CN114222759A - 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 derived 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

This application claims priority to chinese patent application 201910842676.8 filed on 6/9/2019.

Technical Field

The disclosure belongs to the field of biomedicine, and relates to an anti-PD-1 single-domain antibody, a derived protein thereof and application thereof as a medicament for preventing and/or treating PD-1 related diseases.

Background

PD-1(Programmed Cell death-1) belongs to the CD28 receptor family and 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, and has a structure similar to CTLA-4, but PD-1 lacks the MYPPPY sequence that binds to B7-1 and B7-2.

PD-1 is predominantly expressed 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). Neither PD-L1 nor PD-L2 bound to other members of the CD28 receptor family. PD-L1 is widely expressed in lymphocytes (e.g., CD 4)⁺T cells and CD8⁺T cells, macrophages, etc.) as well as, for example, peripheral tissues, various tumor cells, and virus-infected cells, etc.

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

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 (zuo 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, and 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 reverses immunosuppression, while inhibiting the effects of PD-1 and PD-L1, PD-L2 simultaneously results in synergy (Iwai et al 2002, Proc. nat' l.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) with a deleted light chain, and variable region fragments (VHHs) derived from this antibody are called single domain antibodies (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 diameter is 2.5nm, the length is 4nm, and the antibody with the minimum known antibody has complete antigen binding activity.

Single domain antibodies also contain 3 CDRs, of which CDR3 plays a major role for affinity. Compared with the human antibody VH, the CDR3 of the single domain antibody is longer, can form a bulge loop (loop) structure, and can go deep into the antigen, thereby better combining the antigen. Thus, VHH is characterized by high affinity and high specificity. In addition, the hydrophobic residue of FR2 in the single domain antibody is replaced by a hydrophilic residue, which is more water soluble and less prone to aggregate formation. 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, WO 2018127711. Among these, WO2018127709, WO2018127710, WO2018127711 relate to single domain antibodies that do not block the binding of PD-1 and a ligand. At present, single-domain antibodies against PD-1 are in an early development stage on a global scale, and no single-domain antibody drug targeting PD-1 is on the market.

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 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, and more particularly, an anti-PD-1 single domain antibody, a protein derived therefrom, and a medical use thereof.

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 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 binding protein 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 joint is a flexible joint, e.g. G₄S、GS、GAP、ASGS、(G ₄S) _nAnd 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 asvkgrf. In some embodiments, the immunoglobulin single variable domain comprises three complementarity determining regions CDR1, CDR2, and CDR3, with dsvkgrfs or asvkgrfs located in CDR 2.

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

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

CDR1 comprises SEQ ID NO: 62, CDR2 comprises X₁IDSVGX ₂TX ₃YX ₄X ₅SVKG (SEQ ID NO: 115) wherein X₁Selected from S or T, X₂Selected from T or A, X₃Selected from D, N or G, X₄Selected from T or A, X₅Selected from N or D, CDR3 comprises SEQ ID NO: 64; or

CDR1 comprises SEQ ID NO: 81, CDR2 comprises VVDRFFGGX₆IYAX ₇SVKX ₈(SEQ ID NO: 116) wherein X₆Selected from I or T, X₇Selected from A or D, X₈Selected from K or D, CDR3 comprising GSYTX₉X ₁₀X ₁₁SCX ₁₂PDAL (SEQ ID NO: 117) wherein X₉Selected from S or D, X₁₀Selected from A or D, X₁₁Selected from N or G, X₁₂Is selected from Q or H; or

CDR1 comprises YNX₁₃MX ₁₄(SEQ ID NO: 118) wherein X₁₃Selected from F or Y, X₁₄Selected from S or T, CDR2 comprises SEQ ID NO: 66, CDR3 comprises the amino acid sequence shown in SEQ ID NO: 67; or

CDR1 comprises SEQ ID NO: 84, CDR2 comprises VINTGX ₁₅NX ₁₆TYYADSVKG (SEQ ID NO: 119), wherein X₁₅Selected from A or T, X₁₆Selected from S or T, CDR3 comprises SEQ ID NO: 64; or

CDR1 comprises SEQ ID NO: 78, CDR2 comprises X₁₇YPTAGX ₁₈TYX ₁₉X ₂₀DSX ₂₁KG (SEQ ID NO: 120) wherein X₁₇Selected from L or I, X₁₈Selected from R or K, X₁₉Selected from Y or F, X₂₀Selected from G or A, X₂₁Selected from M or V, CDR3 comprises SEQ ID NO: 80; or

The CDR1, CDR2, CDR3 comprise SEQ ID NOs: 59. 60, 61; or

The CDR1, CDR2, CDR3 comprise SEQ ID NOs: 74. 75, 76; or

The CDR1, CDR2, CDR3 comprise SEQ ID NOs: 88. 89 and 90;

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

In some embodiments, the PD-1 binding proteins of the present disclosure comprise any one selected from the group consisting of:

CDR1 comprises SEQ ID NO: 62, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 63. 68, 69, 70, 72, 77, CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 64 or 73; or

CDR1 comprises SEQ ID NO: 81 and CDR2 comprises the amino acid sequence shown in SEQ ID NO: 71. 82, 91, 93, 94, CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 83. 92, 95 or a pharmaceutically acceptable salt thereof; or

CDR1 comprises SEQ ID NO: 65. 113, 114, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 66, CDR3 comprises the amino acid sequence shown in SEQ ID NO: 67; or

CDR1 comprises SEQ ID NO: 84, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 85. 102, CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 86; or

CDR1 comprises SEQ ID NO: 78, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 79. 87, 99, 100, 101, CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 80, or a pharmaceutically acceptable salt thereof.

In some embodiments, the PD-1 binding proteins of the present disclosure comprise any one selected from the group consisting of:

CDR1 comprises SEQ ID NO: 62, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 63. 68, 69, 70, 72, 77, CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 64; or

CDR1, CDR2, CDR3 comprise SEQ ID NOs: 62. 63, 73; or

CDR1 comprises SEQ ID NO: 81 and CDR2 comprises the amino acid sequence shown in SEQ ID NO: 71. 82, CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 83; or

CDR1 comprises SEQ ID NO: 81 and CDR2 comprises the amino acid sequence shown in SEQ ID NO: 91. 93, CDR3 comprises the amino acid sequence shown in SEQ ID NO: 92; or

CDR1, CDR2, CDR3 comprise SEQ ID NOs: 81. 94 and 95, respectively.

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 which has 3, 2, 1 amino acid differences with the amino acid sequence.

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

(i) CDR1 comprises a sequence selected from SEQ ID NO: 59. 62, 65, 74, 78, 81, 84, 88, 93, or an amino acid sequence that differs therefrom by 3, 2, 1 amino acids; and/or

(ii) CDR2 comprises a sequence selected from SEQ ID NO: 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 that differs therefrom by 3, 2, 1 amino acids; and/or

(iii) CDR3 comprises a sequence selected from SEQ ID NO: 61. 64, 67, 73, 76, 80, 83, 86, 90, 92, 95, 98, or an amino acid sequence that differs therefrom by 3, 2, 1 amino acids.

In some embodiments, one or more of the above CDRs are grafted (graft) on 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 such CDR grafting are known in the art.

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

In some embodiments, the antibody or antigen-binding fragment thereof is a camelid antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or a 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 is 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 is identical to SEQ ID NO: any of 7-33, 35-58, 123-128 comprise one or more amino acid substitutions, preferably conservative amino acid substitutions, for example, 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 by affinity maturation, e.g., as described in SEQ ID NO:7-33, 35-58, 123-128. The affinity matured PD-1 binding protein may have one or more alterations in one or more CDRs which result in an increased 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 PD-1 or an epitope thereof.

The inclusion of an Fc region in the PD-1 binding proteins of the present disclosure allows the binding proteins to form dimeric molecules while extending the in vivo half-life of the binding proteins. Fc regions useful in the present disclosure may be from different subtypes of immunoglobulins, e.g., IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM. In general, the Fc region includes the hinge or partial hinge region of the constant region, the CH2 region, and the CH3 region.

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

a) a mutation that alters Fc-mediated CDC activity;

b) mutations that alter Fc-mediated ADCC activity; or

c) A mutation that alters FcRn-mediated half-life in vivo. 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-; paul r. hinton et al J Immunol, 2006, 176: 346-356. For example, mutations of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in the CH2 region may be used to increase or remove Fc-mediated ADCC or CDC activity or to enhance or reduce 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 can be introduced on the Fc sequence, thereby making the mutated Fc more susceptible to the formation of homodimers or heterodimers. The knob-hole model, which exploits steric effects of amino acid side chain groups at the Fc contact interface, as mentioned in Ridgway, Presta et al 1996 and Carter 2001, makes heterodimerization between different Fc mutations easier; for another example, by changing the charges of the amino acids on the Fc contact interface and thus changing the ionic interaction force between the Fc contact interfaces, heterodimers are more easily formed between different Fc mutant pairs (CN 102558355a), or homodimers are more easily formed between Fc with the same mutation (CN 103388013A).

The immunoglobulin Fc region is preferably a human immunoglobulin Fc region, such as the Fc region of human IgG1Fc, human IgG4, 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 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, in the PD-1 binding proteins of the present disclosure, the immunoglobulin single variable domain is linked to the immunoglobulin Fc region by a linker. The linker may be a non-functional amino acid sequence of 1-20 or more amino acids in length without more than secondary structure. For example, the joint is a flexible joint, e.g. G₄S、GS、GAP、(G ₄S) ₂、(G ₄S) ₃、(G ₄S) ₄、(G ₄S) ₅ASGS, and the like.

In some embodiments, a PD-1 binding protein of the present disclosure comprises an immunoglobulin single variable domain linked, directly or through a linker, to an immunoglobulin Fc region. In some embodiments, a PD-1 binding protein of the present disclosure comprises two immunoglobulin single variable domains, which are linked, directly or through a linker, to an immunoglobulin Fc region that allows the PD-1 binding protein to form a dimeric molecule comprising 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, linked to each other, either directly or through a linker, that allows the PD-1 binding protein to form a dimeric 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 of another antigen, e.g., forms a heterodimer.

In some embodiments, a PD-1 binding protein of the present disclosure comprising an immunoglobulin Fc region comprises SEQ ID NO: 34. 104, 109, 112 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% sequence identity thereto.

In some embodiments, the present disclosure provides a PD-1 binding protein capable of binding 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) to be less than or equal to 10^-7Binds to human PD-1 or an epitope thereof;

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

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

(d) inducing CD4+ T cells to secrete IFN- γ;

(e) enhanced activation of PBMCs;

(f) enhancing the activation of T cells;

(g) inhibiting tumor growth.

The PD-1 binding proteins of the present disclosure can bind PD-1 with a KD value of ≤ 1 × 10^-7M, e.g.. ltoreq.1X 10^-8M, or ≤ 1 × 10^-9M, or ≤ 1 × 10^-10M。

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 with treatment at 40 ℃ for up to 30 days, and at least stability at 60 ℃.

In a second aspect, the present disclosure provides nucleic acid molecules encoding the PD-1 binding proteins of the disclosure. The nucleic acids of the present disclosure can 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 especially be an expression vector, i.e. a vector providing for the 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 regulatory elements (e.g., promoters, enhancers, terminators, and the like). The selection of the elements and their sequences for expression in a particular host is within the knowledge of one skilled in the art. Regulatory elements and other elements useful or necessary for expression of the PD-1 binding proteins of the present disclosure are, for example, promoters, enhancers, terminators, integration factors, selection markers, leaders, reporters.

Nucleic acids of the disclosure can 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 a polypeptide of the disclosure, and/or can be isolated from a suitable natural source.

In a third aspect, the present disclosure provides a recombinant host cell that expresses or is capable of expressing one or more PD-1 binding proteins of the present disclosure and/or contains 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 (e.g., Escherichia coli, Proteus, and Pseudomonas strains) and gram-positive bacterial strains (e.g., Bacillus (Bacillus), Streptomyces, Staphylococcus, and Lactococcus strains).

Fungal cells include, for example, cells of species of the genera Trichoderma (Trichoderma), Neurospora (Neurospora), and Aspergillus (Aspergillus); or cells of species including Saccharomyces (Saccharomyces) such as Saccharomyces cerevisiae, Schizosaccharomyces (Schizosaccharomyces pombe), Pichia (Pichia) such as Pichia pastoris and Pichia methanolica, and 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 cells 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, further purification and/or modification of the PD-1 binding protein of the present disclosure.

The PD-1 binding proteins of the present disclosure may be produced intracellularly (e.g., in the cytoplasm, in the periplasm, or in inclusion bodies) in a cell as described above, followed by isolation from the host cell and optionally further purification; or it may be produced extracellularly (e.g. in the medium in which the host cell is cultured), followed by isolation from the medium and optionally further purification.

Methods and reagents for recombinant production of polypeptides, such as specifically adapted 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 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 said PD-1 binding protein, as described above, and one or more pharmaceutically acceptable carriers, diluents, buffers, or excipients.

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

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

In a seventh aspect, the present disclosure provides uses and methods 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 the 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 provides application of the PD-1 binding protein in preparing medicaments for preventing and/or diseases related to PD-1.

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

In some embodiments, the present disclosure provides a method of inhibiting PD-1 activity or promoting T cell proliferation, and in other embodiments, the present disclosure provides a method of benefiting a patient or subject from upregulation of an immune response, 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 present 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 may prevent and/or treat cancer, preferably, but not limited to, cancers that are responsive to immunotherapy.

Non-limiting examples of cancer in the above methods include lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma (e.g., metastatic malignant melanoma), renal cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematologic malignancies, head and neck cancer, glioma, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine body tumor, 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, cutaneous or intraocular malignant melanoma, uterine cancer, cancer of the anal region, testicular cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's disease, non-Hodgkin's lymphoma, carcinoma of the esophagus, carcinoma of the small intestine, carcinoma of the endocrine system, carcinoma of the thyroid gland, carcinoma of the parathyroid gland, carcinoma of the adrenal gland, sarcoma of soft tissue, carcinoma of the urethra, carcinoma of the penis, chronic or acute leukemias including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors of childhood, lymphocytic lymphomas, cancer of the bladder, kidney or ureter, carcinoma of the renal pelvis, Central Nervous System (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, carcinoma of the urinary tract, carcinoma of the colon, carcinoma of the rectum, carcinoma of the Central Nervous System (CNS), primary CNS lymphoma, tumor angiogenesis, spinal column tumor angiogenesis, brain stem glioma, pituitary adenoma, Squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, including asbestos-induced cancers, and combinations thereof. In some embodiments, the cancer or tumor is metastatic.

In some embodiments, the present disclosure provides a method of treating a disease or disorder associated with PD-1, 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, PD-1 binding proteins can be used alone, or in combination with vaccines to stimulate immune responses to pathogens, toxins and autoantigens. Examples of pathogens for which this treatment may be particularly applicable 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 viruses (a, b, c), influenza, herpes, giardia, malaria, leishmania, staphylococcus aureus, pseudomonas aeruginosa.

Some examples of pathogenic 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 viruses), adenovirus, influenza virus, arbovirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papilloma virus, molluscum virus, polio virus, rabies virus, JC virus, and arbovirus encephalitis virus.

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, Absidia, Rhizopus), Sporothrix schenckii, Blastomyces dermatitidis, paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma immitis.

Some examples of pathogenic parasites of infectious diseases that can be treated with the methods of the present disclosure include entamoeba histolytica, dactylopilus coli, formica freudenreichii, acanthamoeba species, giardia lamblia, cryptosporidium species, pneumocystis carinii, plasmodium vivax, babesia frugii, trypanosoma brucei, trypanosoma cruzi, leishmania donovani, toxoplasma gondii, and leiomycinia bracteata.

Drawings

FIG. 1: PD-1 single domain antibodies numbered 7#, 32# hu _3, 106#, 107# activated T cells in vitro and secreted IFN γ.

FIG. 2: PD-1 single domain antibodies numbered 32# hu _3_ IgG4, 7# hu _4_ hIgG4, 106# hu _1_ hIgG4, 107# hu _4_ hIgG4 activated T cells in vitro and secreted IFN γ.

FIG. 3: the PD-1 antibody inhibits the growth of mouse M38 colon cancer tumor.

Detailed Description

Term(s) for

In order that the disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless clearly defined otherwise herein, all other technical and scientific terms used herein have the same meaning as 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", "PDCD 1", and "hPD-1" are used interchangeably and include variants, isoforms, species homologs of human PD-1, and analogs having at least one common epitope with PD-1. The complete PD-1 sequence can be found in GenBank accession No. U64863.

The term "programmed death ligand-1 (PD-L1)" is one of two cell surface glycoprotein ligands of PD-1 (the other being PD-L2) that down-regulates T cell activation and cytokine secretion when bound to PD-1. The term "PD-L1" as used herein includes variants, isoforms, and interspecies homologs of human PD-L1(hPD-L1), hPD-L1, and analogs having at least one common epitope with hPD-L1. The complete hPD-L1 sequence can be found using GenBank accession No. Q9NZQ 7.

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 three letter codes and the one letter codes for amino acids used in this disclosure are as described in j. diol. chem, 243, p3558 (1968).

The term "antibody" or "immunoglobulin" whether referring to a heavy chain antibody or to a conventional tetrapeptide chain antibody made 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, as well as 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 the terms "immunoglobulin sequence", "antibody sequence", "single variable domain sequence", "VHH sequence" or "protein sequence" etc.) should generally be understood to encompass both the relevant amino acid sequences and the nucleic acid or nucleotide sequences encoding said sequences, unless the disclosure requires a 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, proteins can be added, removed, or transferred to other proteins without loss of function of the rest of the protein and/or 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" (FR1), "framework region 2" (FR2), "framework region 3" (FR3), and "framework region 4" (FR4), respectively; wherein the framework regions are separated by three "complementarity determining regions" referred to in the art and hereinafter as "complementarity determining region 1" (CDR1), "complementarity determining region 2" (CDR2), and "complementarity determining region 3" (CDR3), respectively. Thus, the general structure (or sequence) of a variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4. The variable domain confers specificity to the antigen by virtue of having an antigen binding site.

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

The term "immunoglobulin single variable domain" as used in the present disclosure refers to a variable domain that is individually capable of specifically binding 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.

A "VHH domain", also known as a heavy chain single domain antibody, VHH antibody fragment or VHH antibody, is a variable domain in an immunoglobulin called "heavy chain antibody" (i.e. "antibody lacking the light chain") (Hamers-Casterman C, Atarhouch T, Muydermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R., "Natural curing antibodies void of light chains"; Nature363, 446- "448 (1993)). The term "VHH domain" is used with the aim of distinguishing it from the heavy chain variable domain (which is referred to in this disclosure as "VH domain") and the light chain variable domain (which is referred to in this disclosure as "VL domain") present in conventional tetrapeptide chain antibodies. The 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 together with the VH domain recognize an epitope. The VHH domain is a small, stable and efficient antigen recognition unit formed from a single immunoglobulin domain. The terms "heavy chain single domain antibody", "VHH domain", "VHH antibody fragment", "VHH antibody" and "Nanobody" ("Nanobody" is a trademark of Ablynx n.v. company, Ghent, Belgium) are used interchangeably.

For example, as shown in FIG. 2 of Riechmann and Muydermans, J.Immunol.methods 231, 25-38(1999), the amino acid residues employed for the VHH domain are numbered according to the general numbering of the VH domain given by Kabat et al ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, Md., Pub. No. 91). According to the numbering process:

FR1 contains the amino acid residues at positions 1-30,

-CDR1 comprises amino acid residues at positions 31-35,

FR2 contains the amino acids at positions 36-49,

-CDR2 comprises amino acid residues at positions 50-65,

FR3 contains the amino acid residues at positions 66-94,

-CDR3 comprises amino acid residues at positions 95 to 102, and

-FR4 comprises the amino acid residue at position 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 the Kabat numbering, as is well known in the art for VH and VHH domains (i.e., one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat numbering). This means that, in general, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence.

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

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

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

the VHH domain is naturally "designed" such that functional binding to the antigen occurs in the absence and without interaction with the light chain variable domain. The VHH domain may be used as a single and relatively small functional antigen binding unit, domain or polypeptide. VHH domains differ from the VH and VL domains of conventional tetrapeptide chain antibodies in that the VH and VL domains themselves are not suitable for practical use as antigen binding proteins or immunoglobulin single variable domains alone, the VH and VL domains need to be in some form or combined 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 also as part of a larger polypeptide) offers a number of significant advantages over the use of conventional VH and VL domains, scfvs 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 two separate domains need to be present, nor is it required to ensure that the two domains are present in the proper spatial conformation and configuration (e.g. scFv typically require the use of specially 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;

the VHH domain is highly soluble and has no tendency to aggregate;

VHH domains are highly stable to heat, pH, proteases and other denaturants or conditions and therefore can be prepared, stored or transported without the use of refrigeration equipment, thereby achieving cost, time and environmental 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 with a size of conventional IgG), and therefore shows a higher tissue permeability and can be administered at higher doses;

VHH domains may exhibit so-called cavity binding properties (VHHs have extended CDR3 loops compared to conventional VH domains, so that target epitopes not accessible by conventional tetrapeptide chain antibodies and antigen binding fragments thereof may be reached).

Methods for obtaining VHHs that bind to a particular antigen or epitope have been previously disclosed in the following references: 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 WO 94/04678.

VHH domains may be "humanized" (also referred to in this disclosure as "sequence optimization"; in addition to humanization, "sequence optimization" may also encompass other modifications 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 camelidae) with one or more amino acid residues at corresponding positions in the VH domain of a 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 the human framework region sequence of IGHV 3. Humanization methods such as protein surface amino acid humanization (resurfacing) and VHH humanization universal framework grafting (CDR grafting to a universal frame).

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-camelid mammal. In order to bind an epitope in the form of a single antigen binding domain (i.e. without pairing with a VL or VH domain, respectively), the antigen binding properties need to be specifically selected, 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 need to be humanized for e.g. human therapeutic use. As in the case of VHH domains, domain antibodies are also well expressed in prokaryotic expression systems, thereby significantly reducing the overall manufacturing costs.

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

As used in this disclosure, the term "epitope" or the interchangeably used term "antigenic determinant" refers to any antigenic determinant on an antigen to which an antibody binds. Antigenic determinants generally comprise chemically active surface groups of the molecule, such as amino acids or sugar side chains, and generally have specific three-dimensional structural characteristics and/or specific charge characteristics. For example, an epitope typically includes 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 can be a "linear" epitope or a "conformational" epitope. See, e.g., epitopic Mapping Protocols in Methods in Molecular Biology, vol 66, g.e. morris, Ed. (1996). In a linear epitope, all points of interaction 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, the points of interaction exist across amino acid residues that are separated from each other.

Epitopes of a given antigen can be identified using a number of epitope mapping techniques well known in the art. See, e.g., epitopic Mapping Protocols in Methods in Molecular Biology, vol 66, g.e. morris, Ed. (1996). For example, a linear epitope can be determined by, for example, the following methods: a plurality of peptides are simultaneously synthesized on a solid support, wherein the peptides correspond to portions of a protein molecule, and the peptides are reacted with an antibody 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) Molec. Immunol.23: 709- "715. Conformational epitopes can also be identified by determining the spatial configuration of amino acids, such as by x-ray crystallography and two-dimensional nuclear magnetic resonance, for example.

Antibodies can be screened for binding competition with the same epitope 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 the antigen. A high throughput method for obtaining antibodies binding to the same epitope based on their cross-competition is 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 routine techniques known to those skilled in the art.

In general, the term "specificity" refers to the number of different types of antigens or epitopes that a particular antigen binding molecule or antigen binding protein (e.g., a PD-1 binding protein of the present disclosure) can bind. Specificity can be determined based on the affinity (affinity) and/or avidity (avidity) of the antigen binding protein. The 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 the antigen binding site on the 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, affinity can be determined in a known manner depending on the particular antigen of interest. Avidity is a measure of the strength of binding between an antigen binding protein (e.g., an immunoglobulin, an antibody, an immunoglobulin single variable domain, or a polypeptide containing the same) and an associated antigen. Affinity is related to both: affinity to its antigen binding site on the antigen binding protein, and the number of relevant binding sites present on the antigen binding protein.

The term "PD-1 binding protein" as used in this disclosure means any protein capable of specifically binding 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. A "PD-1 binding protein" 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" can comprise 2, 3, 4, or more immunoglobulin single variable domains (e.g., VHHs) that bind PD-1. The PD-1 binding proteins of the present disclosure may also comprise, in addition to an immunoglobulin single variable domain that binds PD-1, a linker and/or a moiety with effector molecule function, e.g., a half-life extending moiety (such as an immunoglobulin single variable domain that binds serum albumin), and/or a fusion partner (such as serum albumin) and/or a conjugated polymer (such as PEG) and/or an Fc region. In some embodiments, the "PD-1 binding proteins" of the present disclosure also encompass bi-/multispecific antibodies containing immunoglobulin single variable domains that bind different antigens.

An "affinity matured" PD-1 antibody, in particular a VHH or domain antibody, has one or more changes in one or more CDRs which result in an increased affinity for PD-1 compared to its respective parent anti-PD-1 antibody. Affinity matured anti-PD-1 antibodies can be prepared by methods known in the art, for example, as described below: marks et al, 1992, Biotechnology 10: 779 783 or Barbas et al, 1994, Proc. Nat. Acad. Sci, USA 91: 3809-3813; shier et al, 1995, Gene 169: 147-; 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, respectively; KS Johnson and RE Hawkins, "Affinity mapping of antibodies using phase display", Oxford University Press 1996.

Generally, the PD-1 binding proteins of the present disclosure will be measured as preferably 10 as measured in Biacore or KinExA or Fortibio assays^-7To 10^-10Mole/liter (M), more preferably 10^-8To 10^-10Mole/liter, even more preferably 10^-9To 10^-10Or a dissociation constant (KD) of less, and/or at least 10-⁷M, preferably at least 10-⁸M, more preferably at least 10^-9M, more preferably at least 10^-10The binding constant (KA) of M binds to the antigen to be bound (i.e., PD-1). Any greater than 10^-4The KD value of M is 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.

The term "compete" when used in the context of antigen binding proteins that compete for the same epitope (e.g., neutralizing antigen binding proteins or neutralizing antibodies) means competition 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 a reference antigen binding protein (e.g., ligand or reference antibody) to a common antigen (e.g., PD-1 antigen or fragment thereof). Numerous types of competitive binding assays are available for determining whether an antigen binding protein competes with another, such as: solid phase direct or indirect Radioimmunoassays (RIA), solid phase direct or indirect Enzyme Immunoassays (EIA), sandwich competition assays (see, e.g., Stahli et al, 1983, methods in Enzymology 9: 242-; solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al, 1986, J.Immunol.137: 3614-), solid phase direct labeling assay, solid phase direct labeling sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); direct labeling of RIA with a solid phase of I-125 label (see, e.g., Morel et al, 1988, mol. 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 (either on a solid surface or on the surface of a cell) that binds to a test antigen binding protein with an unlabeled label and a labeled reference antigen binding protein. Competitive inhibition is measured by measuring the amount of label bound to a solid surface or cells in the presence of the antigen binding protein to be detected. Typically, the antigen binding protein to be tested is present in excess. Antigen binding proteins identified by competitive assays (competing antigen binding proteins) include: an antigen binding protein that binds to the same epitope as a reference antigen binding protein; and an antigen binding protein that binds to an epitope sufficiently close to the epitope to which the reference antigen binding protein binds, said two epitopes sterically hindering the binding from occurring. Additional details regarding methods for determining competitive binding are provided in the disclosed embodiments. Typically, when a competing antigen binding protein is present in excess, it will inhibit (e.g., decrease) the specific binding of at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more of a reference antigen binding protein to a common antigen. In certain instances, 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 a PD-1 binding protein of the present disclosure to bind to PD-1 from a different species or an epitope thereof. For example, a single domain antibody or derivative protein of the present disclosure that binds to human PD-1 may also bind to PD-1 of another species. Cross-reactivity is measured by detecting specific reactivity with purified antigens in binding assays (e.g., SPR and ELISA), or binding or functional interactions with cells that physiologically express PD-1. 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., in relation to a cell) is intended to include any measurable decrease in cell growth.

"homology" or "identity" in this disclosure refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., 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 matching or homologous positions shared by the two sequences divided by the number of positions compared x 100. For example, two sequences are 60% homologous if there are 6 matches or homologies at 10 positions in the two sequences when the sequences are optimally aligned; two sequences are 95% homologous if there are 95 matches or homologies at 100 positions in the two sequences. In general, comparisons are made when aligning two sequences to obtain the greatest percentage of homology.

The term "mutant sequence" refers to a nucleotide sequence and an amino acid sequence having different degrees of percent sequence identity to the nucleotide sequence and the amino acid sequence of the present disclosure, which are obtained by mutational modification such as appropriate substitution, insertion, or deletion of the nucleotide sequence and the amino acid sequence of the present disclosure. 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 performed by the 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, meaning that the amino acid residue is replaced with another amino acid residue that is chemically similar in structure and that has 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, for example conservative amino acid substitutions are preferably made where one amino acid within the following groups (i) - (v) is replaced with another amino acid residue within the same group:

(i) smaller aliphatic nonpolar or weakly polar residues: ala, Ser, Thr, Pro, and Gly;

(ii) polar negatively charged residues and their (uncharged) amides: asp, Asn, Glu and Gln;

(iii) polar positively charged residues: his, Arg and Lys; (iv) larger aliphatic non-polar residues: met, Leu, Ile, Val and Cys; and

(v) aromatic residue: phe, Tyr, and Trp.

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

The term "back mutation" refers to the mutation of an amino acid residue in the FR region from which a human antibody is derived to an amino acid residue in the corresponding position in the original derived antibody, usually in order to avoid the reduction in the activity caused by the humanized antibody while avoiding the reduction in immunogenicity, and the humanized antibody variable region may be subjected to minimal reverse mutation to maintain the activity of the antibody.

The term "nucleic acid molecule" as used in the present disclosure refers to both 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 can 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 having a bacterial origin of replication and episomal mammalian vectors) or can be integrated into the genome of a host cell upon introduction into the host cell so as to be replicated along with the host genome (e.g., non-episomal mammalian vectors).

The expressions "cell," "cell line," and "cell culture" as used in this disclosure are used interchangeably, and all such designations include progeny. Thus, "transformants" and "transformed cells" include the primary test cells and cultures derived therefrom, regardless of the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where different names are intended, 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 susceptible to transformation include members of the enterobacteriaceae family (enterobacteriaceae), such as strains of Escherichia coli (Escherichia coli) or Salmonella (Salmonella); bacillaceae (Bacillus) such as Bacillus subtilis; pneumococcus (Pneumococcus); streptococcus (Streptococcus) and Haemophilus influenzae (Haemophilus influenzae). Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (chinese hamster ovary cell line), NS0 cells, 293 cells.

"pharmaceutical composition" means a mixture containing one or more compounds described in 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 facilitate administration to an organism, facilitate absorption of the active ingredient and exert biological activity.

The terms "cancer" and "cancerous" and "tumor" refer to or describe a physiological condition in mammals that is generally characterized by unregulated cell growth. Examples of cancer 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 cancer (e.g., epithelial squamous cell cancer), lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, 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 carcinoma, salivary gland carcinoma, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma, superficial spreading melanoma, lentigo malignant melanoma, acromegaly melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, Chronic Lymphocytic Leukemia (CLL), Acute Lymphoblastic Leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with scarring nevus (phakomatases), edema (such as associated with brain tumors) and Meigs (Meigs) syndrome, brain tumors and cancers, as well as head and neck cancers, and associated metastases. In certain embodiments, cancers suitable for treatment by 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 carcinoma (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 is 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" when referred to in this disclosure are not mutually exclusive.

"administration," "administering," and "treating," when applied to an animal, human, subject, cell, tissue, organ, or biological fluid, refers to contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "administration," "administering," and "treating" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. The treatment of the cells comprises contacting the reagent with the cells and contacting the reagent with a fluid, wherein the fluid is in contact with the cells. "administering", "administering" and "treating" also mean treating a cell in vitro and ex vivo by an agent, a diagnostic, a 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 condition. An effective amount also means an amount sufficient to allow 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 method and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

By "treating" is meant administering an internal or external therapeutic agent, e.g., a composition comprising any one 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, to a subject who has one or more disease symptoms for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in the 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 development of such symptoms to any clinically measurable degree. The amount of therapeutic agent effective to alleviate any particular disease symptom (also referred to as a "therapeutically effective amount") 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 commonly used by physicians or other health professional to assess the severity or progression of the symptom. Although embodiments of the present disclosure (e.g., methods of treatment or articles of manufacture) may be ineffective in alleviating the symptoms of each target disease, they should alleviate the symptoms of the target disease in a statistically significant number of patients as determined according to any statistical test method known in the art, such as Student's t-test, chi-square test, U-test by Mann and Whitney, Kruskal-Wallis test (H-test), Jonckhere-Terpstra test, and Wilcoxon test.

The term "preventing cancer" refers to delaying, inhibiting or preventing the onset of cancer in a subject in which the onset of cancer or tumorigenesis has not been confirmed, but a susceptibility to cancer has been identified, 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 a malignant tumor or to cause regression thereof.

"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 this disclosure means a mammal, particularly a primate, particularly a human.

Detailed description of the preferred embodiments

The present disclosure is further described below with reference to examples, but these examples do not limit the scope of the present disclosure. The experimental methods of the present disclosure, in which specific conditions are not specified, are generally performed 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 material or the goods. Reagents of specific sources are not indicated, and conventional reagents are purchased in the market.

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 (the PD-1 antigen is not specifically described below and refers to human PD-1).

Human PD-1 full-length protein:

(Note: double horizontal line part is Signal peptide (Signal peptide); single horizontal line part is PD-1 Extracellular region (Extracellular Domain), wherein 35-144 are Ig-like V-type 1 domains (Ig-like V-type 1 domains), 70-77 are the parts that interact with CD 274; dotted line part is Transmembrane region part (Transmembrane Domain); italic part is intracellular region (cytoplastic Domain).)

SEQ ID NO：1

Monkey PD-1 full-length amino acid sequence:

(Note: double horizontal line part is signal peptide; single horizontal line part is PD-1 extracellular region, wherein 38-127 th position is V-Set domain, 39-125 th position is Ig-like V type 1 domain; dotted line part is Transmembrane region part; italic part is intracellular region.)

SEQ ID NO：2

Human PD-1 antigen (a commercial product of Sino Biological Cat.10377-H08H)) for screening and detection:

(Note: Single-horizontal part is PD-1 extracellular region; italic part is His-tag marker.)

SEQ ID NO：3

Human PD-1-Fc antigen (a commercial product (Baiying biology: B3789)) for screening and detection:

(Note: the cross-hatched part is the extracellular region; the italic part is the human Fc marker.)

SEQ ID NO：4

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

(Note: the crossline portion is the extracellular region of PD-L1; the italic portion is the His-tag.)

SEQ ID NO：5

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

(Note: the crossline portion is the extracellular region of PD-L2; the italic portion is the His-tag.)

SEQ ID NO：6

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

Two ends of bactrian camel are respectively immunized by human PD-1 protein (ACRO, Cat # PD-1-H5259 and ACRO, Cat # PD-1-H5221), 5mL of camel serum before immunization is taken, and serum is separated. Freund's complete adjuvant was mixed with antigen volume 1:1 after mixing, camels were immunized subcutaneously in multiple spots (immunization dose of 100. mu.g protein/mouse/each). Boosters were performed every two weeks and titers were measured after four immunizations. Plates (100. mu.L/well) were coated with 5. mu.g/mL PD-1-his protein overnight at 4 ℃. The next day of washing was followed by addition of 4% skimmed milk powder for blocking at 37 deg.C for 2 h. After washing, different dilutions of camel serum were added and incubated at 37 ℃ for 1 h. Negative controls were preimmune serum (1: 1000 dilution) and PBS solution. After incubation, the cells were washed three times with PBST, rabbit anti-camel polyclonal antibody (1: 1000 dilution) was added and incubated at 37 ℃ for 1 hour. After washing again, a secondary goat anti-mouse alkaline phosphatase labeled antibody (1: 1000 dilution) was added and incubated at 37 ℃ for 1 h. Finally, alkaline phosphatase developing solution is added for washing, 2M sulfuric acid is used for termination, and the absorption value is read at the wavelength of 450 nm. 1: titers were detected after 25600-fold dilution. The potency is qualified, and camel peripheral blood is collected for library construction.

Separating lymphocytes from peripheral blood of camel, and counting the number of the cells is 1.2 × 10⁸Resuspend with Trizol reagent (1X 10)⁷Individual cells/mL Trizol) to lyse the cells, left on ice for 5 min; centrifuging at 13000rpm for 3min, collecting supernatant, and removing precipitate; adding 1/5 volume of chloroform, shaking vigorously for 30-60s, and standing in ice bath for 2 min; centrifuging at 13000rpm for 10min, and sucking the upper aqueous phase layer into a new 1.5mL tube; adding isopropanol with equal volume, mixing, and standing at-20 deg.C for 30 min; centrifuging at 13000rpm for 10min, removing supernatant, and retaining precipitate; adding pre-cooled 75% ethanol, washing the precipitate, and standing at room temperature for 5-10 min; adding 600 mu L of deionized water removed by RNase, redissolving to obtain RNA, carrying out reverse transcription to obtain cDNA, and constructing a phage library.

The single domain antibody with high affinity to PD-1 antigenic protein is obtained by screening phage library, 20 mu g PD-1-avi-biotin protein is combined with 1mg Dynabeads MyOne streptavidin T1, after standing for one hour at 37 ℃, 2% skim milk is used for sealing for 2 hours at room temperature, a camel heavy chain single domain antibody phage display library is added, and the reaction is carried out for 1 hour at room temperature. Unbound phage was removed by washing 9 times with PBST (0.05% Tween-20) solution. Phages specifically bound to PD-1 were eluted with 1mg/mL trypsin and infected with E.coli TG1, which was grown in log phase, and phages were generated and purified for the next round of screening. The same screening process was repeated for 2-3 rounds. Positive clones were enriched.

From the screening of enriched positive clones, 96 monoclonal colonies were picked and packaged into phage single chain antibodies for phage ELISA testing. The ELISA plates were coated with 2. mu.g/mL of PD-1-his protein, and the diluted phage supernatant was added and detected with anti-M13 HRP. Clones tested by ELISA binding to an OD450 value greater than 0.5 were sequenced to give 51 specific sequences.

EXAMPLE 3 construction of intact monoclonal antibodies

Complete antibodies are constructed by 51 specific sequences obtained by screening phage libraries in example 2, and 27 antibodies are determined to have strong binding capacity and can inhibit the interaction of PD-1 and PD-L1 by an ELISA binding experiment and an ELISA competition experiment, and the results are shown in Table 1.

TABLE 1 ELISA test results for PD-1 antibody

Antibody numbering	OD450	Antibody numbering	OD450	Antibody numbering	OD450
2#	1.71	56#	1.6633	109#	1.7868
4#	1.7036	59#	1.697	112#	1.6533
6#	1.8356	61#	1.7869	113#	1.4008
7#	1.7844	62#	1.7721	114#	1.5921
11#	1.5262	68#	1.6568	118#	1.5299
19#	1.6879	104#	1.765	122#	1.6316
32#	1.869	106#	1.7502	123#	1.5266
41#	1.4095	107#	1.659	Opdivo (Positive control)	1.7387
54#	1.5173	108#	1.7068	PBS (negative control)	0.161

The 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#

7-33 of the above sequence SEQ ID NO, the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the sequence is in italic FR sequence, and the underlined sequences are respectively CDR1, CDR2 and CDR 3. The numbering conventions for PD-1 single domain antibodies provided in this disclosure are all 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 with the sequence of human IgG1-Fc (CH2-CH3) fragment and constructed into PTT5 expression vector, and the sequence of the linked human IgG1-Fc can be as follows:

the following is the complete protein sequence of the VHH sequence fused to the human Fc (CH2-CH3) stretch, the human IgG1-Fc (CH2-CH3) stretch (SEQ ID NO: 103) is single underlined, and the linker sequence is double underlined. The protein sequences are as follows (taking numbers 32#, 7#, 106#, 107# as examples, and the same is true for other PD-1 single-domain antibodies):

32#-IgG1：

7#-IgG1：

106#-IgG1:

107#-IgG1:

example 4 humanization of Single Domain antibodies

By carrying out three-dimensional structure homology modeling on selected specific PD-1 single-domain antibody molecules and combining the results of comparison with a V-base human germline sequence database and an IMGT human antibody heavy chain variable region germline gene database, selecting a heavy chain variable region germline gene with high homology with the screened antibody as a template, transplanting the CDR of the camelid source single-domain antibody into the corresponding human template to form a variable region sequence with the sequence of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4. And (3) carrying out three-dimensional structure simulation and analysis on the transplanted single-domain antibody again, and carrying out back mutation on a specific site influencing the structural morphology of the CDR region in the FR region. The humanized specific sequences obtained were 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, the CDRs of a portion of the antibody were altered, e.g., 7# Hu-5 with a mutation of T35S, the CDR1 sequence shown in YNFMS (SEQ ID NO: 113); 7# Hu _6 has mutations of F33Y and T35S, and the CDR1 sequence shown by YNYMS (SEQ ID NO: 114); 106_ hu _1 to 6 had a mutation of A61D, resulting in a CDR2 sequence shown in VVDRFGGTIYADSVKG (SEQ ID NO: 71); 112_ hu _1 has a mutation of A61D, resulting in the CDR2 sequence shown in VVDRFGGIIYADSVKG (SEQ ID NO: 93).

The method of example 4 was used to construct a humanized PD-1 single domain antibody with the full protein sequence fused to the Fc (CH2-CH3) segment of hIgG1, the sequence of the hIgG1-Fc (CH2-CH3) segment (SEQ ID NO: 103) being single underlined and the linker sequence being double underlined. The protein sequence is as follows (taking 32_ hu _3-IgG1 as an example, and the same is true for other humanized PD-1 single domain antibodies):

32#_hu_3-hIgG1：

the method of example 4 was used to construct the whole protein sequence of the humanized PD-1 single domain antibody fused to the Fc (CH2-CH3) segment of hIgG4, the sequence of the hIgG4-Fc (CH2-CH3) segment being single underlined (SEQ ID NO: 108).

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

the obtained antibody sequences were as follows:

32#_hu_3_hIgG4：

7#_hu_4_hIgG4：

106#_hu_1_hIgG4：

107#_hu_4_hIgG4：

the plasmid was transfected into HEK293 cells, expression supernatant was collected after 6 days, high speed centrifuged to remove impurities, and purified using Protein a column. Equilibrate with PBS until a280 reading falls to baseline. Eluting the target protein with acidic eluent with pH of 3.0-3.5, and neutralizing with 1M Tris-HCl, pH of 8.0-9.0. After the eluted sample was appropriately concentrated, it was further purified by gel chromatography Superdex200(GE) equilibrated with PBS to remove the aggregates, collect the monomer peak, and split for future use. Upon detection, the PD-1 single domain antibodies of the present disclosure are obtained.

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

To test 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 experiments.

The negative control of this example was PBS, the positive control used Opdivo (from Shanghai Ruizi Chemicals (chempartner) lot: 180612001), and a portion of the experiments used an IgG4 type PD-1 antibody in WO2017054646 (WO2017054646) as a positive control, with the following sequences:

PD-1 antibody heavy chain:

PD-1 antibody light chain:

the protein with the PD-1 antibody was diluted to 2. mu.g/mL with PBS buffer, pH7.4, added to a 96-well plate (corning, 901825/box 96well clear float bottom plate) at a volume of 100. mu.L/well, and left overnight at 4 ℃ for 16-20 hours. After discarding the liquid, the plate was washed three times with PBST (pH7.4, 0.05% Tween-20) buffer, and then 2% BSA blocking solution (300. mu.L/well) diluted with PBS buffer was added and the plate was blocked by incubation in an incubator at 37 ℃ for 2 hours. After blocking was completed, the 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, and the plate was diluted three-fold with PBS buffer for 8 gradients and incubated at 37 ℃ in an incubator for 1 hour. After completion of the incubation, the reaction solution in the microplate was discarded, and the plate was washed 6 times with PBST, 100. mu.L/HRP-labeled secondary antibody against his (Abcam ab1187) (1:5000 dilution) was added to each well, and incubated at 37 ℃ for 1 hour. Washing the plate 6 times with PBST, adding 100 μ LTMB chromogenic substrate, incubating at room temperature for 3-5min, adding 100 μ L1M sulfuric acid to stop the reaction, reading the absorbance at 450nm with a SpectraMax M5 microplate reader, and calculating the antibody binding EC to the antigen₅₀The value is obtained. EC of partial antibody₅₀The results are shown in Table 3. The results show that the polypeptide has better binding force with human and monkey PD-1 antigens.

TABLE 3 binding force EC of different PD-1 antibodies to human and monkey PD-1 antigens₅₀(nM)

Antibody numbering	EC 50 binding to human PD-1	EC 50 binding to monkey PD-1
7#	1.86	2.2
32#	1.99	4.8
32#_hu_1	4.08	6.2
32#_hu_2	3.43	2.3

32#_hu_3	2.98	1.2
61#	1.85	/
106#	2.56	0.67
107#	3.14	2.9
112#	2.51	1.5
Positive control (opsivo)	1.69	2.88
Negative control (PBS)	0	0

(Note: "/" indicates no detection)

In addition, dissociation constants of PD-1 antibody and PD-1 protein were also determined by Biacore 8K (GE healthcare) instrument. Firstly, an anti-human IgG Fc antibody (GE Healthcare, # BR-1008-39) is covalently coupled to a CM5S series chip, a PD-1 antibody to be detected is captured to the surface of the chip through affinity, then PD-1 proteins (SEQ ID NO: 3) with different concentrations flow through the surface of the chip, a Biacore instrument is utilized to detect reaction signals in real time so as to obtain a binding dissociation curve, and a binding force constant is obtained through fitting. The experimental solution used was HBS-P solution (10mM HEPES, 150mM NaCl, 0.005% P20, pH 7.4). At the end of each experimental cycle, 3M MgCl was used₂The chip is cleaned and regenerated by the solution. The affinity results for the partial antibodies are shown in table 4. The results show that the affinity of the antibodies obtained by the screening of the disclosure to PD-1 is equivalent to that of the positive control.

TABLE 4 affinity of different PD-1 antibodies to 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 also examined the affinity of different PD-1 antibodies to human PD-1 by Biacore T200(GE Healthcare), the results of which are shown in Table 5.

TABLE 5 affinity of different PD-1 antibodies to 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 (WO2017054646)	6.18E+04	4.79E-04	7.75E-09

Example 6 blocking of the binding of PD-1 and 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-L1 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, added to a 96-well plate (corning, 901825/box 96well clear float plate) at a volume of 100. mu.L/well, and left overnight at 4 ℃ for 16-20 hours. After discarding the liquid, the plate was washed three times with PBST (pH7.4, 0.05% Tween-20) buffer, and then 300. mu.L/well of 2% BSA blocking solution diluted with PBS buffer was added, and the blocking was performed by incubating in an incubator at 37 ℃ for 2 hours. After blocking was complete, the blocking solution was discarded and the plate was washed 3 times with PBST buffer, then protein with biotinylated PD-L1 and PD-L2 was 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, diluted 6 gradients three-fold in PBS buffer and incubated for 1 hour in an incubator at 37 ℃. After the incubation was completed, the reaction solution in the microplate was discarded, the plate was washed 6 times with PBST, and 100. mu.L/well was addedHRP-labeled secondary anti-SA antibody (Peroxidase-conjugated Streptavidin, Jackson 136861) was diluted (1:500) with PBS (0.5% BSA) and incubated at 37 ℃ for 1 hour. Washing the plate 6 times with PBST, adding 100 μ L/well TMB chromogenic substrate, incubating at room temperature for 3-5min, adding 1M sulfuric acid to stop the reaction, reading the absorbance at 450nm with a SpectraMax M5 microplate reader, and calculating the binding IC of the antibody to the antigen₅₀The value is obtained. IC of partial antibody₅₀The results are shown in Table 4. The results showed that the antibodies were all able to compete with both PD-L1 and PD-L2 for binding to PD-1, PBS was used as negative control, and Opdivo (from Shanghai Ruizi chemical (chempartner) lot: 180612001 was used as positive control). The results of the partial antibodies blocking the binding of PD-1 to PD-L1 are shown in tables 6 and 7.

TABLE 6 IC of different PD-1 antibodies competing for PD-1 antigen with PD-L1 and PD-L2₅₀(nM)

TABLE 7 IC of different PD-1 antibodies competing for PD-1 antigen with PD-L1₅₀(nM)

Antibody numbering	IC 50 for blocking binding of PD-1 to PD-L1
32#_hu_3_hIgG4	2.42
7#_hu_4_hIgG4	1.22
106#_hu_1_hIgG4	3.14
PD-1 antibody (WO2017054646)	2.79
Negative control (PBS)	9999

Example 7 binding of PD-1 Single Domain antibodies to PD-1 on the surface of cells in vitro

Collecting stable high expression PD-1 cell line CHO-PD-1, each tube is 5X 10⁵A cell. The PD-1 antibody was diluted in a gradient of 0.01, 0.1, 1, 10. mu.g/mL and incubated with CHO-PD-1 on ice for 1 hour. After washing with PBS, FITC anti-human IgG (1:100) was added to each tube and incubated for 1 hour on ice in the absence of light. After washing with PBS, 100. mu.L/tube of PBS was resuspended and fluorescence detected on a flow cytometer. Binding of PD-1 antibody to CHO-PD-1 cells was dose dependent by quantitative analysis of the mean fluorescence intensity obtained from each dose treatment of the antibody. Binding capacity of partial antibodies EC₅₀The results are shown in table 8, and show that the binding force of the antibodies (such as 2#, 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) obtained by screening in the present disclosure and PD-1 is significantly better than that of the positive control opsivo.

The negative controls of examples 7-9 were all NC (which has the same constant region IgG4 as the experimental antibody, but the variable region does not recognize the antigen PD-1), and the positive control used Opdivo (from Shanghai Rui Zhi Chemie (chempartner) lot: 180612001).

TABLE 8 binding force EC of different PD-1 antibodies to the cell surface antigen PD-1₅₀(nM)

Antibodies	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

Collecting stable high expression PD-1 cell line CHO-PD-1, each tube is 5X 10⁵A cell. The PD-1 antibody was diluted in a gradient of 50, 16.67, 5.55, 1.85, 0.617, 0.205, 0.069. mu.g/mL and incubated with CHO-PD-1 on ice for 1 hour. After washing with PBS, each tube was incubated for 1 hour on ice with 1. mu.g/mL of PD-L1-mIgG2a protein and washed again with PBS. PE anti-mouse IgG2a (1:300) was added to each tube and incubated on ice for 1 hour. After washing with PBS, 100. mu.L/tube of PBS was resuspended and fluorescence detected on a flow cytometer. The PD-1 antibody blocks the binding of PD-L1 protein to CHO-PD-1 cells as shown in tables 9 and 10, and the binding intensity was dose-dependent by quantitative analysis of the mean fluorescence intensity obtained from each dose treatment of the antibody. The results show that the antibodies obtained by screening in the present disclosure (e.g., 7#, 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 stronger ability to block the binding of PD-L1 and PD-1 than the positive control opsivo.

TABLE 9 IC of different PD-1 antibodies blocking the PD-L1 protein from the cell surface antigen PD-1₅₀(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 IC of different PD-1 antibodies blocking the PD-L1 protein from the cell surface antigen PD-1₅₀(nM)

Antibody numbering	IC	50
32#_hu_3_hIgG4	0.6513
7#_hu_4_hIgG4	0.613
106#_hu_1_hIgG4	2.432
107#hu4hIgG4	4.914
PD-1 antibody (WO2017054646)	0.5667
Negative Control (NC)	9999

Example 9 PD-1 Single Domain antibodies promote cytokine secretion by Mixed lymphocytes in vitro

Isolation of human fresh or resuscitated PBMCs CD14 by EasySep human CD14 Positive screening kit (STEMCELL technologies, 17858)⁺A monocyte. Isolated CD14⁺Dendritic cell differentiation kit (R) derived from cells according to monocytes&D system, CDK004) by adding IL-4 and GM-CSF for 6 days, and then adding TNF-alpha for further 3 days to obtain mature DC.

Human PBMC CD3 was isolated by EasySep human CD3 positive screening kit (STEMCELL technologies, 18051)⁺T cells (different donor source than DCs). Separating the obtained DC from T cells 10: 1 proportion mixed culture, adding low endotoxin controlled PD-1 antibody, culturing for 5 days, using human IFN gamma quantikine ELISA kit (R)&D system, DIF50) to detect secretion of IFN γ by activated T cells.

The amounts of IFN γ secretion after mixed lymph culture are shown in table 11 and table 12 and fig. 1 and fig. 2. The results show that a plurality of PD-1 antibodies obtained by screening can effectively enhance the activation of T cells and secrete IFN gamma.

TABLE 11 IFN γ secretion promoting amounts of different antibodies

Antibody numbering	IFN gamma secretion (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 IFN γ secretion promoting amounts of different antibodies

Antibody numbering	IFN gamma secretion (pg/ml)
32#_hu_3_hIgG4	877.3
7#hu4hIgG4	759.9
106#_hu_1_hIgG4	736.94
PD-1 antibody (WO2017054646)	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 Efisch pharmaceutical science, Inc., using HuPD-1 humanized transgenic mice, female, 6-8 weeks old, purchased from Nanjing Galaxy biomedical Inc.

PBS resuspended mouse colon carcinoma cell line MC38 cells at 5X 10⁵The seeds were inoculated subcutaneously in the right flank of HuPD-1 humanized mouse at a concentration of 0.1mL and a volume of 0.1 mL. When the average tumor volume reaches 100mm³(70-120mm ³) When the method is used, mice with moderate individual tumor volumes are selected and grouped, and the right tumor volume is taken as a grouping basis. The medicine is administered on the grouping day, and the administration dose is 0.3 mg/kg; the administration frequency is from once every three days for a total of three weeks(ii) a The mode of administration is intravenous injection.

The results of the PD-1 antibody inhibiting the growth of colon cancer tumor in mice are shown in Table 13 and FIG. three. The results showed that the positive control had a tumor suppression ratio of 47.3% at day 24; the tumor suppression ratio of 32# hu _3_ hIgG4 was 50.8%; the tumor suppression ratio of 7# hu _4_ hIgG4 was 68.4%; the tumor inhibition ratio of 106# hu _3_ hIgG4 was 64.4%, which was effective in inhibiting tumor growth in mice.

TABLE 13 results of inhibition of Colon cancer tumor growth in mice by PD-1 antibody

Claims (21)

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

   1) CDR1 comprises SEQ ID NO: 62, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 115, CDR3 comprises the amino acid sequence shown in SEQ ID NO: 64; or

   2) CDR1 comprises SEQ ID NO: 81 and CDR2 comprises the amino acid sequence shown in SEQ ID NO: 116, CDR3 comprises the amino acid sequence shown in SEQ ID NO: 117; or

   3) CDR1 comprises SEQ ID NO: 118, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 66, CDR3 comprises the amino acid sequence shown in SEQ ID NO: 67; or

   4) CDR1 comprises SEQ ID NO: 84, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 119 and CDR3 comprises the amino acid sequence of SEQ ID NO: 64; or

   5) CDR1 comprises SEQ ID NO: 78, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 120, CDR3 comprises the amino acid sequence shown in SEQ ID NO: 80; or

   6) CDR1 comprises SEQ ID NO: 59, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 60, CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 61; or

   7) CDR1 comprises SEQ ID NO: 74, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 75, CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 76; or

   8) CDR1 comprises SEQ ID NO: 88, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 89 and CDR3 comprises the amino acid sequence of SEQ ID NO: 90; or

   9) CDR1 comprises SEQ ID NO: 96 and CDR2 comprises the amino acid sequence shown in SEQ ID NO: 97, CDR3 comprises the amino acid sequence of SEQ ID NO: 98, or a pharmaceutically acceptable salt thereof.
2. The PD-1 binding protein of claim 1, wherein:

   1) CDR1 comprises SEQ ID NO: 62, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 63. 68, 69, 70, 72, 77, CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 64 or 73; or

   2) CDR1 comprises SEQ ID NO: 81 and CDR2 comprises the amino acid sequence shown in SEQ ID NO: 71. 82, 91, 93, 94, CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 83. 92, 95 or a pharmaceutically acceptable salt thereof; or

   3) CDR1 comprises SEQ ID NO: 65. 113, 114, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 66, CDR3 comprises the amino acid sequence shown in SEQ ID NO: 67; or

   4) CDR1 comprises SEQ ID NO: 84, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 85. 102, CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 86; or

   5) CDR1 comprises SEQ ID NO: 78, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 79. 87, 99, 100, 101, CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 80, or a pharmaceutically acceptable salt thereof.
3. The PD-1 binding protein of claim 1 or 2, wherein:

   1) CDR1 comprises SEQ ID NO: 62, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 63. 68, 69, 70, 72, 77, CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 64; or

   2) CDR1 comprises SEQ ID NO: 62, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 63, CDR3 comprises the amino acid sequence shown in SEQ ID NO: 73; or

   3) CDR1 comprises SEQ ID NO: 81 and CDR2 comprises the amino acid sequence shown in SEQ ID NO: 71. 82, CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 83; or

   4) CDR1 comprises SEQ ID NO: 81 and CDR2 comprises the amino acid sequence shown in SEQ ID NO: 91. 93, CDR3 comprises the amino acid sequence shown in SEQ ID NO: 92; or

   5) CDR1 comprises SEQ ID NO: 81 and CDR2 comprises the amino acid sequence shown in SEQ ID NO: 94, CDR3 comprises the amino acid sequence shown in SEQ ID NO: 95, or a pharmaceutically acceptable salt thereof.
4. A PD-1 binding protein according to any one of the preceding claims, which is an antibody that binds PD-1 or an epitope thereof;

   preferably, the antibody is a camelid antibody, a chimeric antibody, a humanized antibody, a fully human antibody.
5. A PD-1 binding protein according to any preceding claim, which is an sdAb or a bispecific, multispecific antibody.
6. A PD-1 binding protein according to any one of the preceding claims, wherein said immunoglobulin single variable domain is a VHH, preferably said VHH is a humanized VHH or an affinity matured VHH.
7. The PD-1 binding protein according to any one of the preceding claims, which further comprises an Fc region; preferably, the Fc region is selected from: fc region of human IgG1, IgG2, IgG3, IgG 4; more preferably, the Fc region is human IgG4Fc or human IgG4Fc with a S228P substitution.
8. A PD-1 binding protein according to any one of the preceding claims, which comprises a structure represented by the formula X-L-Y, wherein X represents an immunoglobulin single variable domain, L represents the presence or absence of a linker, and Y represents an Fc region;

   preferably, the PD-1 binding protein is a monomer or dimer;

   more preferably, the PD-1 binding protein forms a homodimer through the Fc region.
9. The PD-1 binding protein of claim 8, wherein L is a linker that is 1-20 amino acids in length; preferably, L is Ala-Ser-Gly-Ser.
10. The PD-1 binding protein according to any one of the preceding claims, which comprises:

    SEQ ID NO:7-33, 35-58, 123-128; or

    And SEQ ID NO:7-33, 35-58, 123-128, or a sequence having at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.
11. The PD-1 binding protein of any one of the preceding claims which:

    as shown in SEQ ID NO: 34. 104, 109, 112; or

    And SEQ ID NO: 34. 104, 109, 112 has at least 80%, at least 90%, at least 95%, at least 98%, at least 99% sequence identity.
12. The PD-1 binding protein according to any one of the preceding claims, which has an activity selected from at least one of the following:

    (a) to be less than or equal to 10^-7Binds to human PD-1 or an epitope thereof;

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

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

    (d) inducing CD4+ T cells to secrete IFN- γ;

    (e) enhanced activation of PBMCs;

    (f) enhancing the activation of T cells;

    (g) inhibiting tumor growth.
13. A nucleic acid molecule encoding the PD-1 binding protein of any one of claims 1-12.
14. A vector comprising the nucleic acid molecule of claim 13.
15. A host cell comprising the vector of claim 14;

    preferably, the host cell is selected from the group consisting of: bacteria, yeast, mammalian cells;

    more preferably, the host cell is selected from the group consisting of: escherichia coli, Pichia pastoris, Chinese hamster ovary cells, human embryonic kidney 293 cells.
16. A method of preparing a PD-1 binding protein according to any one of claims 1 to 12, comprising the steps of:

    culturing the host cell of claim 15;

    recovering the PD-1 binding protein, and

    optionally, purifying and/or modifying the PD-1 binding protein.
17. A pharmaceutical composition comprising:

    a therapeutically or prophylactically effective amount of the PD-1 binding protein of any one of claims 1-12 and/or the nucleic acid molecule of claim 13; and

    one or more pharmaceutically acceptable carriers, diluents, buffers or excipients.
18. A method of preventing and/or treating cancer, comprising administering to a subject a therapeutically effective amount, or a prophylactically effective amount, of the PD-1 binding protein of any one of claims 1-12, or the nucleic acid molecule of claim 13, or the pharmaceutical composition of claim 17;

    preferably, the cancer is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, renal cancer, squamous cell carcinoma, hematological cancer, or a disease or disorder characterized by uncontrolled cell growth.
19. A method of inhibiting PD-1 activity or promoting T cell proliferation or benefiting a subject from upregulation of an immune response, comprising administering to the subject a therapeutically effective amount or a prophylactically effective amount of the PD-1 binding protein of any one of claims 1-12, or the nucleic acid molecule of claim 13, or the pharmaceutical composition of claim 17;

    preferably, the subject's expression of PD-L1 and/or PD-L2 is up-regulated;

    more preferably, the subject has cancer.
20. A method of treating or preventing an infectious disease, comprising administering to a subject a therapeutically effective amount or a prophylactically effective amount of the PD-1 binding protein of any one of claims 1-12, or the nucleic acid molecule of claim 13, or the pharmaceutical composition of claim 17;

    preferably, the infectious disease is caused by bacteria, fungi, viruses or parasites.
21. Use of the PD-1 binding protein of any one of claims 1 to 12 or the nucleic acid molecule of claim 13 or the pharmaceutical composition of claim 17 in the manufacture of a medicament for any one of the following:

    preventing cancer, treating cancer and infection, inhibiting PD-1 activity, and promoting T cell proliferation.

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