CN114222759B - anti-PD-1 single-domain antibody, derived protein and medical application thereof - Google Patents

Abstract

Relates to an anti-PD-1 single domain antibody, a derivative protein thereof and application thereof in treating PD-1 related diseases (such as tumors).

Description

anti-PD-1 single-domain antibody, derived protein and medical application thereof

The present application claims priority from chinese patent application 201910842676.8 filed on 9, 6, 2019.

Technical Field

The present disclosure relates to anti-PD-1 single domain antibodies, their derived proteins, and their use as PD-1 related disease prevention and/or treatment drugs, in the field of biological medicine.

Background

PD-1 (Programmed Cell death-1), which belongs to the CD28 receptor family, is an immunosuppressive receptor (Riley et al 2009, immunol. Rev. 29:114-25). This family also includes CD28, CTLA-4, ICOS, PD-1 and BTLA. PD-1 is a type I transmembrane protein that is very similar in structure to CTLA-4, but PD-1 lacks the MYPPPY sequence that binds to B7-1 and B7-2.

PD-1 is expressed predominantly on activated B cells, T cells and bone marrow cells (Chen et al 2013, nat. Rev. Immunol. 13:227-42), with two cell surface glycoprotein ligands, PD ligand 1 (PD-L1, also known as CD274, B7-H1) and PD ligand 2 (PD-L2, also known as B7-DC), respectively. Neither PD-L1 nor PD-L2 binds to other CD28 receptor family members. PD-L1 is widely expressed in lymphocytes (e.g., CD4+ T cells and CD8 ⁺ T cells, macrophages, etc.) and such as peripheral tissues, various tumor cells, and virus-infected cells.

PD-L2 is expressed predominantly in activated dendritic cells and macrophages (Dong et al 1999, nat. Med. 5:1365-9). After PD-1 binds to its ligand PD-L1 or PD-L2, it down regulates T cell function, including reducing T cell activation, differentiation and proliferation, cytokine secretion, etc.

PD-L1 is highly expressed in a variety of human tumors including melanoma, glioma, non-small cell lung cancer, head and neck cancer, leukemia, pancreatic cancer, renal cancer, and liver cancer, among others (Zou and Chen,2008, nat. Rev. Immunol. 8:467-77). PD-L1, which is highly expressed by tumor cells, down regulates T cell function, increases T cell apoptosis, plays an important role in the immune escape process of tumors ((Freeman et al 2000, J. Exp. Med.192:1027-34; latchman et al 2001, nat. Immunol.2:261-8; cater et al 2002, eur. J. Immunol.32:634-43; ohigashi et al 2005,Clin.Cancer Res.11:2947-53).

Blocking the interaction of PD-1 and PD-L1 can reverse immunosuppression, while simultaneously inhibiting the effects of PD-1 and PD-L1, PD-L2 can act synergistically (Iwai et al 2002, proc.Nat'1.Acad.Sci. USA,99:12293-7; brown et al 2003, J.Immunol.170:1257-66).

Camelids (e.g., camels and alpacas) produce a unique heavy chain antibody (HcAb) lacking the light chain, and variable region fragments (VHHs) derived from such antibodies are known as single domain antibodies (single domain antibody, sdabs). The molecular weight of the single-domain antibody is only 12-15kDa, which is one tenth of that of the traditional antibody (comprising four chains), and the structure of the single-domain antibody is 2.5nm in diameter and 4nm in length, which is the smallest antibody with complete antigen binding activity known at present.

Single domain antibodies also contain 3 CDRs, with CDR3 playing a major role for affinity. Compared with human antibody VH, CDR3 of single domain antibody is longer, can form a raised loop (band loop) structure, can go deep inside antigen, and thus bind antigen better. Thus, VHH has the characteristics of high affinity and high specificity. In addition, the hydrophobic residues of FR2 in single domain antibodies are replaced by hydrophilic residues, which are more water soluble and less prone to form aggregates. Compared with the traditional antibody, the single domain antibody has the advantages of high binding force, high specificity, high solubility, high stability, high expression level and the like.

Related patents for anti-PD-1 single domain antibodies include WO2008071447, US10087251, WO2019032663, CN201610827021.X, CN201711223594.2, WO2019104860, WO2018127709, WO2018127710, WO2018127711. Among them, WO2018127709, WO2018127710, WO2018127711 relate to single domain antibodies that do not block PD-1 and ligand binding. Currently, single domain antibodies to PD-1 are in an early development stage on a global scale, and no single domain antibody drug targeting PD-1 is marketed yet.

There remains a need in the art for PD-1 antibodies, particularly PD-1 single domain antibodies, that are capable of binding to PD-1 with high affinity and that are capable of blocking the binding of PD-1 to PD-L1, PD-L2.

Disclosure of Invention

The present disclosure provides a PD-1 binding protein, more specifically, an anti-PD-1 single domain antibody, its derivative protein and its medical use.

In a first aspect, the present disclosure provides a PD-1 binding protein comprising at least one immunoglobulin single variable domain capable of specifically binding to PD-1. In some embodiments, the PD-1 binding protein comprises an immunoglobulin single variable domain that specifically binds PD-1. In other embodiments, the PD-1 binding protein comprises 2, 3, 4, or more immunoglobulin single variable domains that specifically bind PD-1. In some embodiments, the PD-1 junctionThe synuclein comprises two or more identical immunoglobulin single variable domains that specifically bind PD-1. In other embodiments, the PD-1 binding protein comprises two or more different immunoglobulin single variable domains that specifically bind PD-1. In some embodiments, the two or more immunoglobulin single variable domains that specifically bind PD-1 are directly linked. In other embodiments, the two or more immunoglobulin single variable domains that specifically bind PD-1 are linked by a linker. The linker may comprise 1-20 or more amino acids and does not comprise a non-functional amino acid sequence of secondary or tertiary structure. For example, the linker is a flexible linker, e.g. G ₄ S、GS、GAP、ASGS、(G ₄ S) _n And the like, wherein n is an integer between 1 and 8.

In some embodiments, a PD-1 binding protein of the present disclosure comprises at least one immunoglobulin single variable domain comprising an amino acid sequence as set forth in DSVKGRFT or ASVKGRFA. In some embodiments, the immunoglobulin single variable domain comprises three complementarity determining regions CDR1, CDR2, and CDR3, with DSVKGRFT or ASVKGRFA located in CDR 2.

In some embodiments, a PD-1 binding protein of the present disclosure comprises three mutually spaced complementarity determining regions CDR1, CDR2, and CDR3 in order from amino-terminus to carboxy-terminus.

In some embodiments, an immunoglobulin single variable domain of the present disclosure (in order from amino-terminus to carboxy-terminus) comprises three complementarity determining regions CDR1, CDR2, and CDR3, wherein:

CDR1 comprises SEQ ID NO:62, CDR2 comprises X ₁ IDSVGX ₂ TX ₃ YX ₄ X ₅ An amino acid sequence shown by SVKG (SEQ ID NO: 115), wherein X ₁ Selected from S or T, X ₂ Selected from T or A, X ₃ Selected from D, N or G, X ₄ Selected from T or A, X ₅ Selected from N or D, CDR3 comprises SEQ ID NO:64, an amino acid sequence shown in seq id no; or (b)

CDR1 comprises SEQ ID NO:81, CDR2Comprising VVDRFGGX ₆ IYAX ₇ SVKX ₈ (SEQ ID NO: 116) wherein X is the amino acid sequence shown ₆ Selected from I or T, X ₇ Selected from A or D, X ₈ Selected from K or D, CDR3 comprising GSYTX ₉ X ₁₀ X ₁₁ SCX ₁₂ PDAL (SEQ ID NO: 117) wherein X is the amino acid sequence shown ₉ Selected from S or D, X ₁₀ Selected from A or D, X ₁₁ Selected from N or G, X ₁₂ Selected from Q or H; or (b)

CDR1 contains YNX ₁₃ MX ₁₄ (SEQ ID NO: 118) wherein X is the amino acid sequence shown ₁₃ Selected from F or Y, X ₁₄ Selected from S or T, CDR2 comprises SEQ ID NO:66, CDR3 comprises the amino acid sequence set forth in SEQ ID NO:67, an amino acid sequence shown in seq id no; or (b)

CDR1 comprises SEQ ID NO:84, CDR2 comprises VINTGX ₁₅ NX ₁₆ TYYADSVKG (SEQ ID NO: 119), wherein X ₁₅ Selected from A or T, X ₁₆ Selected from S or T, CDR3 comprises SEQ ID NO:64, an amino acid sequence shown in seq id no; or (b)

CDR1 comprises SEQ ID NO:78, CDR2 comprises X ₁₇ YPTAGX ₁₈ TYX ₁₉ X ₂₀ DSX ₂₁ An amino acid sequence shown as KG (SEQ ID NO: 120), wherein X ₁₇ Selected from L or I, X ₁₈ Selected from R or K, X ₁₉ Selected from Y or F, X ₂₀ Selected from G or A, X ₂₁ Selected from M or V, CDR3 comprises SEQ ID NO:80, an amino acid sequence shown in seq id no; or (b)

CDR1, CDR2, CDR3 comprise SEQ ID NO: 59. 60, 61; or (b)

CDR1, CDR2, CDR3 comprise SEQ ID NO: 74. 75, 76; or (b)

CDR1, CDR2, CDR3 comprise SEQ ID NO: 88. 89, 90;

alternatively, CDR1, CDR2, CDR3 comprise SEQ ID NO: 96. 97, 98.

In some embodiments, a PD-1 binding protein of the present disclosure comprises any one selected from the group consisting of:

CDR1 comprises SEQ ID NO:62, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 63. 68, 69, 70, 72, 77, CDR3 comprises the amino acid sequence of any one of SEQ ID NOs: 64 or 73; or (b)

CDR1 comprises SEQ ID NO:81, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 71. 82, 91, 93, 94, CDR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 83. 92, 95; or (b)

CDR1 comprises SEQ ID NO: 65. 113, 114, CDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 66, CDR3 comprises the amino acid sequence set forth in SEQ ID NO:67, an amino acid sequence shown in seq id no; or (b)

CDR1 comprises SEQ ID NO:84, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 85. 102, CDR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 86, an amino acid sequence shown in seq id no; or (b)

CDR1 comprises SEQ ID NO:78, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 79. 87, 99, 100, 101, CDR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 80, and an amino acid sequence shown in seq id no.

In some embodiments, a PD-1 binding protein of the present disclosure comprises any one selected from the group consisting of:

CDR1 comprises SEQ ID NO:62, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 63. 68, 69, 70, 72, 77, CDR3 comprises the amino acid sequence of any one of SEQ ID NOs: 64, an amino acid sequence shown in seq id no; or (b)

CDR1, CDR2, CDR3 comprise SEQ ID NO: 62. 63, 73; or (b)

CDR1 comprises SEQ ID NO:81, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 71. 82, CDR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 83, an amino acid sequence shown in seq id no; or (b)

CDR1 comprises SEQ ID NO:81, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 91. 93, CDR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 92, an amino acid sequence shown in seq id no; or (b)

CDR1, CDR2, CDR3 comprise SEQ ID NO: 81. 94, 95.

In some embodiments, an immunoglobulin single variable domain of a PD-1 binding protein of the present disclosure comprises three complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR3 is selected from the group consisting of SEQ ID NOs: 61. 64, 67, 73, 76, 80, 83, 86, 90, 92, 95, 98 or an amino acid sequence having a 3, 2, 1 amino acid difference thereto.

In some embodiments, at least one immunoglobulin single variable domain in a PD-1 binding protein of the present disclosure:

(i) CDR1 comprises a sequence selected from SEQ ID NOs: 59. 62, 65, 74, 78, 81, 84, 88, 93, or an amino acid sequence having a 3, 2, 1 amino acid difference thereto; and/or

(ii) CDR2 comprises a sequence selected from SEQ ID NOs: 60. 63, 66, 68, 69, 70, 71, 72, 75, 77, 79, 82, 85, 87, 89, 91, 93, 94, 97, 99, 100, 101, 102, 113, 114, or an amino acid sequence having a 3, 2, 1 amino acid difference thereto; and/or

(iii) CDR3 comprises a sequence selected from SEQ ID NOs: 61. 64, 67, 73, 76, 80, 83, 86, 90, 92, 95, 98, or an amino acid sequence having a 3, 2, 1 amino acid difference thereto.

In some embodiments, one or more of the CDRs described above are grafted (graft) onto a scaffold or FR (including, but not limited to, a scaffold derived from a human, or a non-immunoglobulin scaffold). Scaffolds and techniques suitable for grafting the CDRs are known in the art.

In some embodiments, the PD-1 binding proteins of the present disclosure are antibodies or antigen-binding fragments thereof that bind to PD-1, or conjugates, fusion proteins comprising the antibodies, antigen-binding fragments.

In some embodiments, the antibody or antigen binding fragment thereof is a camelid antibody, chimeric antibody, humanized antibody, fully human antibody, or fragment thereof. In some embodiments, the antigen binding fragment is an sdAb or a bispecific antibody, a multispecific antibody.

In some embodiments, at least one immunoglobulin single variable domain in a PD-1 binding protein of the present disclosure is a VHH.

In some embodiments, the VHH comprises SEQ ID NO:7-33 or an amino acid sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity thereto.

In other embodiments, the VHH is a humanized VHH. The humanized VHH comprises a sequence identical to SEQ ID NO:35-58, 123-128, or an amino acid sequence that has at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity. Alternatively, the amino acid sequence of the VHH hybridizes to SEQ ID NO: any of the 7-33, 35-58, 123-128 comprises one or more amino acid substitutions, preferably conservative amino acid substitutions, e.g., comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions.

In some embodiments, the PD-1 binding proteins of the present disclosure are obtained via affinity maturation, e.g., as set forth in SEQ ID NO: affinity maturation was performed on the basis of 7-33, 35-58, 123-128. Affinity-matured PD-1 binding proteins can have one or more changes in one or more CDRs that result in an increase in affinity for PD-1 as compared to the parent PD-1 binding protein.

In some embodiments, a PD-1 binding protein of the present disclosure comprises an Fc region in addition to at least one immunoglobulin single variable domain capable of specifically binding to PD-1 or an epitope thereof.

The inclusion of an Fc region in the PD-1 binding proteins of the present disclosure may allow the binding proteins to form dimer molecules while extending the in vivo half-life of the binding proteins. The Fc regions useful in the present disclosure may be from immunoglobulins of different subtypes, e.g., igG (e.g., igG1, igG2, igG3, or IgG4 subtypes), igA1, igA2, igD, igE, or IgM. Generally, the Fc region includes the hinge region or a portion of the hinge region, the CH2 region, and the CH3 region of the constant region.

In some embodiments, mutations may be introduced in the wild-type Fc sequence for altering Fc-mediated related activity. Such mutations include, but are not limited to:

a) Mutations that alter Fc-mediated CDC activity;

b) Mutations that alter Fc-mediated ADCC activity; or (b)

c) Mutations that alter FcRn-mediated in vivo half-life. Such mutations are described in the following documents: leonard G Presta, current Opinion in Immunology 2008, 20:460-470; esohe E.Idusogie et al J Immunol 2000, 164:4178-4184; RAPHAEL a.clynes et al Nature Medicine,2000,Volume 6,Number 4:443-446; paul R.Hinton et al J Immunol,2006, 176:346-356. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids on the CH2 region may be mutated for increasing or deleting Fc-mediated ADCC or CDC activity or for increasing or decreasing the affinity of FcRn. In addition, the stability of the protein can be increased by mutating 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of the hinge region.

In some embodiments, mutations may be introduced into the Fc sequence, thereby making the mutant Fc more susceptible to homodimer or heterodimer formation. The knob-hole model, as mentioned in Ridgway, presta et al 1996 and Carter 2001, which uses the steric effect of the amino acid side chain groups of the Fc contact interface, makes heterodimer formation between different Fc mutations easier; for another example, by changing the charge of the amino acids at the Fc contact interface, and thus the ionic interaction force between the Fc contact interfaces, heterodimers are more likely to form between different pairs of Fc mutations (CN 102558355A), or homodimers are more likely to form between Fc with the same mutation (CN 103388013 a).

The immunoglobulin Fc region is preferably a human immunoglobulin Fc region, such as the Fc region of human IgG1 Fc, human IgG4 (S228P). In some embodiments, the immunoglobulin Fc region has an amino acid sequence as set forth in SEQ ID NO: 103. 108 or at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%.

In some embodiments, in a PD-1 binding protein of the present disclosure, an immunoglobulin single variable domain is linked to an immunoglobulin Fc region by a linker. The linker may be a nonfunctional amino acid sequence 1-20 or more amino acids long, without secondary or more structure. For example, the linker is a flexible linker, e.g. G ₄ S、GS、GAP、(G ₄ S) ₂ 、(G ₄ S) ₃ 、(G ₄ S) ₄ 、(G ₄ S) ₅ ASGS, etc.

In some embodiments, the PD-1 binding proteins of the present disclosure comprise an immunoglobulin single variable domain that is linked to an immunoglobulin Fc region directly or through a linker. In some embodiments, a PD-1 binding protein of the present disclosure comprises two immunoglobulin single variable domains that are linked, either directly or through a linker, to an immunoglobulin Fc region that allows the PD-1 binding protein to form a dimeric molecule comprising the two immunoglobulin single variable domains. Such PD-1 binding proteins are also referred to as bivalent PD-1 binding proteins.

In some embodiments, a PD-1 binding protein of the present disclosure comprises three or four immunoglobulin single variable domains and one immunoglobulin Fc region, directly or via a linker, that allows the PD-1 binding protein to form a multimeric molecule comprising three or four immunoglobulin single variable domains. Such PD-1 binding proteins are also referred to as trivalent or tetravalent PD-1 binding proteins.

In other embodiments, the PD-1 binding protein comprises at least one PD-1 binding domain and at least one binding domain for another antigen, e.g., forming a heterodimer.

In some embodiments, a PD-1 binding protein of the present disclosure comprising an immunoglobulin Fc region comprises the amino acid sequence of SEQ ID NO: 34. 104-107, 109-112 or has at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity thereto.

In some embodiments, the disclosure provides PD-1 binding proteins that are capable of hybridizing to a polypeptide consisting of SEQ ID NO: the VHH consisting of the amino acid sequence of any one of 7-33, 35-58, 123-128 binds to the same PD-1 epitope or competes for binding to the same PD-1 epitope.

The PD-1 binding proteins of the present disclosure have at least one of the following characteristics:

(a) At a value of 10 or less ^-7 To human PD-1 or an epitope thereof;

(b) Inhibit the binding of PD-1 to PD-L1;

(c) Inhibit the binding of PD-1 to PD-L2;

(d) Inducing cd4+ T cells to secrete IFN- γ;

(e) Enhancing activation of PBMCs;

(f) Enhancing activation of T cells;

(g) Inhibit tumor growth.

The PD-1 binding proteins of the present disclosure bind PD-1 with KD values of 1×10 or less ^-7 M, e.g..ltoreq.1X10) ^-8 M, or less than or equal to 1X 10 ^-9 M, or less than or equal to 1X 10 ^-10 M。

In some embodiments, the PD-1 binding proteins of the present disclosure are capable of specifically binding to human PD-1 and blocking the interaction of PD-1 and PD-L1, and/or PD-1 and PD-L2.

The PD-1 binding proteins of the present disclosure are capable of inhibiting tumor growth by at least about 10%, e.g., at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%.

In addition, the PD-1 binding proteins of the present disclosure are resistant to heat treatment or have higher stability. For example, no significant aggregation or degradation is seen up to 30 days of treatment at 40 ℃, at least stable at 60 ℃.

In a second aspect, the present disclosure provides nucleic acid molecules encoding the PD-1 binding proteins of the present disclosure. The nucleic acid of the present disclosure may be RNA, DNA, or cDNA. According to some embodiments of the disclosure, the nucleic acid of the disclosure is a substantially isolated nucleic acid.

The nucleic acids of the present disclosure may also be in the form of a vector, may be present in and/or may be part of a vector, such as a plasmid, cosmid, YAC, or viral vector. The vector may in particular be an expression vector, i.e. a vector which provides for expression of the PD-1 binding protein in vitro and/or in vivo (i.e. in a suitable host cell, host organism and/or expression system). The expression vector typically comprises at least one nucleic acid of the present disclosure operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.). The choice of the element and its sequence for expression in a particular host is common knowledge to the skilled person. Regulatory elements useful or necessary for the expression of the PD-1 binding proteins of the present disclosure and other elements are, for example, promoters, enhancers, terminators, integration factors, selectable markers, leader sequences, reporter genes.

The nucleic acids of the present disclosure may be prepared or obtained by known means (e.g., by automated DNA synthesis and/or recombinant DNA techniques) based on information about the amino acid sequence of the polypeptides of the present disclosure, and/or may be isolated from a suitable natural source.

In a third aspect, the present disclosure provides a recombinant host cell expressing or capable of expressing one or more PD-1 binding proteins of the present disclosure and/or containing a nucleic acid or vector of the present disclosure. In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

Bacterial cells include, for example, cells of gram-negative bacterial strains, such as Escherichia coli (Escherichia coli) strains, proteus (Proteus) strains and Pseudomonas (Pseudomonas) strains, and gram-positive bacterial strains, such as Bacillus (Bacillus) strains, streptomyces (Streptomyces) strains, staphylococcus (Staphylococcus) strains and Lactococcus (Lactobacillus) strains.

Fungal cells include, for example, cells of species of Trichoderma (Trichoderma), neurospora (Neurospora) and Aspergillus (Aspergillus); or cells of species including Saccharomyces (e.g., saccharomyces cerevisiae (Saccharomyces cerevisiae)), schizosaccharomyces (Schizosaccharomyces pombe) (e.g., schizosaccharomyces pombe), pichia (Pichia) (e.g., pichia pastoris and Pichia methanolica (Pichia methanolica)), and Hansen (Hansenula).

Mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, heLa cells, COS cells, and the like.

However, the present disclosure may also use amphibian cells, insect cells, plant cells, and any other cell used in the art for expression of heterologous proteins.

In a fourth aspect, the present disclosure provides a method of producing a PD-1 binding protein of the present disclosure, the method generally comprising the steps of:

-culturing a host cell of the disclosure under conditions that allow expression of a PD-1 binding protein of the disclosure; and

-recovering the PD-1 binding protein expressed by the host cell from the culture; and

-optionally, comprising further purification and/or modification of the PD-1 binding proteins of the present disclosure.

The PD-1 binding proteins of the present disclosure can be produced in cells as described above in an intracellular manner (e.g., in the cytoplasm, in the periplasm or in inclusion bodies), then isolated from the host cell and optionally further purified; or it may be produced in an extracellular manner (e.g., in the medium in which the host cells are cultured), then isolated from the medium and optionally further purified.

Methods and reagents for recombinant production of polypeptides, such as specific suitable expression vectors, transformation or transfection methods, selection markers, methods of inducing protein expression, culture conditions, and the like are known in the art. Similarly, protein isolation and purification techniques suitable for use in methods of making the PD-1 binding proteins of the present disclosure are well known to those skilled in the art.

However, the PD-1 binding proteins of the present disclosure may also be obtained by other methods of producing proteins known in the art, such as chemical synthesis, including solid phase or liquid phase synthesis.

In a fifth aspect, the present disclosure provides a composition, e.g., a pharmaceutical composition, comprising a prophylactically or therapeutically effective amount of a PD-1 binding protein of the present disclosure and/or a nucleic acid molecule encoding the PD-1 binding protein as described above, and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients.

In some embodiments, the pharmaceutical composition unit dose may contain from 0.01 to 99% by weight of the PD-1 binding protein. In other embodiments, the unit dose of the pharmaceutical composition comprises from 0.1 to 2000mg of the PD-1 binding protein; in some embodiments 1-1000mg.

In a sixth aspect, the present disclosure provides a kit comprising a PD-1 binding protein of the present disclosure and/or a nucleic acid molecule encoding a PD-1 binding protein of the present disclosure. In some embodiments, diagnostic reagents comprising the PD-1 binding proteins of the present disclosure and/or nucleic acid molecules encoding the PD-1 binding proteins of the present disclosure are also provided, as are the use of the PD-1 binding proteins of the present disclosure and/or encoding the PD-1 binding proteins of the present disclosure in the preparation of a diagnostic for a disease associated with PD-1.

In a seventh aspect, the present disclosure provides the use and method of the PD-1 binding proteins, nucleic acid molecules, host cells and pharmaceutical compositions of the present disclosure in the prevention and/or treatment of diseases, which may or may not be associated with a PD-1 signaling pathway. In some embodiments, the present disclosure provides a method of preventing and/or treating a disease associated with PD-1, the method comprising administering to a subject a prophylactically and/or therapeutically effective amount of a PD-1 binding protein of the present disclosure, or a pharmaceutical composition comprising a PD-1 binding protein of the present disclosure. And, also provided is the use in the manufacture of a medicament for the prevention and/or treatment of a PD-1-related disorder of the present disclosure.

The PD-1 binding proteins of the present disclosure can be used alone or in combination with other anti-tumor therapies (e.g., in combination with other immunogenic agents, standard cancer therapies, or other antibody molecules) to inhibit the growth of cancerous tumors.

In some embodiments, the disclosure provides a method of inhibiting PD-1 activity or promoting T cell proliferation, in other embodiments, the disclosure provides a method of benefiting a patient or subject from an upregulation of immune responses, each comprising administering to the patient or subject a prophylactically and/or therapeutically effective amount of a PD-1 binding protein, nucleic acid, or pharmaceutical composition of the disclosure.

In some embodiments, the present disclosure provides a method of preventing and/or treating cancer comprising administering to a patient or subject a prophylactically and/or therapeutically effective amount of a PD-1 binding protein, nucleic acid, or pharmaceutical composition of the present disclosure, inhibiting tumor cell growth in the patient or subject. In some embodiments, the use of the PD-1 binding proteins of the present disclosure can prevent and/or treat cancer, preferably but not limited to cancer responsive to immunotherapy.

Non-limiting examples of cancers in the above methods include lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma (e.g., metastatic malignant melanoma), kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematological malignancy, head and neck cancer, glioma, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine fibroids, and osteosarcoma. Examples of other cancers that may be treated with the methods of the present disclosure include: bone cancer, pancreatic cancer, skin cancer, prostate cancer, skin or intraocular malignant melanoma, uterine cancer, anal region cancer, testicular cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulval cancer, hodgkin's disease, non-hodgkin's lymphoma, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urinary tract cancer, penile cancer, chronic or acute leukemia including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, childhood solid tumors, lymphocytic lymphomas, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central Nervous System (CNS) tumors, primary CNS lymphomas, tumor angiogenesis, spinal tumors, brain stem glioma, pituitary adenoma, kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T cell lymphoma, environmentally induced cancers including asbestos-induced cancers, and combinations of said cancers. In some embodiments, the cancer or tumor is metastatic.

In some embodiments, the present disclosure provides a method of treating a PD-1-related disorder and disease, including autoimmune diseases, such as Systemic Lupus Erythematosus (SLE), psoriasis, systemic scleroderma, autoimmune diabetes, and the like, comprising administering an effective amount of a PD-1 binding protein, nucleic acid, or pharmaceutical composition of the present disclosure.

In addition, the present disclosure also provides a method of preventing and/or treating an infectious disease in a subject or patient comprising administering to the subject or patient a PD-1 binding protein of the present disclosure such that the infectious disease in the subject is prevented and/or treated. Similar to the use for tumors as described above, the PD-1 binding proteins may be used alone or in combination with a vaccine to stimulate an immune response to pathogens, toxins and autoantigens. Examples of pathogens for which this method of treatment may be particularly useful include pathogens for which no effective vaccine is currently available, or pathogens for which conventional vaccines are not fully effective. Including but not limited to HIV, hepatitis virus (a, b, c), influenza virus, herpes virus, giardia, malaria, leishmania, staphylococcus aureus, pseudomonas aeruginosa.

Some examples of pathogen viruses of infectious diseases that can be treated with the methods of the present disclosure include HIV, hepatitis (a, b, c), herpes viruses (e.g., VZV, HSV-1, HAV-6, HSV-II and CMV, EB virus), adenoviruses, influenza viruses, arboviruses, epox viruses, rhinoviruses, coxsackieviruses, coronaviruses, respiratory syncytial viruses, mumps viruses, rotaviruses, measles viruses, rubella viruses, parvoviruses, vaccinia viruses, HTLV viruses, dengue viruses, papillomaviruses, molluscs, polioviruses, rabies viruses, JC viruses, and arbovirus encephalitis viruses.

Some examples of pathogenic bacteria of infectious diseases that can be treated with the methods of the present disclosure include chlamydia, rickettsia, mycobacteria, staphylococci, streptococci, pneumococci, meningococci and gonococci, klebsiella, proteus, ralstonia, pseudomonas, legionella, diphtheria, salmonella, bacillus, cholera, tetanus, botulinum, bacillus anthracis, plague, leptospira, and lyme disease bacteria.

Some examples of pathogenic fungi of infectious diseases that can be treated with the methods of the present disclosure include candida (candida albicans, candida krusei, candida glabrata, candida tropicalis, etc.), cryptococcus neoformans, aspergillus (aspergillus fumigatus, aspergillus niger, etc.), mucor (mucor, coluba, rhizopus), sporon sampsonii, blastomyces dermatitis, paracoccidiopsis brasiliensis, pachycoccoides crudus, and histoplasma tunica.

Some examples of pathogenic parasites of infectious diseases that can be treated with the methods of the present disclosure include Entamoeba histolytica, coccota sachalinensis, grignard, acanthamoeba species, giardia Cryptosporidium species, pycnoporus carinii, plasmodium vivax, babesia fructicola, trypanosoma brucei, trypanosoma cruzi, leishmania donovani, toxoplasma gondii, brazilian round-robinia.

Drawings

Fig. 1: the results of the in vitro activation of T cells and secretion of IFNγ by PD-1 single domain antibodies numbered 7#, 32#, 32# hu_3, 106#, 107#.

Fig. 2: the results of the in vitro activation of T cells and secretion of IFNγ by PD-1 single domain antibodies numbered 32# hu_3_IgG4, 7# hu_4_hIgG4, 106# hu_1_hIgG4, 107# hu_4_hIgG4 are shown.

Fig. 3: the PD-1 antibody inhibits the growth result of the tumor of the M38 colon cancer of the mice.

Detailed Description

Terminology

For easier understanding of the present disclosure, certain technical and scientific terms are specifically defined below. Unless clearly defined otherwise elsewhere in this disclosure, all other technical and scientific terms used in this disclosure have the meanings commonly understood by one of ordinary skill in the art to which this disclosure belongs.

The terms "programmed death 1", "programmed cell death 1", "protein PD-1", "PDCD1" and "hPD-1" are used interchangeably and include variants, isoforms, species homologs of human PD-1, and analogs having at least one epitope in common with PD-1. Complete PD-1 sequences can be found in GenBank accession U64863.

The term "programmed death ligand-1 (PD-L1)" is one of the two cell surface glycoprotein ligands of PD-1 (the other is PD-L2), which down-regulates T cell activation and cytokine secretion upon binding to PD-1. The term "PD-L1" as used herein includes human PD-L1 (hPD-L1), variants, isoforms, and interspecies homologs of hPD-L1, as well as analogs having at least one epitope in common with hPD-L1. The complete hPD-L1 sequence can be found using GenBank accession number Q9 NZQ.

The term "cytokine" is a general term for proteins released by one cell population that act on other cells as intercellular mediators. Examples of such cytokines include lymphokines, monokines, chemokines, and traditional polypeptide hormones. Exemplary cytokines include: human IL-2, IFN-gamma, IL-6, TNF alpha, IL-17 and IL-5.

The amino acid three-letter codes and one-letter codes used in the present disclosure are as described in j.biol. Chem,243, p3558 (1968).

The term "antibody" or "immunoglobulin", whether referring to a heavy chain antibody or a conventional tetrapeptide chain antibody formed from two identical heavy chains and two identical light chains joined by an interchain disulfide bond, is used as a generic term to include full length antibodies, individual chains thereof, and all portions, domains, or fragments thereof (including but not limited to antigen binding domains or fragments, such as VHH domains or VH/VL domains).

The term "sequence" (e.g., in terms of "immunoglobulin sequence", "antibody sequence", "single variable domain sequence", "VHH sequence" or "protein sequence", etc.) is generally understood to include both the relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding the sequence, unless the disclosure requires further defined interpretation.

The term "domain" of a polypeptide or protein refers to a folded protein structure. In general, domains are responsible for a single function of a protein. In many cases, it is possible to add, remove or transfer to other proteins without losing the function of the rest of the protein and/or the domain.

The term "variable domain" as used in the present disclosure refers to a domain consisting essentially of four "framework regions" referred to in the art and hereinafter as "framework region 1" (FR 1), "framework region 2" (FR 2), "framework region 3" (FR 3), and "framework region 4" (FR 4), respectively; wherein the framework regions are separated by three "complementarity determining regions" referred to in the art and hereinafter as "complementarity determining region 1" (CDR 1), "complementarity determining region 2" (CDR 2), and "complementarity determining region 3" (CDR 3), respectively. Thus, the general structure (or sequence) of a variable domain can be expressed as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The variable domain confers specificity for the antigen by virtue of having an antigen binding site.

The term "Framework Region (FR)" as used in this disclosure refers to a portion of a variable domain that serves as a scaffold for CDRs.

The term "immunoglobulin single variable domain" as used in the present disclosure refers to a variable domain alone capable of specifically binding to an epitope of an antigen. One example of an immunoglobulin single variable domain in the present disclosure is a "domain antibody," e.g., an immunoglobulin single variable domain VH or VL (VH domain or VL domain). Another example is a "VHH domain" (or simply "VHH") as defined below.

"VHH domains", also known as heavy chain single domain antibodies, VHH antibody fragments or VHH antibodies, are variable domains in immunoglobulins known as "heavy chain antibodies" (i.e. "light chain deficient antibodies") (Hamers-Casterman C, atarhouch T, muydermans S, robinson G, hamers C, songa EB, bendahman N, hamers R.: "Naturally occurring antibodies devoid of light chains"; nature363, 446-448 (1993)). The term "VHH domain" is used for the purpose of distinguishing from heavy chain variable domains (which are referred to as "VH domains" in this disclosure) and light chain variable domains (which are referred to as "VL domains" in this disclosure) that are present in conventional tetrapeptide chain antibodies. VHH domain specificity is capable of binding an epitope alone without the need for additional antigen binding domains. In contrast, for conventional tetrapeptide chain antibodies, it is desirable that the VL domain recognizes an epitope along with the VH domain. VHH domains are small stable and efficient antigen recognition units formed from a single immunoglobulin domain. The terms "heavy chain single domain antibody", "VHH domain", "VHH antibody fragment", "VHH antibody" and "Nanobody" are used interchangeably (Nanobody is a trademark of Ablynx N.V, ghent, belgium).

For example, amino acid residues used in VHH domains are numbered according to the general numbering of VH domains given by Kabat et al ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, publication No. 91) as shown in FIG. 2 of Riechmann and Muyledermans, J.Immunol. Methods 231, 25-38 (1999). According to the numbering scheme:

FR1 comprises amino acid residues at positions 1-30,

CDR1 comprising amino acid residues at positions 31-35,

FR2 comprises the amino acids at positions 36-49,

CDR2 comprises amino acid residues at positions 50-65,

FR3 comprises amino acid residues at positions 66-94,

CDR3 comprises amino acid residues at positions 95-102 and

FR4 comprises amino acid residues at positions 103-113.

It should be noted, however, that the total number of amino acid residues in each CDR may be different and may not correspond to the total number of amino acid residues indicated by Kabat numbering (i.e., one or more positions according to Kabat numbering may not be occupied in the actual sequence or the actual sequence may contain more amino acid residues than the Kabat numbering allows), as is well known in the art for VH domains and VHH domains. This means that in general, numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence.

Alternative methods of numbering amino acid residues of VH domains are known in the art, which may also be similarly applied to VHH domains. However, unless otherwise indicated, in the present disclosure, claims and figures, numbering according to Kabat and applicable to VHH domains as described above will be followed.

The total number of amino acid residues in the VHH domain will typically range from 110 to 120, often between 112 and 115. It should be noted, however, that smaller and longer sequences may also be suitable for the purposes described in this disclosure.

Other structural and functional properties of VHH domains, and polypeptides comprising VHH domains, can be summarized as follows:

the VHH domain is naturally "designed" so that it functionally binds to an antigen in the absence of and without interaction with the light chain variable domain. VHH domains can be used as single and relatively small functional antigen binding units, domains or polypeptides. VHH domains differ from the VH and VL domains of conventional tetrapeptide chain antibodies in that VH and VL domains themselves are not suitable for practical use as antigen binding proteins or immunoglobulin single variable domains alone, and VH and VL domains need to be combined in some form or to provide a functional antigen binding unit (e.g., in the form of a Fab or scFv).

Because of these unique properties, the use of VHH domains (alone or as part of a larger polypeptide) provides a number of significant advantages over the use of conventional VH and VL domains, scFv or conventional antibody fragments (e.g., fab or F (ab') 2 fragments):

only a single domain is required to bind antigen with high affinity and high selectivity, so that neither the presence of two separate domains nor the assurance that the two domains are present in the appropriate spatial conformation and configuration is required (e.g. scFv typically require the use of specifically designed linkers);

the VHH domain can be expressed from a single gene and does not require post-translational folding or modification;

VHH domains can be easily engineered into multivalent and multispecific formats;

-VHH domains are highly soluble and have no aggregation tendency;

VHH domains are highly stable to heat, pH, proteases and other denaturants or conditions, and thus can be prepared, stored or transported without the use of refrigeration equipment, thereby achieving cost, time and environment savings;

VHH domains are easy to prepare and relatively inexpensive, even on the scale required for production;

the VHH domain is relatively small compared to conventional tetrapeptide chain antibodies and antigen binding fragments thereof (about 15kDa or 1/10 of the size of conventional IgG), thus exhibiting higher tissue penetration and can be administered at higher doses;

VHH domains may exhibit so-called cavity (cavity) binding properties (VHH has an extended CDR3 loop compared to conventional VH domains, so that target epitopes not reachable by conventional tetrapeptide chain antibodies and antigen binding fragments thereof are reachable).

Methods for obtaining VHH binding to a specific antigen or epitope have previously been disclosed in the following documents: van der Linden et al Journal of Immunological Methods,240 (2000) 185-195; li et al J Biol chem.,287 (2012) 13713-13721; deffar et al African Journal of Biotechnology Vol.8 (12), pp.2645-2652, 17June,2009 and WO94/04678.

The VHH domain can be "humanized" (also referred to herein as "sequence-optimized"; "sequence-optimized" can also encompass other modifications besides humanization that provide improved properties to the VHH, such as removal of potential post-translational modification sites) by replacing one or more amino acid residues in the amino acid sequence of the original VHH (e.g., a VHH from a camelidae) with one or more amino acid residues at corresponding positions in the VH domain of a human conventional tetrapeptide chain antibody. The humanized VHH domain may contain one or more fully human framework region sequences and in a particular embodiment may contain human framework region sequences of IGHV 3. Methods of humanization such as protein surface amino acid humanization (resurfacing) and VHH humanization universal frame grafting (CDR grafting to a universal framework).

As used in this disclosure, the term "domain antibody" (also referred to as "Dab" and "Dab") is used to refer to the VH or VL domain of an antibody (particularly a human tetrapeptide chain antibody) of a non-camelidae mammal. In order to bind an epitope in the form of a single antigen-binding domain (i.e. without pairing with a VL domain or VH domain, respectively), specific selection of the antigen-binding properties is required, for example, by using a library of human single VH or VL domain sequences. Like VHH, domain antibodies have a molecular weight of about 13kDa to about 16kDa and, if derived from fully human sequences, do not require humanization for use in, for example, human therapy. As in the case of VHH domains, domain antibodies are also well expressed in prokaryotic expression systems, thereby significantly reducing overall manufacturing costs.

"domain antibodies" have been disclosed in, for example, the following documents: ward, es., et al: "Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli"; nature 341:544-546 (1989); holt, LJ. et al: "Domain antibodies: proteins for therapy ": TRENDS in Biotechnology 21 (11): 484-490 (2003).

The term "epitope" or the term "epitope" as used interchangeably herein refers to any epitope on an antigen to which an antibody binds. An epitope typically comprises a chemically active surface group of a molecule, such as an amino acid or sugar side chain, and typically has specific three-dimensional structural features and/or specific charge characteristics. For example, an epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique spatial conformation, which may be a "linear" epitope or a "conformational" epitope. See, e.g., epitope Mapping Protocols in Methods in Molecular Biology, volume 66, g.e.Morris, ed. (1996). In linear epitopes, the point of all interactions between an antigen and an interacting molecule (e.g., an antibody) exist linearly along the primary amino acid sequence of the antigen. In conformational epitopes, points of interaction exist across amino acid residues that are separate from each other.

The epitopes of a given antigen can be identified using a number of epitope mapping techniques well known in the art. See, e.g., epitope Mapping Protocols in Methods in Molecular Biology, volume 66, g.e.Morris, ed. (1996). For example, a linear epitope may be determined by, for example, the following methods: a large number of peptides are synthesized simultaneously on a solid support, wherein these peptides correspond to portions of the protein molecule, and these peptides are reacted with antibodies while still attached to the support. Such techniques are known in the art and are described, for example, in U.S. Pat. nos. 4,708,871; geysen et al (1984) Proc.Natl. Acad. Sci. USA 81:3998-4002; geysen et al (1986) molecular immunol.23: 709-715. Conformational epitopes may also be identified by determining the spatial conformation of the amino acid, such as by, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance.

Antibodies can be screened for binding to the same epitope competitively using conventional techniques known to those skilled in the art. For example, competition and cross-competition studies can be performed to obtain antibodies that compete with each other or cross-compete for binding to an antigen. High throughput methods for obtaining antibodies binding to the same epitope based on their cross-competition are described in international patent application WO 03/48731. Thus, antibodies and antigen binding fragments thereof that compete with the antibody molecules of the present disclosure for binding to the same epitope on PD-1 can be obtained using conventional techniques known to those of skill in the art.

In general, the term "specific" refers to the number of different types of antigens or epitopes that a particular antigen binding molecule or antigen binding protein (e.g., PD-1 binding protein of the present disclosure) can bind. The specificity of an antigen binding protein may be determined based on its affinity (avidity) and/or avidity. Affinity, expressed by the dissociation equilibrium constant (KD) of an antigen to an antigen binding protein, is a measure of the strength of binding between an epitope and an antigen binding site on an antigen binding protein: the smaller the KD value, the stronger the binding strength between the epitope and the antigen binding protein (alternatively, affinity can also be expressed as the binding constant (KA), which is 1/KD). As will be appreciated by those skilled in the art, depending on the particular antigen of interest, affinity can be determined in a known manner. Avidity is a measure of the strength of binding between an antigen binding protein (e.g., an immunoglobulin, antibody, immunoglobulin single variable domain, or polypeptide comprising the same) and the antigen of interest. Affinity is related to both: affinity with the antigen binding sites on its antigen binding protein, and the number of relevant binding sites present on the antigen binding protein.

As used in this disclosure, the term "PD-1 binding protein" means any protein capable of specifically binding to PD-1 or an epitope thereof. The PD-1 binding protein may comprise an antibody or antigen-binding fragment thereof as defined in the present disclosure directed against PD-1 or an epitope thereof, or a conjugate, fusion protein comprising said antibody, antigen-binding fragment thereof. Antigen binding fragments are, for example, sdabs or bispecific antibodies, multispecific antibodies. PD-1 binding proteins also encompass immunoglobulin superfamily antibodies (IgSF) or CDR grafted molecules. The "PD-1 binding proteins" of the present disclosure may comprise at least one immunoglobulin single variable domain (e.g., VHH) that binds PD-1. In some embodiments, a "PD-1 binding protein" may comprise 2, 3, 4 or more immunoglobulin single variable domains (e.g., VHH) that bind PD-1. The PD-1 binding proteins of the present disclosure may also comprise a linker and/or a moiety having effector molecule function, e.g., a half-life extending moiety (e.g., an immunoglobulin single variable domain that binds serum albumin), and/or a fusion partner (e.g., serum albumin) and/or a conjugated polymer (e.g., PEG) and/or an Fc region, in addition to an immunoglobulin single variable domain that binds PD-1. In some embodiments, the "PD-1 binding proteins" of the present disclosure also encompass bispecific antibodies that contain immunoglobulin single variable domains that bind different antigens.

An "affinity matured" PD-1 antibody, particularly a VHH or domain antibody, has one or more changes in one or more CDRs that result in an increase in affinity for PD-1 as compared to its respective parent anti-PD-1 antibody. Affinity matured anti-PD-1 antibodies can be prepared, for example, by methods known in the art as described below: marks et al, 1992,Biotechnology 10:779-783 or barbes et al, 1994, proc.nat. Acad.sci, usa91: 3809-3813; shier et al, 1995, gene 169:147-155; yelton et al, 1995, immunol.155:1994-2004; jackson et al, 1995, j.immunol.154 (7): 3310-9; and Hawkins et al, 1992, j.moi.biol.226 (3): 889896; KS Johnson and RE Hawkins, "Affinity maturation of antibodies using phage display", oxford University Press 1996.

In general, the PD-1 binding proteins of the present disclosure will be measured as the preferred 10 as in the Biacore or KinExA or Fortibio assay ^-7 To 10 ^-10 Mol/liter (M), more preferably 10 ^-8 To 10 ^-10 Molar/liter, even more preferably 10 ^-9 To 10 ^-10 Or lower dissociation constant (KD), and/or at least 10- ⁷ M, preferably at least 10- ⁸ M, more preferably at least 10 ^-9 M, more preferably at least 10 ^-10 The binding constant (KA) of M binds to the antigen to which it is bound (i.e., PD-1). Any of more than 10 ^-4 KD values for M are generally considered to indicate non-specific binding. Specific binding of an antigen binding protein to an antigen or epitope can be determined in any suitable manner known, including, for example, surface Plasmon Resonance (SPR) assays, scatchard assays, and/or competitive binding assays (e.g., radioimmunoassays (RIA), enzyme Immunoassays (EIA), and sandwich competitive assays, as described in the present disclosure.

When the term "compete" is used in the context of antigen binding proteins (e.g., neutralizing antigen binding proteins or neutralizing antibodies) that compete for the same epitope, it is meant that competition is between antigen binding proteins, as determined by the following assay: the antigen binding protein (e.g., antibody or immunologically functional fragment thereof) to be detected prevents or inhibits (e.g., reduces) specific binding of the reference antigen binding protein (e.g., ligand or reference antibody) to the cognate antigen (e.g., PD-1 antigen or fragment thereof). Numerous types of competitive binding assays can be used to determine whether one antigen binding protein competes with another, such as: solid phase direct or indirect Radioimmunoassay (RIA), solid phase direct or indirect Enzyme Immunoassay (EIA), sandwich competition assay (see, e.g., stahli et al, 1983,Methodsin Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., kirkland et al, 1986, J.Immunol.137:3614-3619), solid phase direct labeling assay, solid phase direct labeling sandwich assay (see, e.g., harlow and Lane,1988,Antibodies,A Laboratory Manual (antibodies, laboratory Manual), cold Spring Harbor Press); direct labelling of RIA with the solid phase of the I-125 label (see, e.g., morel et al, 1988, molecular. Immunol. 25:7-15); solid phase direct biotin-avidin EIA (see, e.g., cheung, et al, 1990, virology 176:546-552); and directly labeled RIA (Moldenhauer et al, 1990, scand. J. Immunol. 32:77-82). Typically the assay involves the use of a purified antigen (on a solid surface or cell surface) that is capable of binding to a detection antigen binding protein that is unlabeled and a reference antigen binding protein that is labeled. The amount of label bound to the solid surface or cell is measured in the presence of the antigen binding protein to be tested to measure competitive inhibition. Typically, the antigen binding protein to be tested is present in excess. Antigen binding proteins identified by competition assays (competing antigen binding proteins) include: an antigen binding protein that binds to the same epitope as the reference antigen binding protein; and an antigen binding protein that binds to an epitope adjacent to an epitope bound sufficiently close to the reference antigen binding protein, the two epitopes spatially interfering with each other. Additional details regarding methods for assaying competitive binding are provided in the presently disclosed embodiments. Typically, when the competing antigen binding protein is present in excess, it will inhibit (e.g., reduce) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or more of the specific binding of the reference antigen binding protein to the cognate antigen. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

The term "cross-reactive" refers to the ability of the PD-1 binding proteins of the present disclosure to bind to PD-1 or an epitope thereof from a different species. For example, a single domain antibody or derived protein of the disclosure that binds human PD-1 may also bind PD-1 of another species. Cross-reactivity is measured by detecting specific reactivity with purified antigen, or binding or functional interaction with cells physiologically expressing PD-1 in binding assays (e.g., SPR and ELISA). Methods of determining cross-reactivity include standard binding assays as described in the present disclosure, such as Surface Plasmon Resonance (SPR) analysis, or flow cytometry.

The terms "inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition/blocking.

The term "inhibit growth" (e.g., involving a cell) is intended to include any measurable decrease in cell growth.

"homology", "identity" in the present disclosure refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both comparison sequences is occupied by the same base or amino acid monomer subunit, for example if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matched or homologous positions shared by the two sequences divided by the number of compared positions by 100. For example, when sequences are optimally aligned, if there are 6 matches or homologies at 10 positions in the two sequences, then the two sequences are 60% homologous; if there are 95 matches or homologies at 100 positions in the two sequences, then the two sequences are 95% homologous. In general, a comparison is made when two sequences are aligned to give the greatest percent homology.

"mutant sequence" refers to a nucleotide sequence and an amino acid sequence that have different degrees of percent sequence identity with the nucleotide sequence and the amino acid sequence of the present disclosure when subjected to mutation modification such as appropriate substitution, insertion, or deletion. The sequence identity may be at least 85%, 90%, or 95%, non-limiting examples include at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. Sequence comparison and percent identity determination between two sequences can be made by default settings of the BLASTN/BLASTP algorithm available on the National Center For Biotechnology Institute website.

In the case of amino acid substitutions, the substitution will preferably be a conservative amino acid substitution, which refers to the substitution of an amino acid residue with another amino acid residue of similar chemical structure, with little or no effect on the function, activity, or other biological property of the polypeptide. Such conservative amino acid substitutions are well known in the art, e.g. conservative amino acid substitutions are preferably those wherein one amino acid in the following groups (i) - (v) is replaced by another amino acid residue in the same group:

(i) Smaller aliphatic nonpolar or weakly polar residues: ala, ser, thr, pro and Gly;

(ii) Polar negatively charged residues and (uncharged) amides: asp, asn, glu and Gln;

(iii) Polar positively charged residues: his, arg and Lys; (iv) a larger aliphatic nonpolar residue: met, leu, ile, val and Cys; and

(v) Aromatic residues: phe, tyr and Trp.

Particularly preferred conservative amino acid substitutions are as follows: substitution of Ala with Gly or Ser; arg is replaced by Lys; asn is substituted with Gln or His; asp is substituted with Glu; cys is replaced by Ser; gln is substituted with Asn; glu is substituted with Asp; substitution of Gly with Ala or Pro; his is substituted with Asn or Gln; lie is substituted with Leu or Val; leu is substituted with Ile or Val; lys is substituted with Arg, gin or Glu; met is substituted with Leu, tyr or Ile; phe is substituted with Met, leu or Tyr; ser is substituted by Thr; thr is replaced by Ser; trp is substituted with Tyr; tyr is substituted by Trp or Phe; val is replaced by Ile or Leu.

The term "back mutation" refers to the mutation of an amino acid residue in the FR region of human antibody to an amino acid residue at a position corresponding to the original source antibody, typically in order to avoid the reduced immunogenicity caused by the humanized antibody while at the same time causing reduced activity, the variable region of the humanized antibody may be subjected to minimal back mutation to maintain the activity of the antibody.

The term "nucleic acid molecule" as used in this disclosure refers to DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In one embodiment, the vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. In another embodiment, the vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. The vectors of the present disclosure are capable of autonomous replication in a host cell into which they have been introduced (e.g., bacterial vectors and episomal mammalian vectors having a bacterial origin of replication) or may integrate into the genome of a host cell upon introduction into the host cell so as to replicate with the host genome (e.g., non-episomal mammalian vectors).

The expressions "cell", "cell line" and "cell culture" are used interchangeably herein and all such designations include progeny. Thus, "transformant" and "transformed cell" include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that all offspring may not be exactly identical in terms of DNA content due to deliberate or unintentional mutations. Including mutant progeny having the same function or biological activity as screened in the original transformed cell. Where different names are meant, they are clearly visible from the context.

The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include microbial (e.g., bacterial), plant, or animal cells. Bacteria that are susceptible to transformation include members of the Enterobacteriaceae family, such as strains of Escherichia coli (Escherichia coli) or Salmonella (Salmonella); the family of bacillus (bacillus) such as bacillus subtilis (Bacillus subtilis); pneumococci (pneumococci); streptococcus (Streptococcus) and haemophilus influenzae (Haemophilus influenzae). Suitable microorganisms include Saccharomyces cerevisiae (Saccharomyces cerevisiae) and Pichia pastoris (Pichia pastoris). Suitable animal host cell lines include CHO (chinese hamster ovary cell line), NS0 cells, 293 cells.

"pharmaceutical composition" means a mixture comprising one or more compounds of the present disclosure or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to organisms, facilitate the absorption of active ingredients and thus exert biological activity.

The terms "cancer" and "cancerous" and "tumor" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include, but are not limited to, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, superficial diffuse melanoma, malignant lentigo melanoma, acromelanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), hairy cell leukemia, chronic myelogenous leukemia, and post-transplant lymphoproliferative disorders (PTLD), and associated with komat (phagos), brain metastases and brain metastases, brain and brain associated vascular and head and neck cancer, brain metastases, and head and neck cancer associated with brain metastases. In certain embodiments, cancers suitable for treatment by the PD-1 binding proteins of the present disclosure include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, non-hodgkin's lymphoma (NHL), renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, kaposi's sarcoma, carcinoid cancer (carcinoid carcinoma), head and neck cancer, ovarian cancer, mesothelioma, and multiple myeloma. In some embodiments, the cancer is selected from: non-small cell lung cancer, glioblastoma, neuroblastoma, melanoma, breast cancer (e.g., triple negative breast cancer), gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma. Also, in some embodiments, the cancer is selected from: non-small cell lung cancer, colorectal cancer, glioblastoma, and breast cancer (e.g., triple negative breast cancer), including metastatic forms of those cancers.

The term "proliferative disorder" refers to a disorder associated with a degree of abnormal cell proliferation. In one embodiment, the proliferative disorder refers to cancer.

The term "tumor" refers to all neoplastic (neoplastic) cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer", "cancerous", "proliferative disorder" and "tumor" are not mutually exclusive when referred to in this disclosure.

"administration," "administering," and "treatment," when applied to an animal, human, subject, cell, tissue, organ, or biological fluid, refers to the contact of an exogenous drug, therapeutic, diagnostic, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "administration," "administration," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell includes contacting a reagent with the cell, and contacting the reagent with a fluid, wherein the fluid is in contact with the cell. "administration," "administering," and "treatment" also mean treating a cell in vitro and ex vivo by an agent, diagnosis, binding composition, or by another cell. "treatment" when applied to a human, veterinary or research subject refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

An "effective amount" comprises an amount sufficient to ameliorate or prevent a symptom or condition of a medical disorder. An effective amount is also meant to be an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on the following factors: for example, the condition to be treated, the general health of the patient, the route and dosage of administration, and the severity of the side effects. An effective amount may be the maximum dose or regimen that avoids significant side effects or toxic effects.

By "treating" is meant administering to a subject an internally or externally used therapeutic agent, such as a composition comprising any of the antibodies or antigen-binding fragments thereof of the present disclosure or a nucleic acid molecule encoding an antibody or antigen-binding fragment thereof, that has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered to a subject patient or population in an amount effective to alleviate one or more symptoms of the disease to induce regression of such symptoms or to inhibit the development of such symptoms to any clinically measurable extent. The amount of therapeutic agent (also referred to as a "therapeutically effective amount") effective to alleviate any particular disease symptom can vary depending on a variety of factors, such as the disease state, age, and weight of the patient, and the ability of the drug to produce a desired therapeutic effect in the patient. Whether a disease symptom has been reduced can be assessed by any clinical test method that a physician or other healthcare professional typically uses to assess the severity or progression of the symptom. While embodiments of the present disclosure (e.g., therapeutic methods or articles of manufacture) may be ineffective in alleviating each target disease symptom, it should be determined according to any statistical test method known in the art, such as Student t-test, chi-square test, U-test according to Mann and Whitney, kruskal-Wallis test (H test), jonckheere-Terpstra test, and Wilcoxon test, that the target disease symptom should be alleviated in a statistically significant number of patients.

The term "preventing cancer" refers to delaying, inhibiting or preventing the onset of cancer in a subject in which the onset of carcinogenesis or tumorigenesis has not been confirmed, but has been identified as a susceptibility to cancer, e.g., by genetic screening or other methods. The term also includes treating a subject having a pre-cancerous condition to terminate progression of the pre-cancerous condition to, or cause regression of, the malignancy.

"optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not.

The terms "subject", "patient" as used in the present disclosure mean a mammal, particularly a primate, particularly a human.

Detailed description of the preferred embodiments

The present disclosure is further described below in conjunction with the examples, which are not intended to limit the scope of the present disclosure. The experimental methods without specific conditions noted in the examples of the present disclosure are generally according to conventional conditions, such as the antibody technical laboratory manual of cold spring harbor, molecular cloning manual; or according to the conditions recommended by the manufacturer of the raw materials or goods. The reagents of specific origin are not noted and are commercially available conventional reagents.

Example 1: preparation of PD-1 antigen and protein for detection

PD-1 antigen design:

the amino acid sequences of the PD-1 antigen and the protein for detection were designed using human PD-1 as a PD-1 template (hereinafter, the PD-1 antigen is not specifically described and refers to human PD-1).

Human PD-1 full-length protein:

( Annotation: the double transversal line part is Signal peptide (Signal peptide); the single transverse line portion is the PD-1 extracellular region (Extracellular Domain) in which positions 35-144 are the Ig-like V-type 1 Domain (Ig-like V-type 1 Domain) and positions 70-77 are the portions that interact with CD 274; the dash-dot line portion is a transmembrane region portion (Transmembrane domain); the italic portion is an intracellular area (Cytoplasmic domain). )

SEQ ID NO：1

Monkey PD-1 full-length amino acid sequence:

( Annotation: the double transverse line part is a signal peptide; the single transverse line portion is the PD-1 extracellular region, wherein positions 38-127 are the V-Set domain and positions 39-125 are the Ig-like V-type 1 domain; the dash-dot line portion is a transmembrane region portion (Transmembrane domain); the italic portion is an intracellular area (Cytoplasmic domain). )

SEQ ID NO：2

Screening and detection of human PD-1 antigen (as a commercial product (Sino Biological Cat.10377-H08H)):

( Annotation: the single transverse line part is PD-1 extracellular region; the italic part is His-tag. )

SEQ ID NO：3

Screening and detection of human PD-1-Fc antigen (commercial product (Baiying organism: B3789)):

( Annotation: the cross line is marked as extracellular region; the italic part is a human Fc marker. )

SEQ ID NO：4

Human PD-L1 antigen for detection (commercial product (Sino Biological cat: 10084-H08H-B)):

( Annotation: the cross line is divided into PD-L1 extracellular region; the italic part is His-tag. )

SEQ ID NO：5

Human PD-L2 antigen for detection (commercial product (Sino Biological cat: 10292-H08H-B)):

( Annotation: the cross line is divided into PD-L2 extracellular region; the italic part is His-tag. )

SEQ ID NO：6

Example 2 screening of Positive sequences specifically binding to human PD-1

Human PD-1 proteins (ACRO, cat#PD-1-H5259 and ACRO, cat#PD-1-H5221) were immunized against two-head Bactrian camels, 5mL of serum from a camel before immunization was taken and serum was isolated. After mixing Freund's complete adjuvant with antigen volume 1:1, the camels were subjected to subcutaneous multipoint immunization (immunization dose 100. Mu.g protein/one/each). Boosting was performed every two weeks, and titers were determined four times after immunization. Plates (100. Mu.L/well) were coated with 5. Mu.g/mL PD-1-his protein overnight at 4 ℃. After the next day of washing, 4% skim milk powder was added for blocking at 37 ℃ for 2h. After washing, serum from different gradient dilutions of camel was added and incubated for 1h at 37 ℃. Negative controls were preimmune serum (1:1000 dilution) and PBS solution. After the incubation, the cells were washed three times with PBST, and rabbit anti-camel polyclonal antibody (1:1000 dilution) was added and incubated at 37℃for 1 hour. After washing again, secondary goat anti-mouse alkaline phosphatase-labeled antibody (1:1000 dilution) was added and incubated at 37℃for 1 h. Finally, alkaline phosphatase color development solution is added for washing, and 2M sulfuric acid is used for stopping, and the absorption value is read at the wavelength of 450 nm. Titers were detected after 1:25600 dilution. And collecting camel peripheral blood for stock building, wherein the potency is qualified.

Camel peripheral blood isolated lymphocytes with a cell count of 1.2X10 ⁸ Trizol reagent was added to resuspend (1X 10) ⁷ Individual cells/mL Trizol) to lyse the cells, left on ice for 5min; centrifuging at 13000rpm for 3min, collecting supernatant, and discarding precipitate; adding 1/5 volume of chloroform, shaking vigorously for 30-60s, and standing in ice bath for 2min; centrifuging at 13000rpm for 10min, and sucking the upper aqueous phase layer into a new 1.5mL tube; adding isopropanol with equal volume, mixing, standing at-20deg.C for 30min; centrifuging at 13000rpm for 10min, removing supernatant, and retaining precipitate; adding pre-cooled 75% ethanol to wash precipitate, and standing at room temperature for 5-10min; adding 600 mu L of deionized water removed by RNase, re-dissolving to obtain RNA, carrying out reverse transcription to obtain cDNA, and constructing a phage library.

Single domain antibodies with high affinity for PD-1 antigen protein were obtained by screening phage libraries, binding to 1mg Dynabeads MyOne streptavidin T1 with 20 μg PD-1-avi-biotin protein, standing at 37 ℃ for one hour, blocking with 2% skim milk at room temperature for 2 hours, adding camel heavy chain single domain antibody phage display library, and allowing to act at room temperature for 1 hour. The unbound phage were removed by washing 9 times with PBST (0.05% Tween-20) solution. Phage that specifically bound to PD-1 were eluted with 1mg/mL trypsin and infected with E.coli TG1 grown in log phase, phage were generated and purified for the next round of screening. The same screening process was repeated 2-3 times later. Positive clones were enriched.

96 monoclonal colonies were picked from screening the enriched positive clones and packaged as phage single chain antibodies for phage ELISA testing. ELISA plates were coated with 2. Mu.g/mL of PD-1-his protein, and phage supernatants diluted with blocking solution were added and detected with anti-M13 HRP. Clones with OD450 values greater than 0.5 were sequenced for ELISA binding assays yielding 51 specific sequences.

EXAMPLE 3 construction of intact monoclonal antibodies

The 51 specific sequences obtained in the phage library screening of example 2 were used to construct complete antibodies, and then 27 of them were determined to have strong binding ability and to inhibit the interaction of PD-1 with PD-L1 by ELISA binding experiments and ELISA competition experiments, and the results are shown in Table 1.

TABLE 1 ELISA detection results of PD-1 antibodies

Its complete VHH sequence is as follows:

＞2#

＞4#

＞6#

＞7#

＞11#

＞19#

＞32#

＞41#

＞54#

＞56#

＞59#

＞61#

＞62#

＞68#

＞104#

＞106#

＞107#

＞108#

＞109#

＞112#

＞113#

＞114#

＞116#

＞118#

＞119#

＞122#

＞123#

the above sequence SEQ ID NO:7-33, the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the sequence is italic FR sequence, and the sequences of CDR1, CDR2 and CDR3 are underlined. The numbering convention for PD-1 single domain antibodies provided by the present disclosure is Kabat, and the CDR sequences are summarized in Table 2.

TABLE 2 CDR sequences of PD-1 Single-Domain antibodies

The VHH sequence was fused to a human IgG1-Fc (CH 2-CH 3) segment sequence and constructed into a PTT5 expression vector, and the sequence of the linked human IgG1-Fc can be as follows:

The following is the fusion of the VHH sequence to the full protein sequence of the human Fc (CH 2-CH 3) segment, the single underlined sequence of the human IgG1-Fc (CH 2-CH 3) segment (SEQ ID NO: 103), and the double underlined sequence of the linker sequence. The protein sequences are as follows (for example, 32#, 7#, 106#, 107# as are other PD-1 single domain antibodies):

32#-IgG1：

7#-IgG1：

106#-IgG1：

107#-IgG1：

example 4 humanized engineering of Single Domain antibodies

Through carrying out three-dimensional structural homology modeling on the selected specific PD-1 single domain antibody molecule, combining with the result of comparison with a V-base human germline sequence database, carrying out IMGT human antibody heavy chain variable region germline gene database, selecting heavy chain variable region germline genes with high homology with the screened antibodies as templates, and transplanting CDRs of camel-derived single domain antibodies into corresponding human templates to form variable region sequences with the sequence of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4. And carrying out three-dimensional structural simulation and analysis again on the transplanted single-domain antibody, and carrying out back mutation on specific sites in the FR region, which influence the structural morphology of the CDR region. The specific sequence of humanization obtained is as follows:

2#_Hu_1：

7#_Hu_1：

7#_Hu_2：

7#_Hu_3

7#_Hu_4

7#_Hu_5

7#_Hu_6

32#_Hu_1

32#_Hu_2

32#_Hu_3

32#_Hu_4

32#_Hu_5

32#_Hu_6

32#_Hu_7

61#_Hu_1

61#_Hu_2

106#_Hu_1

106#_Hu_2

106#_Hu_3

106#_Hu_4

106#_Hu_5

106#_Hu_6

106#_Hu_7

106#_Hu_8

106#_Hu_9

107#_Hu_1

107#_Hu_2

107#_Hu_3

107#_Hu_4

112#_Hu_1

as shown in the above sequence, during humanization and back mutation, part of the CDRs of the antibody are changed, such as the CDR1 sequence shown in the YNMMS (SEQ ID NO: 113) obtained by mutation of T35S in 7# Hu_5; 7# Hu_6 has F33Y and T35S mutations, and the obtained YNYMS (SEQ ID NO: 114) shows the CDR1 sequence; 106_hu_1 to 6 had a61D mutation, and the obtained CDR2 sequence shown in VVDRFGGTIYADSVKG (SEQ ID NO: 71); 112_hu_1 had a mutation of A61D, resulting in a CDR2 sequence shown in VVDRFGGIIYADSVKG (SEQ ID NO: 93).

A full protein sequence was constructed using the method of example 4, in which a humanized PD-1 single domain antibody was fused to the Fc (CH 2-CH 3) segment of hIgG1, with the hIgG1-Fc (CH 2-CH 3) segment sequence (shown in SEQ ID NO: 103) being underlined as a linker sequence. The protein sequence is as follows (for example, 32_hu_3-IgG1, as are other humanized PD-1 single domain antibodies):

32#_hu_3-hIgG1：

the method of example 4 was used to construct a full protein sequence of a humanized PD-1 single domain antibody fused to the Fc (CH 2-CH 3) segment of hIgG4, with the hIgG4-Fc (CH 2-CH 3) segment sequence (shown in SEQ ID NO: 108) underlined.

The sequence of the linked human IgG4-Fc is shown below:

the sequences of the antibodies obtained were as follows:

32#_hu_3_hIgG4：

7#_hu_4_hIgG4：

106#_hu_1_hIgG4：

107#_hu_4_hIgG4：

HEK293 cells were transfected with the plasmid, and after 6 days, the expression supernatant was collected, centrifuged at high speed to remove impurities, and purified with Protein A column. Equilibrated with PBS until a280 reading drops to baseline. Eluting target protein with acidic eluent with pH of 3.0-3.5, and neutralizing with 1M Tris-HCl with pH of 8.0-9.0. After proper concentration of the eluted sample, the eluted sample was further purified by gel chromatography Superdex200 (GE) equilibrated with PBS to remove the polymer, and the monomer peak was collected and split-charged for use. The PD-1 single domain antibodies of the present disclosure were obtained upon detection.

Example 5 affinity assay of PD-1 Single-Domain antibodies with PD-1

To examine the in vitro binding capacity of the screened PD-1 single domain antibodies to human PD-1 protein and monkey PD-1, human PD-1 (Sino Biological Cat.10377-H08H) and monkey PD-1 (Sino Biological Cat.90311-C08H) were used for in vitro binding assays by ELISA binding assays.

The negative control of this example was PBS, the positive control was Opdivo (available from Shanghai Yersi Chemie (chempatner) lot: 180612001), and part of the experiment used the IgG4 type PD-1 antibody in WO2017054646 (WO 2017054646) as the positive control, the sequence was as follows:

PD-1 antibody heavy chain:

PD-1 antibody light chain:

PD-1 antibody-carrying protein was diluted to 2. Mu.g/mL with PBS buffer at pH7.4 to 100. Mu.gThe volume of L/well was added to a 96-well ELISA plate (burning, 9018 25/box 96well clear flat bottom plate) and left to stand overnight at 4℃for 16-20 hours. After discarding the liquid, the plate was washed three times with PBST (pH 7.4,0.05% Tween-20) buffer, and then 2% BSA blocking solution (300. Mu.L/well) diluted with PBS buffer was added thereto, and the plate was incubated at 37℃for 2 hours for blocking. After blocking was completed, blocking solution was discarded, and after washing the plate 3 times with PBST buffer, PD-1 antigen (Sino Biological Cat.10377-H08H) protein was added at an initial concentration of 30. Mu.g/mL, diluted by a three-fold ratio of PBS buffer for 8 gradients, and incubated in an incubator at 37℃for 1 hour. After the incubation, the reaction solution in the ELISA plate was discarded, the plate was washed 6 times with PBST, and 100. Mu.L/HRP-labeled anti-his secondary antibody (Abcam ab 1187) (1:5000 dilution) was added to each well, and incubated at 37℃for 1 hour. After washing the plate 6 times with PBST, 100 μLTMB chromogenic substrate was added, incubated at room temperature for 3-5min, 100 μL of 1M sulfuric acid was added to terminate the reaction, absorbance was read at 450nm using a SpectraMax M5 microplate reader, and antibody binding to antigen was calculated ₅₀ Values. EC of partial antibodies ₅₀ The results are shown in Table 3. The results show that the two antibodies can be combined with human and monkey PD-1 antigens well.

TABLE 3 binding force of different PD-1 antibodies to human, monkey PD-1 antigen EC ₅₀ (nM)

(note: "/" indicates undetected)

In addition, the dissociation constants of the PD-1 antibodies and the PD-1 proteins were also determined by a Biacore 8K (GE Healthcare) instrument. The method comprises the steps of firstly, covalently coupling an anti-human IgG Fc antibody (GE Healthcare, cat. # BR-1008-39) to a CM5S series chip, capturing the PD-1 antibody to be detected to the surface of the chip through affinity, then flowing PD-1 proteins (SEQ ID NO: 3) with different concentrations on the surface of the chip, detecting reaction signals in real time by using a Biacore instrument to obtain a binding dissociation curve, and obtaining the binding dissociation curve through fittingThe binding force constant is obtained. The experimental use solution was HBS-P solution (10mM HEPES,150mM NaCl,0.005%P20,pH 7.4). At the end of each experimental cycle, 3M MgCl was used ₂ The chip is cleaned and regenerated by the solution. The affinity results of the partial antibodies are shown in table 4. The results show that the antibodies obtained by the screening of the present disclosure have an affinity for PD-1 comparable to the positive control.

TABLE 4 affinity of different PD-1 antibodies for human PD-1

Antibody numbering	Antigens	k a (1/Ms)	k d (1/s)	K D (M)
					7#	PD-1	1.36E+05	2.81E-04	2.06E-09
32#	PD-1	3.25E+05	2.07E-03	6.35E-09
					32#_hu_1	PD-1	1.79E+05	2.91E-03	1.63E-08
32#_hu_2	PD-1	1.67E+05	1.56E-03	9.36E-09
					32#_hu_3	PD-1	2.20E+05	2.01E-03	9.11E-09
32#_hu_4	PD-1	1.75E+05	3.53E-03	2.02E-08
					32#_hu_5	PD-1	1.62E+05	3.19E-03	1.96E-08
61#	PD-1	1.54E+05	8.19E-04	5.33E-09
					61#_hu_1	PD-1	2.26E+05	4.61E-03	2.04E-08
106#	PD-1	7.94E+04	4.77E-04	6.01E-09
					107#	PD-1	9.65E+04	7.82E-04	8.10E-09
Opdivo	PD-1	5.91E+05	1.45E-03	2.45E-09

We have also examined the affinity of different PD-1 antibodies for human PD-1 by Biacore T200 (GE Healthcare) and the results are shown in Table 5.

TABLE 5 affinity of different PD-1 antibodies for human PD-1

Antibody numbering	k a (1/Ms)	k d (1/s)	K D (M)
				32#_hu_3_hIgG4	1.05E+05	2.01E-03	1.92E-08
7#_hu_4_hIgG4	4.72E+04	5.84E-03	1.24E-07
				106#_hu_1_hIgG4	8.17E+03	7.05E-04	8.63E-08
107#_hu_4_hIgG4	9.40E+03	1.20E-03	1.28E-07
				PD-1 antibody (WO 2017054646)	6.18E+04	4.79E-04	7.75E-09

Example 6 blocking the binding of PD-1 to PD-L1, PD-L2 by PD-1 Single-Domain antibodies

Functional assays for PD-1 antibodies were tested by ELISA competition assays that blocked binding between PD-1 and PD-L2.

The Fc-tagged PD-1 fusion protein was diluted to a concentration of 2. Mu.g/mL with PBS buffer at pH7.4 and 96-well ELISA plates were added at a volume of 100. Mu.L/well (burning, 9018)25/box 96well clear flat bottom plate), left to stand overnight at 4℃for 16-20 hours. After discarding the liquid, the plate was washed three times with PBST (pH 7.4,0.05% Tween-20) buffer, and then 300. Mu.L/well of 2% BSA blocking solution diluted with PBS buffer was added thereto, and the plate was incubated at 37℃for 2 hours for blocking. After blocking was completed, blocking solution was discarded, and after washing the plate 3 times with PBST buffer, PD-L1 and PD-L2 proteins with biotinylation were added at a protein concentration of 6. Mu.g/ml, 50. Mu.L was added per well, followed by addition of PD-1 antibody protein at an initial concentration of 30. Mu.g/ml, three-fold dilution of 6 gradients with PBS buffer, and incubation in an incubator at 37℃for 1 hour. After the incubation, the reaction solution in the ELISA plate was discarded, the plate was washed 6 times with PBST, 100. Mu.L/well of HRP-labeled anti-SA secondary antibody (Peroxidase-conjugated Streptavidin, jackson 136861) diluted (1:500) with PBS (0.5% BSA) was added, and incubated at 37℃for 1 hour. After washing the plate 6 times with PBST, 100. Mu.L/well TMB chromogenic substrate was added, incubated at room temperature for 3-5min, 1M sulfuric acid was added to terminate the reaction, absorbance was read at 450nm using a SpectraMax M5 microplate reader, and the binding IC of the antibody to the antigen was calculated ₅₀ Values. IC of partial antibody ₅₀ The results are shown in Table 4. The results show that the antibodies compete with both PD-L1 and PD-L2 for binding to PD-1, the negative control is PBS, and the positive control is Opdivo (available from Shanghai Zhi Chemie (chemhartner)) lot: 180612001. The results of blocking the binding of PD-1 to PD-L1 by a portion of the antibodies are shown in tables 6 and 7.

TABLE 6 competition of different PD-1 antibodies for PD-1 antigen with the IC of PD-L1 and PD-L2 ₅₀ (nM)

TABLE 7 competition of different PD-1 antibodies for PD-1 antigen and PD-L1 IC ₅₀ (nM)

Antibody numbering	IC blocking binding of PD-1 to PD-L1 50
		32#_hu_3_hIgG4	2.42
7#_hu_4_hIgG4	1.22
		106#_hu_1_hIgG4	3.14
PD-1 antibody (WO 2017054646)	2.79
		Negative control (PBS)	9999

EXAMPLE 7 binding of PD-1 Single-Domain antibodies to in vitro cell surface PD-1

Cell line CHO-PD-1 with stable high expression of PD-1 is collected, and each tube is 5 multiplied by 10 ⁵ And (3) cells. The PD-1 antibody was diluted in a gradient of 0.01,0.1,1, 10. Mu.g/mL and incubated on CHO-PD-1 ice for 1 hour. After washing with PBS, FITC anti-human IgG (1:100) was added to each tube and incubated in the dark for 1 hour. After washing with PBS, the tube was resuspended in 100. Mu.L of PBS and fluorescence detected on a flow cytometer. Binding of PD-1 antibodies to CHO-PD-1 cells was dose dependent by quantitative analysis of the average fluorescence intensity obtained by each dose of antibody treatment. Binding force of partial antibodies EC ₅₀ The results are shown in table 8, and the results show that the binding force of the antibodies obtained by the screening of the present disclosure (such as 2#, 32#, 32# hu_1, 32# hu_2, 32# hu_3, 61#, 32# hu_3_higg4, 7# hu_4_higg4, 106# hu_1_higg4, 107# hu_4_higg4) with PD-1 is significantly better than that of the positive control Opdivo.

The negative controls of examples 7-9 were NC (which had the same constant region IgG4 as the subject antibody, but the variable region did not recognize antigen PD-1), and the positive controls were Opdivo (available from Shanghai intelligent chemistry (chempatner) lot: 180612001).

TABLE 8 binding force of different PD-1 antibodies to cell surface antigen PD-1 EC ₅₀ (nM)

Antibodies to	EC 50
		Negative Control (NC)	637030
Positive control (Opdivo)	66.3
		2#	18.5
32#	2.9
		32_hu_1#	7.3
32_hu_2#	4.8
		32_hu_3#	6.1
61#	16.7
		32#_hu_3_hIgG4	3.912
7#_hu_4_hIgG4	3.614
		106#_hu_1_hIgG4	8.926
107#_hu_4_hIgG4	11.95

Example 8 blocking of PD-1 binding to PD-L1 on cells by PD-1 Single-Domain antibodies

Cell line CHO-PD-1 with stable high expression of PD-1 is collected, and each tube is 5 multiplied by 10 ⁵ And (3) cells. The PD-1 antibodies were gradient diluted to 50, 16.67, 5.55, 1.85, 0.617, 0.205, 0.069 μg/mL and incubated on CHO-PD-1 ice for 1 hour. After washing with PBS, each tube was incubated on ice with 1. Mu.g/mL PD-L1-mIgG2a protein for 1 hour and PBS was again washed. PE anti-mouse IgG2a (1:300) was added to each tube and incubated on ice for 1 hour. After washing with PBS, the tube was resuspended in 100. Mu.L of PBS and fluorescence detected on a flow cytometer. The blocking of binding of PD-L1 protein to CHO-PD-1 cells by PD-1 antibodies is shown in tables 9 and 10, and the binding strength is dose dependent by quantitative analysis of the average fluorescence intensity obtained by each dose of antibody treatment. The results show that the antibodies obtained by the screening of the present disclosure (e.g., 7#, 32#, 32# hu_1, 32# hu_2, 32# hu_3, 106#, 107# 112#, 32# hu_3_hIgG4, 7# hu_4_hIgG4, 106# hu_1_hIgG4, 107# hu_4_hIgG4) have a greater ability to block PD-L1 binding to PD-1 than the positive control opdivo.

TABLE 9 blocking the ICs of PD-L1 proteins with cell surface antigen PD-1 by different PD-1 antibodies ₅₀ (nM)

Antibody numbering	IC	50
			7#	3.8
32#	2.3
		32#_hu_1	6.2
32#_hu_2	4.3
		32#_hu_3	4.3
61#	5.0
		106#	4.8
107#	5.0
		112#	6.1
Positive control (Opdivo)	43.9
		Negative Control (NC)	9999

TABLE 10 blocking PD-L1 proteins by different PD-1 antibodiesIC with cell surface antigen PD-1 ₅₀ (nM)

Antibody numbering	IC	50
			32#_hu_3_hIgG4	0.6513
7#_hu_4_hIgG4	0.613
		106#_hu_1_hIgG4	2.432
107#_hu_4_hIgG4	4.914
		PD-1 antibody (WO 2017054646)	0.5667
Negative Control (NC)	9999

Example 9 PD-1 Single-Domain antibodies promote Mixed lymphocyte secretion of cytokines in vitro

Isolation of CD14 from fresh or resuscitated human PBMCs by EasySep human CD14 positive screening kit (STEMCELL technologies, 17858) ⁺ Monocytes. Isolated CD14 ⁺ Cell differentiation kit according to monocyte derived dendritic cell (R&D system, CDK 004) by addition of IL-4 and GM-CSF factor for 6 days followed by addition of TNF- αStep 3, induction is carried out for 3 days to become mature DC.

Isolation of CD3 by human PBMC Using EasySep human CD3 Positive screening kit (STEMCELL technologies, 18051) ⁺ T cells (different donor source than DCs). The isolated DCs were compared to T cells 10:1, while adding low endotoxin control PD-1 antibody, 5 days after culturing, using human IFNgamma quaternikine ELISA kit (R&D system, DIF 50) detects ifnγ secretion by activated T cells.

After mixed lymphocyte culture, ifnγ secretion amounts are shown in table 11, table 12, and fig. 1 and 2. The results show that the plurality of PD-1 antibodies obtained by screening are effective in enhancing T cell activation and secreting IFNγ.

TABLE 11 different antibodies promote IFN gamma secretion

Antibody numbering	IFNγ secretion amount (pg/ml)
		7#	963.5
32#	555.3
		32#hu_3	1031.5
106#	1164.2
		107#	1776.6
Negative Control (NC)	49
		Positive control (Opdivo)	1181.5

TABLE 12 different antibodies promote IFN gamma secretion

Antibody numbering	IFNγ secretion amount (pg/ml)
		32#_hu_3_hIgG4	877.3
7#_hu_4_hIgG4	759.9
		106#_hu_1_hIgG4	736.94
PD-1 antibody (WO 2017054646)	549.8
		Negative Control (NC)	163

Example 10 inhibition of tumor growth by PD-1 Single-Domain antibodies in a mouse colon cancer model

Animal experiments were performed by Shanghai Ai Fei pharmaceutical technologies Inc., using HuPD-1 humanized transgenic mice, female, 6-8 weeks old purchased from Nanjing Galaxy biological medicine Inc.

PBS resuspended mouse colon cancer cell line MC38 cells at 5×10 ⁵ A concentration of 0.1mL, 0.1 mL/volume was inoculated subcutaneously in the right flank of HuPD-1 humanized mice. When the average tumor volume reaches 100mm ³ (70-120mm ³ ) At the time, mice with moderate tumor volumes of individuals are selected to be grouped, and the tumor volumes on the right side are used as grouping basis. The administration is started on the day of grouping, and the administration dosage is 0.3mg/kg; the frequency of administration is from once every three days for a total of three weeks; the administration is intravenous injection.

The PD-1 antibodies inhibited colon cancer tumor growth in mice as shown in table 13 and figure three. The results showed that on day 24, the tumor inhibition ratio of the positive control was 47.3%; the tumor inhibition ratio of 32# hu_3_hIgG4 is 50.8%; the tumor inhibition ratio of 7# hu_4_hIgG4 is 68.4%; the tumor inhibition ratio of 106# hu_3_hIgG4 is 64.4%, and the growth of tumors in mice can be effectively inhibited.

TABLE 13 PD-1 antibody inhibition of mouse colon cancer tumor growth results

Claims (25)

1. A pd-1 binding protein comprising at least one immunoglobulin single variable domain comprising three complementarity determining regions CDR1, CDR2, and CDR3, wherein:

   the amino acid sequence of CDR1 is shown in SEQ ID NO:62, the amino acid sequence of CDR2 is set forth in SEQ ID NO: 63. 68, 69, 70, 72, 77, the amino acid sequence of CDR3 is as set forth in any one of SEQ ID NOs: shown at 64.
2. 2. The PD-1 binding protein according to claim 1, which is an antibody that binds to PD-1 or an epitope thereof.
3. 3. The PD-1 binding protein according to claim 1, which is a camelid antibody, a chimeric antibody or a humanized antibody.
4. 4. The PD-1 binding protein according to claim 1, which is an sdAb or a bispecific, multispecific antibody.
5. 5. The PD-1 binding protein according to claim 1, wherein the immunoglobulin single variable domain is a VHH.
6. 6. The PD-1 binding protein of claim 5, wherein the VHH is a humanized VHH or an affinity-matured VHH.
7. 7. The PD-1 binding protein according to claim 1, further comprising an Fc region.
8. 8. The PD-1 binding protein according to claim 7, wherein the Fc region is selected from the group consisting of: human IgG1, igG2, igG3, igG4 Fc region.
9. 9. The PD-1 binding protein of claim 7, wherein the Fc region is human IgG4 Fc or human IgG4 Fc with S228P substitution.
10. 10. The PD-1 binding protein according to claim 1, comprising a structure represented by the formula X-L-Y, wherein X represents an immunoglobulin single variable domain, L represents a linker present or absent, and Y represents an Fc region.
11. 11. The PD-1 binding protein according to claim 10, which is a monomer or dimer.
12. 12. The PD-1 binding protein of claim 10, which forms a homodimer through the Fc region.
13. 13. The PD-1 binding protein according to claim 10, wherein L is a linker that is 1-20 amino acids in length.
14. 14. The PD-1 binding protein of claim 13, wherein L is Ala-Ser-Gly-Ser.
15. 15. The PD-1 binding protein according to any one of claims 1-14, which comprises the amino acid sequence of SEQ ID NO:8-9, 11-16, 19-20, 42-46, 123-124.
16. 16. The PD-1 binding protein according to any one of claims 1-14, which is set forth in SEQ ID NO: 34. 107, 109.
17. 17. A nucleic acid molecule encoding the PD-1 binding protein of any one of claims 1-16.
18. 18. A vector comprising the nucleic acid molecule of claim 17.
19. 19. A host cell comprising the vector of claim 18.
20. 20. The host cell of claim 19, wherein the host cell is selected from the group consisting of: bacteria, yeast, mammalian cells.
21. 21. The host cell of claim 19, wherein the host cell is selected from the group consisting of: coli, pichia pastoris, chinese hamster ovary cells, human embryonic kidney 293 cells.
22. 22. A method of preparing the PD-1 binding protein of any one of claims 1-16, comprising the steps of:

    culturing the host cell of claim 19;

    recovering the PD-1 binding protein.
23. 23. The method of claim 22, comprising the steps of: purifying and/or modifying the PD-1 binding protein.
24. 24. A pharmaceutical composition comprising:

    a therapeutically effective amount or a prophylactically effective amount of the PD-1 binding protein of any one of claims 1-16 and/or the nucleic acid molecule of claim 17; and

    one or more pharmaceutically acceptable carriers.
25. 25. Use of the PD-1 binding protein of any one of claims 1-16 or the nucleic acid molecule of claim 17 or the pharmaceutical composition of claim 24 in the manufacture of a medicament for the prevention and/or treatment of a cancer selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, renal cancer or squamous cell carcinoma.

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