin single variable domain (ISVD) fused with a cytokine, wherein an internal fusion site of the ISVD is linked to the cytokine, wherein, in the ISVD, the internal fusion site is located in a loop or turn between two secondary structure elements.
• ISVD immunoglobulin single variable domain
• cytokine is a circularly permuted cytokine, wherein the internal fusion site of the ISVD is linked to an internal fusion site of the cytokine and wherein, in the cytokine, the internal fusion site is located in a loop or turn between two secondary structure elements.
• chimeric protein according to any one of items 3 to 4, wherein the tertiary structure of the ISVD and of the cytokine in the chimeric protein is maintained except for the structure of the internal fusion sites which link the ISVD and the cytokine.
• chimeric protein according to any one of items 1 to 5, wherein, in the chimeric protein, the amino acids positioned /V-terminally from the internal fusion site of the ISVD are connected at the C-terminal ending to the /V-terminus of the cytokine (or circularly permuted cytokine) protein, and the amino acids positioned C-terminally of the internal fusion site are connected with the C-terminus of the cytokine (or circularly permuted cytokine), to form the continuous amino acid sequence.
• chimeric protein according to any one of items 1 to 6, wherein the /V- and C- terminal sequences preceding or following the internal fusion site of the ISVD, respectively and /V- and C-terminal sequences preceding or following the internal fusion site of the cytokine correspond to at least a part of the sequence of the loop or turn between two secondary structure elements of the respective internal fusion sites.
• N- and C- terminal sequences preceding or following the internal fusion sites of the ISVD and/or of the cytokine correspond to the sequence of the loop or turn between two secondary structure elements of the respective internal fusion sites in the original sequences of the ISVD and/or of the cytokine, with O to 10, continuous amino acids missing from the original sequences.
• chimeric protein according to any one of items 1 to 8, wherein the /V- and C- termini of the chimeric protein correspond to the /V- and C- termini of the ISVD, respectively.
• the continuous amino acid sequence comprises (i) the /V-terminal part of the ISVD sequence, followed by (ii) the sequence of the cytokine (or circularly permuted cytokine), followed by (iii) the rest of the sequence of the ISVD (i.e., the C-terminal part of the ISVD).
• chimeric protein according to any one of items 1 to 12, wherein the chimeric protein first comprises the /V-terminal amino acids of the ISVD, followed by the C-terminus of the amino acid at the internal fusion site of the ISVD, which is linked to the /V-terminus of the cytokine or circularly permuted cytokine, and wherein the amino acid sequence of the chimeric protein continues with the rest of the sequence of the cytokine or circularly permuted cytokine, ending in its C-terminus, and finally linked to the /V-terminus of the C- terminally located amino acid at the internal fusion site of the ISVD and the rest of the sequence of the ISVD (the C-terminal part of the ISVD).
• chimeric protein according to any one of items 1 to 13, wherein internal fusion site of the ISVD and the internal fusion site of the cytokine are linked to each other by first removing from 0 to 10, preferably from 0 to 5, more preferably from 0 to 3, such as 0, 1, 2 or 3 (continuous) amino acids from the internal fusion site of the cytokine and/or from the internal fusion site of the ISVD, , optionally though a peptide linker.
• chimeric protein according to any one of items 1 to 16, wherein the original /V- and C-termini of the cytokine (when it undergoes circular permutation) are linked to each other directly or through a peptide linker.
• hEPO human erythropoietin
• hGCSF human granulocyte colony-stimulating factor
• cytokine is an interleukin
• the interleukin is lnterleukin-2 (IL-2) or Interleukin-18 (IL-18).
• cytokine is an interferon
• interferon is Interferon (INF) alpha 2 (IFNA2a).
• chimeric protein according to any one of items 1 to 23, wherein the cytokine is fused with the ISVD at an internal fusion site located in one of the following turns in the ISVD, according to IMGT classification: a. In the first R-turn that connects R-strand A and B of the ISVD; or b. In the R-turn that connects R-strand C and C' of the ISVD; or c. In the R-turn that connects R-strand C" and D of the ISVD; or d. In the R-turn that connects R-strand D and E of the ISVD; or e. In the R-turn that connects R-strand E and F of the ISVD.
• chimeric protein according to any one of items 1 to 26, wherein said cytokine is an interleukin and wherein the internal fusion site of the cytokine is an exposed R-turn of the interleukin R-barrel core motif.
• the chimeric protein comprises an ISVD comprising a sequence as defined in SEQ ID NO.: 1, 2 or 55, or a sequence with at least 80% identity with SEQ ID NO.: 1, 2 or 55.
• chimeric protein according to any one of items 1 to 28, wherein the chimeric protein comprises a cytokine comprising a sequence as defined in 4, 58, 59, 64, 66, 68, 70, 172-195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 220, 222, 225, 227, 244-246,
• chimeric protein according to any one of items 1 to 27, wherein the chimeric protein comprises or consists of a sequence as defined in SEQ ID NO.: 7-25, 36-54, 60-63, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218-219, 221, 223-224, 226, 230-237, 261,
• polypeptide according to item 31 wherein the polypeptide further comprises one or more further groups, residues, moieties or binding units.
• polypeptide according to any one of items 31 to 32, wherein the polypeptide further comprises one or more ISVDs.
• a vector comprising the nucleic acid molecule as defined in item 34.
• 36. The vector according to item 35 for surface display in yeast, phages, bacteria, or viruses.
• a host cell comprising the chimeric protein as defined in any one of items 1 to 30 or the polypeptide as defined in any one of items 31 to 33.
• the method further comprises a step (iv) of screening for chimeric proteins which bind to at least one of the cytokine receptors or receptor subunits with increased or decreased affinity as compared to the binding of the wild-type cytokine, or screening for chimeric proteins wherein the cytokine comprised therein shows modified cytokine signaling as compared with the cytokine not fused to an ISVD, or screening for chimeric proteins which affect receptor or receptor's subunit oligomerization upon binding of the cytokine comprised therein to at least one of its receptors or receptor's subunits. 40.
• a chimeric protein comprising a cytokine fused to an ISVD, directly or by means of a linker, for modulating the binding affinity of the cytokine comprised in the chimeric protein to its receptor.
• ISVD strand A (SEQ ID NO.: 240) 3SG,. is.a short peptide. li.nke IL-2(K35E,C125S) sequence is bold (SEQ ID NO.: 173), circular permutation linker in italics (GG), ISVD
• ISVD strand A (SEQ ID NO.: 240) t GSGG or GGSG, is a i s h o rt : eptide linker, the IL- 2(K35E,C125S) sequence is bold (SEQ ID NO.: 175), circular permutation linker in italics, ISVD
• ISVD strand A (SEQ ID NO.: 240) t GSGG or GGSG, is a i short : peptide li nker, the IL- 2(K35E,C125S) sequence is bold (SEQ ID NO.: 176), circular permutation linker in italics, ISVD
• ISVD strand A (SEQ ID NO.: 240) t GSGG or :.GGSG is a i short : peptide li nker, the IL- 2(K35E,C125S) sequence is bold (SEQ ID NO.: 177), circular permutation linker in italics, ISVD
• ISVD strand A (SEQ ID NO.: 240) t GSG,_ is a. shprtpeptjde.li.nker,. the IL-2(K35E,C125S) sequence is bold (SEQ ID NO.: 178), circular permutation linker in italics, ISVD
• ISVD strand A (SEP ID NO.: 240) 3SG, is a short .peptide. linker, the IL-2(K35E,C125S) sequence is bold (SEQ ID NO.: 179), circular permutation linker in italics, ISVD
• ISVD strand A (SEQ ID NO.: 240) t GSGG or :.GGSG, is a i s h o rt : peptide. linker, the IL- 2(K35E,C125S) sequence is bold (SEQ ID NO.: 180), circular permutation linker in italics, ISVD
• ISVD strand A (SEQ ID NO.: 240) 3SG s a .short .peptide.linker, the IL-2(K35E,C125S) sequence is bold (SEQ ID NO.: 181), circular permutation linker in italics, ISVD
• ISVD strand A (SEQ ID NO.: 240) 3SG s a .short .peptide.linker, the IL-2(K35E,C125S) sequence is bold (SEQ ID NO.: 182), circular permutation linker in italics, ISVD
• ISVD strand A (SEQ ID NO.: 240) t GSGG or :.GGSG, is a i s h o rt :. peptide linker, the IL- 2(K35E,C125S) sequence is bold (SEQ ID NO.: 183), circular permutation linker in italics, ISVD
• ISVD strand A (SEP ID NO.: 240) 3SG, is a short .peptide. linker, the IL-2(K35E,C125S) sequence is bold (SEQ ID NO.: 184), circular permutation linker in italics, ISVD
• ISVD strand A (SEQ ID NO.: 240) t GSGG or :.GGSG, is a i s h o rt : peptide. linker, the IL- 2(K35E,C125S) sequence is bold (SEQ ID NO.: 184), circular permutation linker in italics, ISVD
• ISVD strand A (SEQ ID NO.: 240) t GSGG or :.GGSG, is a i s h o rt :. peptide linker, the IL- 2(K35E,C125S) sequence is bold (SEQ ID NO.: 186), circular permutation linker in italics, ISVD
• ISVD strand A (SEQ ID NO.: 240) t GSGG or GGSG, is a i s h o rt :. peptide linker, the IL- 2(K35E,C125S) sequence is bold (SEQ ID NO.: 187), circular permutation linker in italics, ISVD
• ISVD strand A (SEQ ID NO.: 240) t GSGG or GGSG, is a i s h o rt :. peptide linker, the IL- 2(K35E,C125S) sequence is bold (SEQ ID NO.: 188), circular permutation linker in italics, ISVD
• ISVD strand A (SEQ ID NO.: 240) 2 _GSG or GGSG t is a .short .peptide linker, the IL- 2(K35E,C125S) sequence is bold (SEQ ID NO.: 189), ISVD
• ISVD strand A (SEQ ID NO.: 240) 3SG is .a sh ort.pe.pt ide.l in IL-2(K35E,C125S) sequence is bold (SEQ ID NO.: 185), ISVD
• SEQ ID NO: 35 amino acid sequence of the FLAG3HIS6 tag GAADYKDHDGDYKDHDIDYKDDDDKGAAHHHH or AAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH (SEQ ID NO.: 252)
• SEQ ID NO: 36 IL-2(K35E,C125S)_ALB23OO2 Megabody protein (TP018)
• ISVD strand A (SEQ ID NO: 253, GSG or GGSG, is a short peptide linker, the IL-2 sequence is bold (SEQ ID NO.: 189), ISVD
• SEQ ID NO: 37 IL-2(K35E,C125S)[P34-Y31]_ALB23OO2 Megabody protein (TP019)
• ISVD strand A SEQ ID NO.: 253, GSG or GGSG, is a short peptide linker, the IL-2 sequence is bold (SEQ ID NO.: 173), circular permutation linker in italics, ISVD
• SEQ ID NO: 38 IL-2(K35E,C125S)[N77-S75]_ALB23002 Megabody protein (TP020) (ISVD strand A, SEQ ID NO.: 253, GSG, is a short peptide linker, the IL-2 sequence is bold (SEQ ID NO.: 178), circular permutation linker in italics, ISVD
• SEQ ID NO: 39 IL-2(K35E,C125S)[T102-E100]_ALB23002 Megabody protein (TP021) (ISVD strand A, SEQ ID NO.: 253, GSG, is a short peptide linker, the IL-2 sequence is bold (SEQ ID NO.: 182), circular permutation linker in italics, ISVD B-strands B to G are underlined, SEQ ID NO.: 254) pyQLyESGGGyVGSGTFMCEYADETATIVEFLNRWITFSQSIISTLTGGSSSTKKTQLQLEHLLLDLQMIL NGINNYKNPELTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLEL KGSEGSGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNT LYLQMNSLRPEDTALYYCTIGGSLSRSS
• SEQ ID NO: 40 IL-2(K35E,C125S)[T1O2-E1OO]_ISVD1OF11 (TP048)
• ISVD strand A (SEQ ID NO.: 255), GSG, is a short peptide linker, the IL-2 sequence is bold (SEQ ID NO.: 182), circular permutation linker in italics, ISVD B-strands B to G are underlined, SEQ ID NO.: 298)
• ISVD strand A (SEQ ID NO.: 255), GSG i s a i sh i o rt pe pt i d e I i n ke r, G G GG SGG GS is t he 9GS linker, SEQ ID NO.: 154, the IL-2 sequence is bold (SEQ ID NO.: 182), circular permutation linker in italics, ISVD B-strands B to G are underlined, SEQ ID NO.: 254) EVQLVESGGGVVQPGGSLRLSCAASGRTFSGNTMGWFRQAPGKEREFVAAISSTGRNTNYADSVEGRF
• SEQ ID NO: 42 ALB23002-9GS-IL-2(K35E,C125S)[T102-E100]_ISVD10Fll ISVD-Megabody fusion protein (TP050)
• ALB23002 (SEQ ID NO,: 55), (ISVD strand A (SEQ ID NO.: 255), GSG, is .a.sho rt _pe pt ide .1 i n ke r, GGGGSGGGS is the 9GS linker, SEQ ID NO.: 156, the IL-2 sequence is bold (SEQ ID NO.: 182), circular permutation linker in italics, ISVD B-strands B to G are underlined, SEQ ID NO.: 298) EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFT ISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGVVGSG
• SEQ ID NO: 43 IL-2(K35E,C125S)[T102-E100]_ISVD10Fll-9GS-ALB23002 ISVD-Megabody fusion protein (TP051)
• ISVD strand A (SEQ ID NO.: 255), GSG, is a short peptide linker, the IL-2 sequence is bold (SEQ ID NO.: 182), circular permutation linker in italics, ISVD B-strands B to G are underlined, SEQ ID NO.: 298)
• SEQ ID NO: 44 ISVD1OF11-9GS-IL-2(K35E,C125S)-9GS-ALB23OO2 ISVD-Megabody fusion protein (TP052) EVQLVESGGGVVQPGGSLRLSCAASGRTFSGNTMGWFRQAPGKEREFVAAISSTGRNTNYADSVEGRF TISRDNSKNTVYLQMNSLRPEDTALYYCAASSWAAAAGTIANIYDYWGQGGTLVTVSSGGGGSGGGSA PTSSSTKKTQLQLEHLLLDLQMILNGI NNYKNPELTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA QSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSI ISTLTGGGGSGGGSEVQL VESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSDTLYADSVKGRFTISRD NSKNTLYLQMNS
• SEQ ID NO: 45 IL-2(K35E,C125S)[S75-N71]_ALB23002 Megabody protein (TP056)
• ISVD strand A SEQ ID NO.: 253, GSGG or GGSG is a short peptide linker, the IL-2 sequence is bold (SEQ ID NO.: 177), circular permutation linker in italics, ISVD
• SEQ ID NO: 46 IL-2(K35E,C125S)[F78-Q74]_ALB23002 Megabody protein (TP057)
• ISVD strand A SEQ ID NO.: 253, GSG, is a short peptide linker, the IL-2 sequence is bold (SEQ ID NO.: 179), circular permutation linker in italics, ISVD
• SEQ ID NO: 47 IL-2(K35E,C125S)[L85-P82]_ALB23OO2 Megabody protein (TP058)
• ISVD strand A SEQ ID NO.: 253, GSGG or GGSG, is a short peptide linker, the IL-2 sequence is bold (SEQ ID NO.: 180), circular permutation linker in italics, ISVD
• SEQ ID NO : 48 IL-2(K35E,C125S)[T101-G98]_ALB23002 Megabody protein (TP059) (ISVD strand A, SEQ ID NO.: 253, GSG, is a short peptide linker, the IL-2 sequence is bold (SEQ ID NO.: 181), circular permutation linker in italics, ISVD
• SEQ ID NO: 50 IL-2(K35E,C125S)[L132-I129]_ALB23OO2 Megabody protein (TP063)
• ISVD strand A SEQ ID NO.: 253, GSGG or GGSG, is a short peptide linker, the IL-2 sequence is bold (SEQ ID NO.: 184), circular permutation linker in italics, ISVD
• SEQ ID NO: 51 IL-2(K35E,C125S)[L132-I129]_ALB23OO2 Megabody protein (TP064)
• ISVD strand A SEQ ID NO.: 253, GSG, is a short peptide linker, the IL-2 sequence is bold (SEQ ID NO.: 184), circular permutation linker in italics, ISVD
• SEQ ID NO: 52 IL-2(K35E,C125S)[F42-M39]_ALB23002 Megabody protein (TP065)
• ISVD strand A SEQ ID NO.: 253, GSGG or GGSG is a short peptide linker, the IL-2 sequence is bold (SEQ ID NO.: 174), circular permutation linker in italics, ISVD
• SEQ ID NO: 53 IL-2_ALB23002 Megabody protein (TP072)
• ISVD strand A (SEQ ID NO.: 255), GSG or GGSG, is a short peptide linker, the IL-2 sequence is bold (SEQ ID NO.: 190), ISVD
• SEQ ID NO: 54 IL-2[P34-Y31]_ALB23002 Megabody protein (TP075) (I SVP strand A (SEQ ID NO.: 255), GSG or GGSG, is a short peptide linker, the IL-2 sequence is bold (SEQ ID NO.: 191), circular permutation linker in italics, ISVD
• IFNA2a[D77- W76]V4 the protein sequence of the circular permuted IFNA2a, called IFNA2a[D77- W76]V4 (SA18060; TP088) DETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIM RSFSLSTNLQESLRSGGSCDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQEEFGNQFQKAETI PVLHEMIQQIFNLFSTKDSSAAW
• ISVD strand A (SEQ ID NO.: 255), GSG, is a short peptide linker, the IFNA2a sequence is bold (SEQ ID NO.: 192), circular permutation linker in italics, ISVD
• ISVD strand A (SEQ ID NO.: 255), GSG, is a short peptide linker, the IFNA2a sequence is bold (SEQ ID NO.: 193), circular permutation linker in italics, ISVD
• ISVD strand A (SEQ ID NO.: 255), GSG, is a short peptide linker, the IFNA2a sequence is bold (SEQ ID NO.: 194), circular permutation linker in italics, ISVD
• SEQ ID NO: 63 the sequence of IFNA2[P109-T106]_ALB23002 Megabody protein (TP109) (ISVD strand A (SEQ ID NO.: 255), GSG, is a short peptide linker, the IFNA2a sequence is bold (SEQ ID NO.: 195), circular permutation linker in italics, ISVD
• SEQ ID NO: 70 the protein sequence of IL18[K70-E69]V7 (SA18067)
• Serum albumin binding ISVD sequences (“ID” refers to the SEQ ID NO as used herein)

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