Anti-PD-1 monoclonal antibody and application thereof

Abstract

The invention relates to the technical field of antibody drugs, in particular to an anti-PD-1 monoclonal antibody and application thereof. Specifically, the invention provides an antibody specifically binding to PD-1 or an antigen binding fragment thereof, a pharmaceutical composition containing the antibody or the antigen binding fragment thereof, a nucleic acid molecule encoding the antibody or the antigen binding fragment thereof, and a host cell containing the nucleic acid molecule, and therapeutic and diagnostic uses of the antibodies or antigen-binding fragments thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application 202311307741.X filed on October 10, 2023 and Chinese patent application 202410131767.1 filed on January 30, 2024, and the entire contents of the above patent applications are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to the technical field of antibody drugs, and in particular to anti-PD-1 monoclonal antibodies and applications thereof. Technical Background

[0004] Programmed death-1 (PD-1, also known as CD279) is an inhibitory receptor that is primarily expressed on the surface of activated T cells. Certain subsets of B cells, monocytes, and dendritic cells also express PD-1 after activation.

[0005] PD-1 is a member of the immunoglobulin superfamily. It is 288 amino acids long and contains an extracellular domain, a transmembrane domain, and a cytoplasmic domain. The extracellular domain contains an immunoglobulin-like domain (IgV-likedomain), which is responsible for binding to its ligand. The cytoplasmic domain contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM), which are responsible for signal transduction.

[0006] PD-1 has two ligands, namely programmed death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2). PD-1 is not expressed on the surface of naive T cells. When the TCR / CD3 complex and CD28 on the surface of naive T cells bind to MHC-antigen peptides and CD80 / CD86, respectively, the intracellular regions of the TCR / CD3 complex and CD28 are phosphorylated by protein kinases, thereby recruiting a series of signal transduction proteins and initiating a series of phosphorylation events, which ultimately lead to the activation of naive T cells, and then PD-1 begins to be upregulated on its surface. When PD-1 binds to PD-L1 or PD-L2 on the surface of antigen-presenting cells, PD-1 is activated, and its cytoplasmic domain is phosphorylated by protein kinases, thereby recruiting protein phosphatase SHP-2. SHP-2 can dephosphorylate CD28 and TCR / CD3 complex intracellularly, thereby inhibiting the activation of T cells.

[0007] As an inhibitory receptor, PD-1 plays an important role in the suppression of immune responses and the maintenance of peripheral tolerance. PD-1 gene defects can lead to the occurrence of autoimmune diseases. C57BL / 6 mice with PD-1 gene knockout spontaneously develop lupus-like glomerulonephritis and arthritis symptoms (H Nishimura et al., Immunity. 1999 Aug; 11 (2): 141-51.). NZB / W F1 mice spontaneously develop symptoms similar to human systemic lupus erythematosus and are the most commonly used mouse model for human systemic lupus erythematosus research. Treatment with mouse PD-L1 fusion protein can alleviate proteinuria symptoms in NZB / W F1 mice and prolong mouse survival time (Wenjun Liao et al., Am J Nephrol. 2017; 46 (5): 371-379.). Chicken type II collagen can induce arthritis symptoms in mice. The collagen-induced mouse arthritis model is the most commonly used disease model for human rheumatoid arthritis. Treatment with mouse PD-L1 fusion protein can alleviate arthritis symptoms in C57BL / 6 mice (Amalia P Raptopoulou et al., Arthritis Rheum. 2010 Jul; 62(7): 1870-80.).

[0008] Although there are currently several PD-1 antagonist antibodies (such as pembrolizumab, also known as Keytruda) that block the PD-1 signaling pathway on the market and are used to treat a variety of tumors, it is still necessary to develop PD-1 agonist antibodies that can activate the PD-1 signaling pathway to inhibit T cell responses and thus treat inflammatory and autoimmune diseases. Summary of the invention

[0009] Through extensive research, the inventors of the present application have obtained a mouse antibody that specifically binds to PD-1, which has a significant effect of activating the PD-1 signaling pathway, and further obtained a humanized antibody with PD-1 binding activity and PD-1 signaling pathway activation based on the mouse antibody. The antibody of the present invention can effectively inhibit T cell activity, inhibit immune response or inflammatory response by activating the PD-1 signaling pathway, thereby playing a role in preventing and treating inflammation, allergies and autoimmune-related diseases. In addition, the antibody of the present invention also has species cross-binding activity and activation activity for human and monkey PD-1, which is beneficial to carry out preclinical evaluation research in animal models and other work.

[0010] Antibodies or antigen-binding fragments thereof

[0011] In a first aspect, the present application provides an antibody or an antigen-binding fragment thereof that can specifically bind to PD-1, wherein the antibody or the antigen-binding fragment thereof comprises:

[0012] VH CDR1 or variants thereof, VH CDR2 or variants thereof, and VH CDR3 or variants thereof contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 7 or 15; and / or, VL CDR1 or variants thereof, VL CDR2 or variants thereof, and VL CDR3 or variants thereof contained in the light chain variable region (VL) as shown in SEQ ID NO: 8 or 16;

[0013] Wherein, the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions, e.g., conservative substitutions) compared to the sequence from which it is derived. In certain embodiments, the substitutions are conservative substitutions.

[0014] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0015] The three CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 7 or 15; and / or, the three CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO: 8 or 16.

[0016] In certain embodiments, the three CDRs contained in the VH and / or the three CDRs contained in the VL are defined by the Kabat, IMGT, or Chothia numbering systems.

[0017] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0018] A heavy chain variable region (VH) comprising the following three complementary determining regions (CDRs): VHCDR1 with a sequence of SEQ ID NO: 1, VH CDR2 with a sequence of SEQ ID NO: 2, and VH CDR3 with a sequence of SEQ ID NO: 3; and / or, a light chain variable region (VL) comprising the following three complementary determining regions (CDRs): VL CDR1 with a sequence of SEQ ID NO: 4, VL CDR2 with a sequence of SEQ ID NO: 5, and VL CDR3 with a sequence of SEQ ID NO: 6; wherein the CDRs are defined by the Kabat numbering system.

[0019] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a framework region sequence derived from a murine immunoglobulin.

[0020] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0021] A heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 8 or a variant thereof;

[0022] Wherein, the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, or has at least 80%, at least 85%, 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. Preferably, the substitutions, deletions or additions are not present in the CDR regions.

[0023] In certain embodiments, the substitutions are conservative substitutions.

[0024] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO:7 and a VL comprising the sequence shown in SEQ ID NO:8.

[0025] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a framework region sequence derived from a human immunoglobulin.

[0026] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a framework region contained in an amino acid sequence encoded by a human germline antibody gene. In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain framework region of a human heavy chain germline sequence, and / or, a light chain framework region of a human light chain germline sequence. In certain embodiments, the heavy chain framework region of the human heavy chain germline sequence and / or the light chain framework region of the human light chain germline sequence may optionally comprise a back mutation from a human residue to a mouse residue.

[0027] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 15 or a variant thereof; and / or, a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 16 or a variant thereof;

[0028] Wherein, the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, or has at least 80%, at least 85%, 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. Preferably, the substitutions, deletions or additions are not present in the CDR regions.

[0029] In certain embodiments, the substitutions are conservative substitutions.

[0030] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) as shown in SEQ ID NO:15; and / or, a light chain variable region (VL) as shown in SEQ ID NO:16.

[0031] In a second aspect, the present application provides an antibody or an antigen-binding fragment thereof that can specifically bind to PD-1, wherein the antibody or the antigen-binding fragment thereof comprises:

[0032] VH CDR1 or variants thereof, VH CDR2 or variants thereof, and VH CDR3 or variants thereof contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 31 or 39; and / or, VL CDR1 or variants thereof, VL CDR2 or variants thereof, and VL CDR3 or variants thereof contained in the light chain variable region (VL) as shown in SEQ ID NO: 32 or 40;

[0033] Wherein, the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions, e.g., conservative substitutions) compared to the sequence from which it is derived. In certain embodiments, the substitutions are conservative substitutions.

[0034] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0035] The three CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 31 or 39; and / or, the three CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO: 32 or 40.

[0036] In certain embodiments, the three CDRs contained in the VH and / or the three CDRs contained in the VL are defined by the Kabat, IMGT, or Chothia numbering systems.

[0037] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0038] A heavy chain variable region (VH) comprising the following three complementary determining regions (CDRs): VHCDR1 with a sequence of SEQ ID NO: 25, VH CDR2 with a sequence of SEQ ID NO: 26, and VH CDR3 with a sequence of SEQ ID NO: 27; and / or, a light chain variable region (VL) comprising the following three complementary determining regions (CDRs): VL CDR1 with a sequence of SEQ ID NO: 28, VL CDR2 with a sequence of SEQ ID NO: 29, and VL CDR3 with a sequence of SEQ ID NO: 30; wherein the CDRs are defined by the Kabat numbering system.

[0039] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a framework region sequence derived from a murine immunoglobulin.

[0040] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0041] a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 31 or a variant thereof; and / or a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 32 or a variant thereof;

[0042] Wherein, the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, or has at least 80%, at least 85%, 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. Preferably, the substitutions, deletions or additions are not present in the CDR regions.

[0043] In certain embodiments, the substitutions are conservative substitutions.

[0044] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO:31 and a VL comprising the sequence shown in SEQ ID NO:32.

[0045] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a framework region sequence derived from a human immunoglobulin.

[0046] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a framework region contained in an amino acid sequence encoded by a human germline antibody gene. In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain framework region of a human heavy chain germline sequence, and / or, a light chain framework region of a human light chain germline sequence. In certain embodiments, the heavy chain framework region of the human heavy chain germline sequence and / or the light chain framework region of the human light chain germline sequence may optionally comprise a back mutation from a human residue to a mouse residue.

[0047] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 39 or a variant thereof; and / or, a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 40 or a variant thereof;

[0048] Wherein, the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, or has at least 80%, at least 85%, 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. Preferably, the substitutions, deletions or additions are not present in the CDR regions.

[0049] In certain embodiments, the substitutions are conservative substitutions.

[0050] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) as shown in SEQ ID NO:39; and / or, a light chain variable region (VL) as shown in SEQ ID NO:40.

[0051] In certain embodiments, in the first aspect or the second aspect, the antibody or antigen-binding fragment thereof further comprises a constant region derived from a mammalian (eg, human or mouse) immunoglobulin.

[0052] In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin (e.g., IgG, such as IgG1, IgG2, IgG3, or IgG4), and / or the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from a human immunoglobulin (e.g., κ or λ).

[0053] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region that has binding activity to an Fc receptor (eg, any one or more of CD32A, CD32B, and CD16A).

[0054] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) of a wild-type human immunoglobulin. In certain exemplary embodiments, the heavy chain constant region sequence of the wild-type human immunoglobulin is shown in SEQ ID NO:51.

[0055] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) of a wild-type human immunoglobulin. In certain exemplary embodiments, the sequence of the light chain constant region of the wild-type human immunoglobulin is as shown in SEQ ID NO:53.

[0056] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region set forth in SEQ ID NO:51, and / or a light chain constant region set forth in SEQ ID NO:53.

[0057] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises:

[0058] (1) a heavy chain comprising the sequence shown in SEQ ID NO: 11 and a light chain comprising the sequence shown in SEQ ID NO: 12;

[0059] (2) a heavy chain comprising the sequence shown in SEQ ID NO: 19 and a light chain comprising the sequence shown in SEQ ID NO: 20;

[0060] (3) a heavy chain comprising the sequence shown in SEQ ID NO: 35 and a light chain comprising the sequence shown in SEQ ID NO: 36; or,

[0061] (4) A heavy chain comprising the sequence shown in SEQ ID NO:43 and a light chain comprising the sequence shown in SEQ ID NO:44.

[0062] In other embodiments, the antibody or antigen-binding fragment thereof comprises a variant of a human immunoglobulin heavy chain constant region (CH), and the variant of the heavy chain constant region (CH) may comprise one or more amino acid mutations or chemical modifications to change one or more of the following properties of the antibody of the invention: Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function or complement function, etc. Functional changes can be produced by replacing at least one amino acid residue in the antibody constant region with a different residue or chemical modification, for example, changing the affinity of the antibody for effector ligands (such as FcR or complement C1q), thereby changing the effector function (e.g., reducing or enhancing). The Fc region of an antibody mediates several important effector functions, such as ADCC, phagocytosis, CDC, etc.

[0063] In certain embodiments, the variant may have enhanced binding to an Fc receptor (e.g., any one or more of CD32A, CD32B, CD16A) compared to the wild-type sequence from which it is derived, for example, at least 1.5-fold, at least 2-fold, or at least 2.5-fold, as detected by surface plasmon resonance technology.

[0064] In certain embodiments, the variant is a human IgG1 or IgG4 heavy chain constant region variant comprising a substitution at one or more of the following positions according to EU numbering: 239, 268. In certain embodiments, the substitution is S239D / H268D. In certain embodiments, the substitution is S239D / Q268D.

[0065] In certain exemplary embodiments, the human immunoglobulin heavy chain constant region variant has the sequence shown in SEQ ID NO:52.

[0066] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region set forth in SEQ ID NO:52, and / or a light chain constant region set forth in SEQ ID NO:53.

[0067] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises:

[0068] (1) a heavy chain comprising the sequence shown in SEQ ID NO: 23 and a light chain comprising the sequence shown in SEQ ID NO: 20; or

[0069] (2) A heavy chain comprising the sequence shown in SEQ ID NO:47 and a light chain comprising the sequence shown in SEQ ID NO:44.

[0070] In certain embodiments, in the first aspect or the second aspect, the antigen binding fragment is selected from Fab, Fab', F(ab') 2 、Fd、Fv、disulfide-linked Fv、scFv、di-scFv、(scFv) 2 , diabodies and single domain antibodies (sdAb); and / or, the antibody is a murine antibody, a humanized antibody, a chimeric antibody, a bispecific antibody or a multispecific antibody.

[0071] In a third aspect, the present application also provides an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof as described in the first aspect or the second aspect, or its heavy chain variable region and / or light chain variable region, or its heavy chain and / or light chain.

[0072] In certain embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding a heavy chain or a heavy chain variable region of an antibody or its antigen-binding fragment of the present invention and a second nucleotide sequence encoding a light chain or a light chain variable region of the antibody or its antigen-binding fragment, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different isolated nucleic acid molecules. When the first nucleotide sequence and the second nucleotide sequence are present on different isolated nucleic acid molecules, the isolated nucleic acid molecule of the present invention comprises a first nucleic acid molecule containing the first nucleotide sequence and a second nucleic acid molecule containing the second nucleotide sequence.

[0073] In certain embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding the heavy chain variable region of an antibody or antigen-binding fragment thereof of the present invention, and a second nucleotide sequence encoding the light chain variable region thereof, wherein:

[0074] (i) the first nucleotide sequence comprises: the sequence shown in SEQ ID NO: 9 or a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity thereto, or a degenerate sequence thereof; the second nucleotide sequence comprises: the sequence shown in SEQ ID NO: 10 or a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity thereto, or a degenerate sequence thereof;

[0075] (ii) the first nucleotide sequence comprises: a sequence as set forth in SEQ ID NO: 17, or a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity thereto, or a degenerate sequence thereof; the second nucleotide sequence comprises: a sequence as set forth in SEQ ID NO: 18, or a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity thereto, or a degenerate sequence thereof;

[0076] (iii) the first nucleotide sequence comprises: the sequence shown in SEQ ID NO:33 or a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity thereto, or a degenerate sequence thereof; the second nucleotide sequence comprises: the sequence shown in SEQ ID NO:34 or a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity thereto, or a degenerate sequence thereof; or,

[0077] (iv) the first nucleotide sequence comprises: the sequence shown in SEQ ID NO:41, or a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity thereto, or a degenerate sequence thereof; the second nucleotide sequence comprises: the sequence shown in SEQ ID NO:42, or a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity thereto, or a degenerate sequence thereof.

[0078] In certain embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding the heavy chain of an antibody or antigen-binding fragment thereof of the present invention, and a second nucleotide sequence encoding the light chain thereof, wherein:

[0079] (i) the first nucleotide sequence comprises: the sequence shown in SEQ ID NO: 13 or a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity thereto, or a degenerate sequence thereof; the second nucleotide sequence comprises: the sequence shown in SEQ ID NO: 14 or a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity thereto, or a degenerate sequence thereof;

[0080] (ii) the first nucleotide sequence comprises: the sequence shown in SEQ ID NO: 21, or a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity thereto, or a degenerate sequence thereof; the second nucleotide sequence comprises: the sequence shown in SEQ ID NO: 22, or a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity thereto, or a degenerate sequence thereof;

[0081] (iii) the first nucleotide sequence comprises: the sequence shown in SEQ ID NO: 24, or a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity thereto, or a degenerate sequence thereof; the second nucleotide sequence comprises: the sequence shown in SEQ ID NO: 22, or a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity thereto, or a degenerate sequence thereof;

[0082] (iv) the first nucleotide sequence comprises: a sequence as set forth in SEQ ID NO:37, or a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity thereto, or a degenerate sequence thereof; the second nucleotide sequence comprises: a sequence as set forth in SEQ ID NO:38, or a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity thereto, or a degenerate sequence thereof;

[0083] (v) the first nucleotide sequence comprises: the sequence set forth in SEQ ID NO:45 or a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity thereto, or a degenerate sequence thereof; the second nucleotide sequence comprises: the sequence set forth in SEQ ID NO:46 or a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity thereto, or a degenerate sequence thereof; or,

[0084] (vi) the first nucleotide sequence comprises: the sequence shown in SEQ ID NO:48 or a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity thereto, or a degenerate sequence thereof; the second nucleotide sequence comprises: the sequence shown in SEQ ID NO:46 or a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity thereto, or a degenerate sequence thereof.

[0085] In a fourth aspect, the present application also provides a vector comprising the isolated nucleic acid molecule as described above. In certain embodiments, the vector is a cloning vector or an expression vector.

[0086] In certain embodiments, the vector comprises a first nucleotide sequence encoding a heavy chain or a heavy chain variable region of an antibody or an antigen-binding fragment thereof of the present invention and a second nucleotide sequence encoding a light chain or a light chain variable region of the antibody or an antigen-binding fragment thereof, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different vectors. When the first nucleotide sequence and the second nucleotide sequence are present on different vectors, the vector of the present invention comprises a first vector comprising the first nucleotide sequence and a second vector comprising the second nucleotide sequence.

[0087] In a fifth aspect, the present application also provides a host cell, which comprises the isolated nucleic acid molecule or vector as described above.

[0088] Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (such as E. coli cells), and eukaryotic cells such as fungal cells (such as yeast cells), insect cells, plant cells and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). In certain embodiments, the host cell is a mammalian cell, such as a CHO cell.

[0089] The antibodies of the present invention can be prepared by various methods known in the art, such as by genetic engineering recombinant technology. For example, DNA molecules encoding the heavy chain and light chain genes of the antibodies of the present invention are obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into expression vectors and then transfected into host cells. Then, the transfected host cells are cultured under specific conditions and express the antibodies of the present invention.

[0090] The antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24: 107-117 (1992) and Brennan et al., Science 229: 81 (1985)). In addition, these antigen-binding fragments can also be directly produced by recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11: 548-557 (1999); Little et al., Immunol. Today, 21: 364-370 (2000)). For example, Fab' fragments can be directly obtained from host cells; Fab' fragments can be chemically coupled to form F(ab') 2 Fragments (Carter et al., Bio / Technology, 10:163-167 (1992)). In addition, Fv, Fab or F(ab') 2 Fragments can also be directly isolated from the culture medium of recombinant host cells. Other techniques for preparing these antigen-binding fragments are well known to those of ordinary skill in the art.

[0091] In a sixth aspect, the present application also provides a method for preparing the antibody or antigen-binding fragment thereof as described in the first aspect or the second aspect, comprising culturing the host cell as described above under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

[0092] Antibody derivatization

[0093] The antibodies or antigen-binding fragments thereof of the present invention may be derivatized, for example, linked to another molecule (e.g., another polypeptide or protein). Typically, the derivatization (e.g., labeling) of an antibody or antigen-binding fragment thereof does not adversely affect its binding to PD-1. Therefore, the antibodies or antigen-binding fragments thereof of the present invention are also intended to include such derivatized forms. For example, the antibodies or antigen-binding fragments thereof of the present invention may be functionally linked (by chemical coupling, genetic fusion, non-covalent linkage or other means) to one or more other molecular groups, such as another antibody (e.g., to form a bispecific antibody), a detection agent, a pharmaceutical agent, and / or a protein or polypeptide (e.g., avidin or a polyhistidine tag) that can mediate the binding of an antibody or antigen-binding fragment to another molecule.

[0094] In a seventh aspect, the present application also provides a bispecific or multispecific molecule, which comprises the antibody or antigen-binding fragment thereof as described in the first aspect or the second aspect.

[0095] In certain embodiments, the bispecific or multispecific molecule specifically binds PD-1 and additionally specifically binds one or more other targets.

[0096] In certain embodiments, the bispecific or multispecific molecule further comprises at least one molecule with a second binding specificity for a second target (eg, a second antibody).

[0097] In an eighth aspect, the present application also provides an immunoconjugate, which comprises the antibody or antigen-binding fragment thereof as described in the first aspect or the second aspect and a therapeutic agent linked to the antibody or antigen-binding fragment thereof.

[0098] In certain embodiments, the therapeutic agent is selected from a cytotoxic agent.

[0099] Pharmaceutical composition

[0100] In the ninth aspect, the present application also provides a pharmaceutical composition, which comprises the antibody or antigen-binding fragment thereof as described in the first or second aspect, the isolated nucleic acid molecule as described in the third aspect, the vector as described in the fourth aspect, the host cell as described in the fifth aspect, the bispecific or multispecific molecule as described in the seventh aspect, or the immunoconjugate as described in the eighth aspect, and a pharmaceutically acceptable carrier and / or excipient.

[0101] In certain embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent.

[0102] In certain embodiments, the additional pharmaceutically active agent is an immunosuppressant.

[0103] In certain embodiments, the additional pharmaceutically active agent is a drug used to treat an autoimmune disease, an allergic disease, graft-versus-host disease, transplant rejection, or a lymphoma (such as a T-cell lymphoma).

[0104] In certain embodiments, in the pharmaceutical composition, the antibody or antigen-binding fragment thereof, isolated nucleic acid molecule, vector, host cell, bispecific or multispecific molecule or immunoconjugate of the present invention and the additional pharmaceutically active agent can be provided as separate components or as mixed components. Therefore, the antibody or antigen-binding fragment thereof, isolated nucleic acid molecule, vector, host cell, bispecific or multispecific molecule or immunoconjugate of the present invention and the additional pharmaceutically active agent can be administered simultaneously, separately or sequentially.

[0105] In certain embodiments, the pharmaceutical composition may include a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody or antigen-binding fragment thereof, an isolated nucleic acid molecule, a vector, a host cell, a bispecific or multispecific molecule, or an immunoconjugate of the invention. A "prophylactically effective amount" refers to an amount sufficient to prevent, prevent, or delay the onset of a disease. A "therapeutically effective amount" refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount may vary depending on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0106] Therapeutic Uses

[0107] The antibody or antigen-binding fragment thereof of the present invention can enhance the binding of PD-1 to PD-L1 / PD-L2, activate the PD-1 signaling pathway, and then inhibit T cell activity. The following therapeutic applications are provided.

[0108] In one aspect, the present application provides a method for activating the PD-1 signaling pathway, which comprises using an antibody or antigen-binding fragment thereof as described in the first or second aspect, an isolated nucleic acid molecule as described in the third aspect, a vector as described in the fourth aspect, a host cell as described in the fifth aspect, a bispecific or multispecific molecule as described in the seventh aspect, an immunoconjugate as described in the eighth aspect, or a pharmaceutical composition as described in the ninth aspect. In certain embodiments, the method comprises contacting the antibody or antigen-binding fragment thereof with a cell in vitro to activate the PD-1 signaling pathway in the cell. In certain embodiments, the method comprises administering the antibody or antigen-binding fragment thereof to a subject in need thereof to activate the PD-1 signaling pathway in the body, thereby inhibiting an immune response or inflammatory response.

[0109] On the other hand, the present application provides use of the antibody or antigen-binding fragment thereof as described in the first or second aspect, the isolated nucleic acid molecule as described in the third aspect, the vector as described in the fourth aspect, the host cell as described in the fifth aspect, the bispecific or multispecific molecule as described in the seventh aspect, the immunoconjugate as described in the eighth aspect, or the pharmaceutical composition as described in the ninth aspect in the preparation of a PD-1 signaling pathway activator.

[0110] In one aspect, the present application provides a method for inhibiting the activity of immune cells, which includes using an antibody or antigen binding fragment thereof as described in the first or second aspect, an isolated nucleic acid molecule as described in the third aspect, a vector as described in the fourth aspect, a host cell as described in the fifth aspect, a bispecific or multispecific molecule as described in the seventh aspect, an immunoconjugate as described in the eighth aspect, or a pharmaceutical composition as described in the ninth aspect. In certain embodiments, the method includes contacting the antibody or antigen binding fragment thereof with a cell (e.g., a T cell) in vitro to inhibit its immune activity. In certain embodiments, the method includes administering the antibody or antigen binding fragment thereof to a subject in need thereof to inhibit the activity of immune cells in the body, thereby inhibiting an immune response or inflammatory response.

[0111] On the other hand, the present application provides use of the antibody or antigen-binding fragment thereof as described in the first or second aspect, the isolated nucleic acid molecule as described in the third aspect, the vector as described in the fourth aspect, the host cell as described in the fifth aspect, the bispecific or multispecific molecule as described in the seventh aspect, the immunoconjugate as described in the eighth aspect, or the pharmaceutical composition as described in the ninth aspect in the preparation of an immunosuppressant (e.g., a T cell activity inhibitor).

[0112] In one aspect, the present application provides a method for preventing and / or treating a disease in a subject, comprising: administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof as described in the first or second aspect, the isolated nucleic acid molecule as described in the third aspect, the vector as described in the fourth aspect, the host cell as described in the fifth aspect, the bispecific or multispecific molecule as described in the seventh aspect, the immunoconjugate as described in the eighth aspect, or the pharmaceutical composition as described in the ninth aspect.

[0113] On the other hand, the present application provides the use of the antibody or its antigen-binding fragment, isolated nucleic acid molecule, vector, host cell, bispecific or multispecific molecule, immunoconjugate or pharmaceutical composition in the preparation of a medicament for preventing and / or treating a disease in a subject.

[0114] In certain embodiments, the disease will benefit from activation of the PD-1 signaling pathway. In certain embodiments, the disease is characterized by inactivation of the PD-1 / PD-L1 signaling pathway. In certain embodiments, the disease will benefit from inhibition of an immune response or inflammatory response.

[0115] In certain embodiments, the disease is selected from an autoimmune disease, an allergic disease, graft-versus-host disease, transplant rejection, lymphoma, or any combination thereof.

[0116] In certain embodiments, the autoimmune disease is selected from arthritis, rheumatoid arthritis, psoriatic arthritis, lupus, lupus nephritis, systemic lupus erythematosus, psoriasis, vitiligo, alopecia areata, inflammatory bowel disease, ulcerative colitis, Crohn's disease, type I diabetes, multiple sclerosis, autoimmune hepatitis, primary biliary cirrhosis, celiac disease, scleroderma, Graves' disease, Hashimoto's thyroiditis, ankylosing spondylitis, myasthenia gravis, Sjögren's syndrome, IgA nephropathy, IgG4-Related Disease, vasculitis, ANCA-associated vasculitis, uveitis, pemphigus, pemphigoid, or autoimmune hemolytic anemia.

[0117] In certain embodiments, the allergic disease is selected from idiopathic thrombocytopenic purpura, atopic dermatitis, asthma, allergic rhinitis, drug allergy, food allergy, anaphylaxis, allergic conjunctivitis, urticaria, eosinophilic sinusitis, eosinophilic digestive organ disease, or allergic bronchopulmonary aspergillosis.

[0118] In certain embodiments, the lymphoma is a T-cell lymphoma.

[0119] In certain embodiments, the subject in any of the above aspects is a mammal. In certain embodiments, the subject is a human. In certain embodiments, the subject is a non-human primate, such as a cynomolgus monkey.

[0120] In certain embodiments, the antibody or antigen-binding fragment thereof, isolated nucleic acid molecule, vector, host cell, bispecific or multispecific molecule, immunoconjugate or pharmaceutical composition described in any of the above aspects is used alone or in combination with another pharmaceutically active agent, such as administered simultaneously or sequentially. In certain embodiments, the other pharmaceutically active agent is an immunosuppressant. In certain embodiments, the other pharmaceutically active agent is a drug for treating autoimmune diseases, allergic diseases, graft-versus-host disease, transplant rejection or lymphoma (such as T-cell lymphoma).

[0121] In any of the above aspects, the antibody or antigen-binding fragment thereof or pharmaceutical composition of the present application can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The antibody or antigen-binding fragment thereof or pharmaceutical composition of the present invention should be sterile and stable under production and storage conditions. A preferred dosage form is an injection. Such an injection can be a sterile injection solution. For example, a sterile injection solution can be prepared by the following method: incorporating the necessary dose of the antibody or antigen-binding fragment thereof of the present invention into an appropriate solvent, and optionally, incorporating other desired ingredients (including but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filtration and sterilization. In addition, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze drying) for storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0122] In any of the above aspects, the antibody or its Fab or pharmaceutical composition of the present application can be applied by any suitable method known in the art, including but not limited to, oral, oral, sublingual, eyeball, topical, parenteral, rectal, intrathecal, inguinal, intravesical, topical (such as, powder, ointment or drops), or nasal route. However, for many therapeutic uses, preferred route of administration / mode is parenteral administration (such as intravenous injection or push injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). Technicians should understand that route of administration and / or mode will change according to the intended purpose. In certain embodiments, the antibody or its Fab or pharmaceutical composition of the present invention is given by intravenous injection or push injection.

[0123] Detection purpose

[0124] The antibody or antigen-binding fragment thereof of the present invention can specifically bind to PD-1, thereby providing the following detection applications.

[0125] In one aspect, the present application provides a conjugate, which comprises the antibody or antigen-binding fragment thereof as described in the first aspect or the second aspect, and a detectable label linked to the antibody or antigen-binding fragment thereof.

[0126] In certain embodiments, the detectable label is selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin.

[0127] In another aspect, the present application provides a kit, which includes the antibody or antigen-binding fragment thereof as described in the first aspect or the second aspect, or the conjugate as described above.

[0128] In certain embodiments, the kit comprises a conjugate as described above.

[0129] In certain embodiments, the kit comprises the antibody or antigen-binding fragment thereof as described in the first aspect or the second aspect, and a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof; optionally, the second antibody further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin.

[0130] On the other hand, the present application also provides a method for detecting the presence or level of PD-1 in a sample, which comprises using the antibody or antigen-binding fragment thereof as described in the first aspect or the second aspect or the conjugate as described above.

[0131] In certain embodiments, the methods are used for therapeutic purposes, diagnostic purposes, or non-therapeutic, non-diagnostic purposes.

[0132] In certain embodiments, the method is an immunological assay, such as an immunoblot, an enzyme immunoassay (eg, ELISA), a chemiluminescent immunoassay, a fluorescent immunoassay, or a radioimmunoassay.

[0133] In certain embodiments, the method comprises: (1) contacting the sample with an antibody or antigen-binding fragment thereof of the present invention; (2) detecting the formation of an antigen-antibody immune complex or detecting the amount of the immune complex. The formation of the immune complex indicates the presence of PD-1 or cells expressing PD-1. In certain embodiments, the antibody or antigen-binding fragment thereof carries a detectable label, or the antibody or antigen-binding fragment thereof is detected using a second antibody carrying a detectable label.

[0134] On the other hand, the present application also provides a method for diagnosing a disease associated with PD-1, comprising detecting the level of PD-1 in a sample from a subject using the method described above. In certain embodiments, the method further comprises comparing the level of PD-1 in a sample from a subject with a reference value (e.g., a healthy control) to diagnose whether the subject suffers from the disease.

[0135] In certain embodiments, the PD-1-related disease is characterized by inactivation of the PD-1 / PD-L1 signaling pathway, and when the level of PD-1 is reduced compared to a reference value (e.g., compared to a healthy control), it indicates that the subject suffers from the PD-1-related disease. In certain embodiments, the PD-1-related disease is an autoimmune disease, an allergic disease, a graft-versus-host disease, a transplant rejection, a lymphoma, or a combination thereof. In certain embodiments, the lymphoma is a T-cell lymphoma.

[0136] In certain embodiments, the PD-1-related disease is characterized by overactivation of the PD-1 / PD-L1 signaling pathway, and when the level of PD-1 is increased compared to a reference value (e.g., compared to a healthy control), it indicates that the subject suffers from the PD-1-related disease. In certain embodiments, the PD-1-related disease is a tumor.

[0137] In certain embodiments, the sample can be selected from urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, non-tissue associated cells (i.e., free cells), tissue (e.g., surgically resected tumor tissue, biopsy tissue or fine needle aspirate tissue), histological preparations, etc.

[0138] In certain embodiments, the subject is a mammal, such as a human or a non-human primate (eg, cynomolgus monkey).

[0139] On the other hand, the present application also provides the use of the antibody or antigen-binding fragment thereof as described in the first aspect or the second aspect or the conjugate as described above in the preparation of a detection reagent, wherein the detection reagent is used to detect the presence or level of PD-1 in a sample and / or diagnose a disease associated with PD-1.

[0140] In certain embodiments, the detection reagent detects the presence or level of PD-1 in a sample by the detection method described above.

[0141] In certain embodiments, the detection reagent detects the presence or level of PD-1 in a sample by the detection method described above to diagnose a disease associated with PD-1.

[0142] Definition of terms

[0143] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell culture, molecular biology, biochemistry, nucleic acid chemistry, immunology and other operating steps used herein are conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0144] When the terms "for example," "such as," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[0145] The terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0146] As used herein, the term "programmed death-1 (PD-1)" refers to an inhibitory receptor that is primarily expressed on the surface of activated T cells and is responsible for signal transduction. Activation of the PD-1 signaling pathway inhibits the activity of T cells. The human PD-1 protein encoding gene sequence can be found in NCBI accession number: NM_005018.3.

[0147] As used herein, the term "antibody" refers to an immunoglobulin-derived molecule that can specifically bind to a target antigen, and the immunoglobulin-derived molecule binds to the target antigen through at least one antigen binding site located in its variable region. When referring to the term "antibody", unless the context clearly indicates, it includes not only complete antibodies, but also antigen binding fragments that can specifically bind to a target antigen. "Complete antibodies" are typically composed of two pairs of polypeptide chains (each pair has a light chain (LC) and a heavy chain (HC)). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA and IgE, respectively. In the light chain and heavy chain, the variable region and the constant region are connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant domain is not directly involved in the binding of antibodies to antigens, but exhibits a variety of effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be subdivided into highly variable regions (called complementary determining regions (CDRs)), interspersed with more conservative regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form antigen binding sites, respectively. The distribution of amino acids in various regions or domains can follow the definition of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0148] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. There are three CDRs in each of the variable regions of the heavy and light chains, designated as CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883) or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003). For a given antibody, a person skilled in the art will easily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those skilled in the art (eg, see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0149] In the present invention, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention can be identified according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention are preferably identified by the Kabat numbering system.

[0150] As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

[0151] The term "antibody" is not limited to any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibody can be an antibody of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibody.

[0152] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab'), 2 , Fd, Fv, disulfide-linked Fv, scFv, di-scFv, diabody, single domain antibody and such polypeptides, which contain at least a portion of an antibody sufficient to confer specific antigen binding ability to the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0153] As used herein, the term "Fd" means an antibody fragment consisting of the VH and CH1 domains; the term "Fab fragment" means an antibody fragment consisting of the VL, VH, CL and CH1 domains; the term "F(ab') 2 The term "Fab' fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab' fragment" means a F(ab') fragment linked by reduction. 2 The fragment obtained after the disulfide bond between the two heavy chain fragments in the fragment consists of a complete light chain and the Fd fragment of the heavy chain (consisting of the VH and CH1 domains).

[0154] As used herein, the term "Fv" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen binding site. It is generally believed that the six CDRs confer antigen binding specificity to an antibody. However, even a variable region (e.g., a Fd fragment, which contains only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of a complete binding site.

[0155] As used herein, the term "Fc" means an antibody fragment formed by disulfide bonding of the second and third constant regions of the first heavy chain of an antibody to the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has a variety of different functions, but does not participate in the binding of an antigen. The "effector functions" mediated by the Fc domain include Fc receptor binding; Clq binding and complement-dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation, etc. The Fc domain may include both a native Fc region and a variant Fc region. A native Fc region comprises an amino acid sequence consistent with the amino acid sequence of an Fc region found in nature, for example, a native sequence human Fc region comprises a native sequence human IgG1 Fc region; a native sequence human IgG2 Fc region; a native sequence human IgG3 Fc region; and a native sequence human IgG4 Fc region, as well as naturally occurring variants thereof. A variant Fc region comprises an amino acid sequence that is different from the amino acid sequence of a native sequence Fc region due to at least one amino acid modification. In some embodiments, a variant Fc region may possess altered effector function (eg, Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function) compared to a native Fc region.

[0156] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and a VH domain, wherein the VL and VH are connected by a linker. Such scFv molecules may have a general structure: NH 2 -VL-Linker-VH-COOH or NH 2 -VH-Linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) may be used. 4 The linker of scFv can be used, but its variants can also be used. In some cases, there can also be a disulfide bond between the VH and VL of the scFv. In certain embodiments of the present invention, the scFv can form a di-scFv, which refers to two or more single scFvs connected in series to form an antibody. In certain embodiments of the present invention, the scFv can form (scFv) 2 , which refers to two or more single scFvs in parallel to form an antibody.

[0157] As used herein, the term "diabody" means that its VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0158] As used herein, the term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art, and refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind to the same antigen bound by the full-length antibody.

[0159] As used herein, the term "bispecific antibody" refers to an antibody that has binding specificity for two different antigens (or epitopes). The term "multispecific antibody" refers to an antibody that has binding specificity for at least two or more (e.g., three or four) different antigens (or epitopes). A bispecific antibody or multispecific antibody comprises multiple antigen binding domains that have binding specificity for different antigens (or epitopes), thereby being able to bind to at least two different binding sites and / or target molecules. Each antigen binding domain comprised by a bispecific antibody or multispecific antibody can be independently selected from a full-length antibody (e.g., IgG antibody) or an antigen binding fragment thereof (e.g., Fv fragment, Fab fragment, F(ab') 2 In some cases, the individual antigen binding domains are connected by a peptide linker.

[0160] Each of the above antibody fragments retains the ability to specifically bind to the same antigen as the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen.

[0161] Antibody antigen-binding fragments (e.g., the antibody fragments described above) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and the antibody antigen-binding fragments can be screened for specificity in the same manner as for intact antibodies.

[0162] As used herein, the term "humanized antibody" refers to a non-human antibody that has been genetically engineered, and its amino acid sequence has been modified to improve the homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). In certain embodiments, the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain the expected properties of donor antibodies, including but not limited to, antigen specificity, affinity, reactivity, etc. In the present application, the donor antibody can be a mouse antibody with expected properties (e.g., antigen specificity, affinity, reactivity, etc.). To prepare a humanized antibody, the CDR region of the donor antibody can be inserted into a human framework sequence using methods known in the art. In some cases, the humanized framework sequence may include amino acid mutations replaced by corresponding non-human residues. In addition, the humanized antibody may also include residues not found in the original donor antibody variable region (e.g., light chain variable region or heavy chain variable region) or humanized framework sequence to further improve or optimize the performance of the humanized antibody.

[0163] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of its light chain or / and heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of the light chain or / and heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains binding activity to the target antigen. In certain embodiments, the term "chimeric antibody" may include an antibody in which the heavy chain variable region and light chain variable region of the antibody are derived from a first antibody, and the heavy chain constant region and light chain constant region of the antibody are derived from a second antibody.

[0164] As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. In order to determine the percentage identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (for example, a gap can be introduced in the first amino acid sequence or nucleic acid sequence to optimally align with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percentage identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percentage identity = number of identical overlapping positions / total number of positions × 100%). In certain embodiments, the two sequences are the same length.

[0165] The determination of the percent identity between two sequences can also be achieved using a mathematical algorithm. A non-limiting example of a mathematical algorithm for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2264-2268, as improved in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90: 5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215: 403.

[0166] As used herein, the term "variant", in the context of a polypeptide (including a polypeptide), also refers to a polypeptide or peptide comprising an amino acid sequence that has been changed by introducing amino acid residue substitutions, deletions or additions. In some cases, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to a polypeptide or peptide). For example, but not limiting, a polypeptide can be modified, such as by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage, connection to a cell ligand or other protein, etc. Derivatized polypeptides or peptides can be produced by chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, a variant has a function similar, identical or improved to the polypeptide or peptide from which it is derived.

[0167] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its antigen. The strength or affinity of a specific binding interaction can be measured by the equilibrium dissociation constant (K) of the interaction. DIn the present invention, the term "K D ” refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

[0168] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rate of formation and dissociation of the antigen binding site / antigen complex. Both the "association rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation. The ratio of kdis / kon is equal to the dissociation constant K D K can be measured by any valid method. D , kon and kdis values. In certain embodiments, the dissociation constant can be measured in Biacore using surface plasmon resonance (SPR).

[0169] As used herein, the detectable label of the present invention can be any substance that can be detected by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Such labels are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3 H. 125 I. 35 S. 14 C or 32 P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), fluorescent proteins (e.g., phycoerythrin (PE)), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa750)), luminescent substances (e.g., chemiluminescent agents, such as acridinium ester compounds, luminol and its derivatives, ruthenium derivatives such as terpyridine ruthenium), magnetic beads (e.g., ), calorimetric markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above markers. In certain embodiments, the detectable label is selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin. In certain embodiments, the detectable label as described above can be connected to the antibody or antigen-binding fragment thereof of the present invention by linkers of different lengths to reduce potential steric hindrance.

[0170] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

[0171] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0172] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0173] The compilation of the twenty conventional amino acids involved in this article follows conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0174] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with a subject and an active ingredient, which is well known in the art and includes, but is not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, preservatives, stabilizers. For example, pH regulators include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning generally understood by those skilled in the art, which can stabilize the desired activity of the active ingredient in the drug, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate), etc. In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient includes a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (such as 0.9% NaCl), glucose solution (such as 5% glucose), a solution containing a surfactant (such as 0.01% polysorbate 20), a pH buffer solution (such as a phosphate buffer solution), Ringer's solution, and any combination thereof.

[0175] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease or disorder or symptom in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical result for a subject who exhibits symptoms of a disease or is diagnosed with a disease. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partially or completely), whether detectable or undetectable. In addition, "treatment" can also refer to extending survival compared to the expected survival (e.g., survival without treatment).

[0176] As used herein, the term "subject" refers to a mammal, such as a human or non-human primate (e.g., cynomolgus monkey). In certain embodiments, the subject suffers from an autoimmune disease, an allergic disease, a graft-versus-host disease, a transplant rejection, or a lymphoma (e.g., a T-cell lymphoma), or has the risk of suffering from the above diseases.

[0177] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, an effective amount for preventing a disease (e.g., an autoimmune disease, an allergic disease, a graft-versus-host disease) refers to an amount sufficient to prevent, prevent, or delay the occurrence of the disease; an effective amount for treating a disease refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient who already has the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0178] Advantageous Effects of the Invention

[0179] The anti-PD-1 monoclonal antibody provided by the present invention not only does not block the binding of PD-1 to PD-L1 / PD-L2, but also can enhance the binding of PD-1 to PD-L1 / PD-L2, activate the PD-1 signaling pathway, inhibit T cell activity, and thus inhibit immune response or inflammatory response. Therefore, the anti-PD-1 monoclonal antibody of the present invention has important clinical significance for the treatment of inflammation, allergy and autoimmune related diseases. In addition, the antibody of the present invention has species cross-binding activity and activation activity for human and monkey PD-1, which is beneficial to carry out preclinical evaluation research in animal models and other work.

[0180] Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. According to the following detailed description of the accompanying drawings and preferred embodiments, various objects and advantages of the present invention will become practicable to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0181] Figure 1 : Anti-PD-1 chimeric antibody inhibits Jurkat-PD1-NFAT reporter cell activation assay results.

[0182] Figure 2 : Binding assay results of anti-PD-1 humanized antibody and CHOS-hPD-1.

[0183] Figure 3: Binding assay results of anti-PD-1 humanized antibody and CHOS-cPD-1.

[0184] Figure 4 : Anti-PD-1 humanized antibody inhibits Jurkat-PD1-NFAT reporter cell activation assay results.

[0185] Figure 5 :The results of the assay on the inhibition of IFN-γ secretion activity of primary T cells by anti-PD-1 humanized antibody.

[0186] Figure 6 : ADCC activity assay results of anti-PD-1 humanized antibody.

[0187] Figure 7 : Assay results of anti-PD-1 humanized antibodies blocking or enhancing the binding of hPD-L1 to CHOS-hPD-1.

[0188] Figure 8 : Assay results of anti-PD-1 humanized antibodies blocking or enhancing the binding of hPD-L2 to CHOS-hPD-1.

[0189] Fig. 9 :The results of efficacy test of anti-PD-1 humanized antibody in mouse GVHD model.

[0190] Fig.10 :Pharmacokinetic results of anti-PD-1 humanized antibody in human FcRn transgenic mice.

[0191] Fig.11 :The results of efficacy test of anti-PD-1 humanized antibody in mouse SLE model.

[0192] Fig.12 :The results of efficacy test of anti-PD-1 humanized antibody in mouse TNBS-IBD model.

[0193] Sequence information

[0194] Table 1: The information of the sequences involved in the present invention is described in the table below:

[0195]

[0196] Example

[0197] The invention is now described in the following non-limiting examples.

[0198] Those skilled in the art will appreciate that the embodiments describe the present invention by way of example and are not intended to limit the scope of protection claimed in the present application. The experimental methods in the embodiments are conventional methods unless otherwise specified. If specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0199] Example 1: Preparation of antigen protein

[0200] 1.1 Transient expression of antigens

[0201] Using FreeStyle TM hPD-1ECD mIgG1Fc and cPD-1ECD mIgG1Fc were transiently expressed in 293F cells (Gibco) in Expi293 Expression Medium (Gibco). 24 hours before transfection, 2.5-3×10 6 100 mL of Expi293F cells / mL was placed in the culture flask at 37°C and 8% CO 2 , cultured in a shaker at 120 rpm. The cell density was measured before transfection and reached 4.5-5.5×10 6 cells / mL, the cell density was adjusted to 3×10 6 cells / mL and transfect. When transfecting, first take 320μL of ExpiFectamine TM 293 Reagent (Gibco) was added to 5.68 mL Opti-MEM (Life) and mixed well. The mixture was incubated at room temperature for 5 minutes. At the same time, 100 μg of hPD-1ECD mIgG1Fc and cPD-1ECD mIgG1Fc expression plasmids were diluted to 6 mL using Opti-MEM. The diluted transfection reagent and plasmids were fully mixed and incubated at room temperature for 10-20 minutes. Then, the mixture was added to the cells and mixed well. The culture flask was placed at 37°C and 8% CO 2 , and cultured in a shaker at 120 rpm for 5 days. 18-22 hours after cell transfection, 600 μL ExpiFectamin293 enhancer 1 and 6 mL enhancer 2 (Gibco) were added.

[0202] 1.2 Antigen purification

[0203] The cell culture medium was centrifuged at 2000g for 20 minutes, and the supernatant was collected and filtered with a 0.22 micron filter membrane. After PrismA (Cytiva) affinity chromatography (protein purification liquid chromatography system: Cytiva, AKTA avant 25), eluted with 20mM citric acid-sodium citrate (pH 3.2), and then adjusted to neutral pH with 1M Tris base. The purified sample was subjected to SDS-PAGE using 4-20% gradient gel to determine the protein purity.

[0204] Example 2: Production of anti-PD-1 monoclonal antibodies based on single B cell cloning technology

[0205] 2.1 Immunization of mice

[0206] Female BALB / c mice aged 6-8 weeks (Beijing Biotechnology Co., Ltd., Animal License No.: SCXK (Beijing) 2019-0010) were housed in an SPF-grade laboratory with a 12 / 12 hour light / dark cycle, a temperature of 20-26°C, and a humidity of 40-70%. Before immunization, 10 μg of hPD-1ECD mIgG1Fc or cPD-1ECD mIgG1Fc protein was mixed with adjuvant in a volume ratio of 1:1. Ten mice were immunized in the foot. Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for the second immunization 2 days later. The volume injected at each injection point was 25 μL. After 3 weeks of immunization, blood was collected from the mouse orbits, and the serum titer was detected by indirect ELISA. Mice with a titer of 1:200,000 or above were boosted.

[0207] 2.2 Plasma cell enrichment

[0208] 3-5 days after the booster immunization, the mice were euthanized by cervical dislocation and soaked in 75% alcohol for 5 minutes. The abdominal skin and leg muscles were cut open, and the lymph nodes at the corresponding positions in the abdominal skin and leg muscles with rich blood vessels were lifted up with small forceps, cut off, and non-lymph node tissues were removed, and transferred to Gibco RPMI1640 medium. The skin and peritoneum at the junction of the left abdomen and chest of the mouse were cut open to expose the spleen, and the spleen was lifted up with small forceps, cut off, and non-spleen tissues were removed, and transferred to Gibco RPMI1640 medium.

[0209] Carefully peel off the leg muscles of the mouse, cut off the thigh bone and tibia respectively, cut off the cartilage at both ends to expose the red bone marrow cavity, and use a dust-free cloth to remove the muscles on the bones. Take a 1mL sterile syringe, draw 1mL RPMI1640 culture medium, and gently insert the sterile needle into the bone marrow cavity to flush the bone marrow cavity to obtain bone marrow. Place a 40μm filter in a 50mL centrifuge tube, use a pipette to draw the bone marrow fluid, and slowly inject the bone marrow fluid into the centrifuge tube through the filter to remove the residue. Place the centrifuge tube in a centrifuge and centrifuge for 10 minutes (400g, 4°C). Remove the supernatant and add EasySep TM Buffer (STEMCELL Technologies), after thorough mixing, bone marrow cell counting was performed.

[0210] Grind the spleen and lymph nodes with a syringe piston, filter the cells through a 40μm filter, transfer the cell suspension into sterile centrifuge tubes, discard the supernatant after centrifugation, resuspend the lymphocytes in 5mL Gibco RPMI1640 for later use; take 10μL of the cell suspension to count the spleen and lymph node cells. Then use EasySep TM Plasma cells were enriched using the Mouse CD138 Positive Isolation Kit (STEMCELL Technologies).

[0211] 2.3 Screening of plasma cells that specifically bind to PD-1 antigen using the Beacon single-cell photoconductive system

[0212] Biotin-labeled hPD-1 or cPD-1 protein was coated with assay beads (Berkeley Lights), and then the coated antigen-beads were washed with PBS for later use. The density of enriched plasma cells was adjusted to 7×10 6 / mL was then introduced into the NanoPen of the OptoSelect 14K chip (Berkeley Lights). The hPD-1 or cPD-1 protein-coated assay beads and 5μg / mL PE Goat Anti-Mouse IgG secondary antibody (Jackson) were introduced into the Channel of the chip. The PE fluorescence channel exposure time was set to 1000ms, with a total of 7 cycles, each cycle of 6min. After the experiment, plasma cells that were positive for both hPD-1 and cPD-1 protein screening were exported to a 96-well plate containing lysis buffer and mineral oil.

[0213] 2.4 Plasma cell sequencing and expression vector construction

[0214] Opto Plasma B Discovery cDNA Synthesis Kit (Berkeley Lights) was used for cDNA synthesis. Then, Opto Plasma B Discovery Sanger Prep Kit (Berkeley Lights) was used to amplify the IgG heavy chain variable region gene and the Kappa chain variable region gene. The heavy chain variable region and light chain variable region fragments obtained after amplification were homologously recombined, transformed and sequenced with the vector pcDNA3.4(EBV)-hIgG1CH-S1 containing the human IgG1 heavy chain constant region sequence (SEQ ID NO: 51) and the vector pcDNA3.4(EBV)-hIgKCL-S1 containing the human IgK light chain constant region sequence (SEQ ID NO: 53).

[0215] Finally, two anti-PD-1 chimeric antibodies B1-1B5-H1L1 and B1-1C4-H1L1 were obtained, and their sequence information is shown in the following table.

[0216] Table 2: Anti-PD-1 chimeric antibody sequence information

[0217]

[0218] Example 3: Preparation of recombinant anti-PD-1 chimeric antibodies

[0219] 3.1 Antibody transient expression

[0220] 24 hours before transfection, inoculate 2.5-3×10 6 100 mL of Expi293F cells / mL was added and the culture flask was placed at 37°C with 8% CO 2 , cultured in a shaker at 120 rpm. The cell density was measured before transfection and reached 4.5-5.5×10 6 cells / mL, the cell density was adjusted to 3×10 6 Cells / mL and transfected. The plasmid was diluted to 20 ng / μL using Opti-MEM (Gibco) in a 96-well PCR plate, and 40 μL of the diluted plasmid was slowly added to a U-bottom 96-well plate pre-filled with 50 μL / well Opti-MEM (total plasmid amount was 0.8 μg, 0.4 μg each of light and heavy chain plasmids); during transfection, 2.5 μL of ExpiFectamine TM293 Reagent (Gibco) was added to 50 μL Opti-MEM and mixed, and incubated at room temperature for 5 minutes. The diluted transfection reagent and plasmid were fully mixed and incubated at room temperature for 10-20 minutes; 0.8 mL of cell suspension was added to a 96-well cell culture plate, and 0.8 mL of PBS was added to the remaining wells. The prepared transfection system solution was slowly added to the cells, and the cell culture plate was placed at 37°C, 8% CO 2 The cells were shaken at 900 rpm in a cell shaker at 80-85% humidity for 5 days. 18-22 hours after cell transfection, 5 μL ExpiFectamin 293 enhancer 1 and 50 μL ExpiFectamin 293 enhancer 2 (Gibco) were added.

[0221] 3.2 Antibody Purification

[0222] The supernatant was collected into a 96-well plate, and PrismA affinity chromatography medium (Cytiva) was added to the supernatant, and incubated for 2 hours for binding. Equilibrated with 20mM PB-150mM NaCl, eluted with 20mM citric acid-sodium citrate (pH 3.2), and then adjusted to neutral pH with 1M Tris base. The purified sample was subjected to SDS-PAGE using 4-20% gradient gel to determine the purity of the antibody.

[0223] Example 4: Binding activity assay of anti-PD-1 chimeric antibodies to PD-1 overexpressing cell lines

[0224] 4.1 Preparation of PD-1 overexpressing cell lines

[0225] The Homo sapiens PD-1 protein encoding gene (gene number: NM_005018.3) was connected to the pLVX-hPD-1-IRES-Neo plasmid to construct the hPD-1 protein lentiviral plasmid. The Mus musculus PD-1 protein encoding gene (gene number: NM_008798.3) was connected to the pLVX-mPD-1-IRES-Hyg plasmid to construct the mPD-1 protein lentiviral plasmid. The Macaca fascicularis PD-1 protein encoding gene (gene number: EF443145.1) was connected to the pLVX-cPD-1-IRES-Hyg plasmid to construct the cPD-1 protein lentiviral plasmid.

[0226] The three-plasmid system (envelope plasmid pCMV-VSVG, packaging plasmid pCMV-dr8.91 and target plasmid pLvx main plasmid) was used for lentiviral packaging, and then concentrated to obtain lentiviral solution. CHOS cells were infected with lentiviral solution. After infection, the infected cells were pressure screened by 500μg / mL GENETICIN (Gibco) or 500μg / mL Hygromycin B (Invitrogen) to obtain resistant Pool. The expression of hPD-1, mPD-1, and cPD-1 proteins on the cell surface was determined by FACS. The identified positive Pool cells were monocloned by limiting dilution method, and CHOS-hPD-1, CHOS-mPD-1, and CHOS-cPD-1 cell lines overexpressing hPD-1, mPD-1, and cPD-1 proteins were obtained after expansion and identification.

[0227] 4.2 Determination of the binding activity of antibodies to PD-1 overexpressing cell lines

[0228] CHOS-hPD-1, CHOS-mPD-1, CHOS-cPD-1, and CHOS empty cells were resuspended in 1% BSA / 1×PBS and then added to a 96-well U-shaped plate at 2×10 cells per well. 5 cells. The antibodies were diluted to 10 μg / mL with 1% BSA / 1×PBS, 100 μL per well, 2 replicates for each antibody, NC and Blank were set at the same time, and incubated at 4°C for 30 minutes. Washed twice with 1% BSA / 1×PBS, and then Goat Anti-Human IgG (Jackson) secondary antibody diluted at a ratio of 1:200 was added, 100 μL per well, and incubated at 4°C for 30 minutes. After washing twice with 1% BSA / 1×PBS, resuspended with 50 μL 1% BSA / 1×PBS. The cells were analyzed on a flow cytometer (BD Canto II). The measurement results were analyzed using FlowJo software, and the mean fluorescence intensity (MFI) was calculated. Antibodies with MFI values ​​greater than 10 times NC for binding to CHOS-hPD-1 or CHOS-cPD-1 were selected for subsequent experiments.

[0229] Example 5: Activity assay of anti-PD-1 chimeric antibodies inhibiting activation of Jurkat-PD1-NFAT reporter cells

[0230] Jurkat-PD1-NFAT cells (purchased from China Food and Drug Administration) were cultured in RPMI-1640 (Gibco, C11875500BT) medium containing 1% FBS (Gibco, 10091-148) to adjust the density to 2×10 6 / mL, 25μL / well was inoculated in a 96-well all-white plate (CORNING, 3917), i.e. 5×10 4 The antibody to be tested was diluted in the experimental culture medium and added to a 96-well plate at 25 μL / well. The highest concentration was 75 nM, 4-fold dilution, and 9 gradients. THP-1 cells (purchased from BNCC) were adjusted to a density of 1.6×10 6 / mL, 25μL / well was inoculated in a 96-well plate, i.e. 4×10 4 CD3 antibody (self-made) was diluted in experimental culture medium and added to the corresponding experimental wells at a final concentration of 0.01 nM. The total experimental system was 100 μL / well and placed at 37°C and 5% CO 2 Incubate in an incubator for 6-24 hours. Add 100 μL / well of Bright-Lite (Novozyme, DD1204-02), protect from light, and stand at room temperature for 3 minutes. SpectraMax iD5 multi-function microplate reader (Molecular Devices) detects the luminescence value. The reporter gene inhibition rate is calculated as follows: Reporter gene inhibition (%) = 100% × [1-(RLUsample-RLUcell) / (RLUcontrol-RLUcell)].

[0231] The results are as follows Figure 1 As shown in Table 3, anti-PD-1 chimeric antibodies B1-1B5-H1L1 and B1-1C4-H1L1 showed dose-dependent inhibition of Jurkat-PD1-NFAT reporter cell activation, and their inhibitory activity was superior to that of the positive controls P01-hIgG1 (reference patent US20190270818A1, heavy chain sequence: SEQ ID NO: 1; light chain sequence: SEQ ID NO: 2) and P02-hIgG1 (reference patent WO2020247648A2, heavy chain sequence: SEQ ID NO: 51; light chain sequence: SEQ ID NO: 52), as reflected in that EC50 was 2-3 times smaller than that of the positive controls P01-hIgG1 and P02-hIgG1. The heavy chain constant region sequences of the two positive control antibodies are the same as SEQ ID NO: 51 in this application, and the hIgG1 isotype is an unrelated negative control antibody.

[0232] Table 3: Anti-PD-1 chimeric antibody inhibits Jurkat-PD1-NFAT reporter cell activation assay results

[0233] Antibody Name EC50(nM) hIgG1 isotype NA P01-hIgG1 0.30 P02-hIgG1 0.33 B1-1B5-H1L1 0.12 B1-1C4-H1L1 0.14

[0234] Example 6: Humanization of anti-PD-1 antibody

[0235] The screened mouse antibodies B1-1B5-H1L1 and B1-1C4-H1L1 were compared and analyzed with known human antibody databases to find the germline gene with the highest homology, and its framework region was selected for use (using Kabat to define CDR and framework region). Then, with the help of computer prediction simulation, the CDR was replaced with the corresponding CDR sequence in the parent antibody, and the mouse amino acids in the parent antibody framework region that have an important effect on antigen binding were retained by back mutation. Finally, the variable regions 1B5-zu1 (heavy chain variable region sequence: SEQ ID NO: 15; light chain variable region sequence: SEQ ID NO: 16) and 1C4-zu1 (heavy chain variable region sequence: SEQ ID NO: 39; light chain variable region sequence: SEQ ID NO: 40) of the humanized antibodies of B1-1B5-H1L1 and B1-1C4-H1L1 were generated respectively.

[0236] Example 7: Preparation of anti-PD-1 humanized antibodies

[0237] 7.1 Transient expression of humanized antibodies

[0238] The heavy chain variable region and light chain variable region of the humanized antibody described in Example 6 were connected to the heavy chain constant region and the light chain constant region, respectively, to obtain 4 heavy chain expression vectors and 4 light chain expression vectors, which were combined and transfected to express antibodies, and the antibody amino acid sequence and nucleotide sequence are shown in Table 4. Among them, 1B5-zu1-hIgG1 DD and 1C4-zu1-hIgG1 DD are Fc region modified mutants of 1B5-zu1-hIgG1 and 1C4-zu1-hIgG1, respectively, DD represents S239D / H268D mutant (EU numbering system), and the sequence of the Fc variant is shown in SEQ ID NO:52.

[0239] Table 4: Anti-PD-1 humanized antibody amino acid and nucleotide sequences

[0240]

[0241] 24 hours before transfection, inoculate 3-4×10 6 100 mL of ExpiCHO-S cells at 100 cells / mL was placed in a 37°C, 8% CO 2 , cultured in a shaker at 120 rpm. Before transfection, the cell density was measured to reach 6-8×10 6 cells / mL, the cell density was adjusted to 6×10 6Cells / mL and transfect. During transfection, first dilute 100μg of the total expression plasmids used for 1B5-zu1-hIgG1, 1B5-zu1-hIgG1 DD, 1C4-zu1-hIgG1 and 1C4-zu1-hIgG1 DD molecules (the ratio of light and heavy chain expression plasmids is 1:1) to 4mL with Opti-PRO SFM; then take 320μL of ExpiFectamine CHO Reagent and add it to 3.68mL OptiPRO SFM, mix well, and incubate at room temperature for 2 minutes. Mix the diluted transfection reagent and plasmid thoroughly, incubate at room temperature for 2 to 5 minutes, and then add all the mixtures to the cells and mix well. Place the culture flask at 37°C, 8% CO 2 , and cultured in a shaker at 120 rpm. 18-22 h after cell transfection, add 600 μl ExpiFectamine CHO Enhancer and 16 mL ExpiCHO Feed to the culture system and adjust the culture conditions to 32°C, 5% CO 2 , 120rpm. On the fifth day after transfection, add 16mL of ExpiCHO Feed and continue culturing for 12 to 14 days.

[0242] 7.2 Humanized Antibody Purification

[0243] The cell culture medium was centrifuged at 2000g for 20 minutes, and the supernatant was collected and filtered with a 0.22 micron filter membrane. After PrismA (Cytiva) affinity chromatography (protein purification liquid chromatography system: Cytiva, AKTA avant 25), eluted with 20mM citric acid-sodium citrate (pH 3.2), and then adjusted to neutral pH with 1M Tris base. The purified sample was subjected to SDS-PAGE using 4-20% gradient gel to determine the purity of the antibody.

[0244] Example 8: Determination of affinity between anti-PD-1 humanized antibody and PD-1 antigen

[0245] Using Sensor Chip Protein A biosensor chip (Cytiva, 29-1275-56), 1B5-zu1-hIgG1, 1B5-zu1-hIgG1 DD, 1C4-zu1-hIgG1, and 1C4-zu1-hIgG1 DD were fixed on the chip as ligands, and different concentrations of antigens hPD-1ECD-Avi-His and cPD-1ECD-Avi-His were flowed through the chip at a flow rate of 30 μL / min. The antigen and antibody bound with a binding time of 180 s and a dissociation time of 180 s. Kinetic fitting was performed using Biacore 8K Evaluation (Cytiva), and the affinity constants obtained are shown in Tables 5 and 6. The affinity of 1B5-zu1-hIgG1 and 1B5-zu1-hIgG1 DD to hPD-1ECD-Avi-His is comparable; the affinity of 1C4-zu1-hIgG1 and 1C4-zu1-hIgG1 DD to hPD-1ECD-Avi-His is comparable. The affinity of 1B5-zu1-hIgG1 and 1B5-zu1-hIgG1 DD to cPD-1ECD-Avi-His is comparable; the affinity of 1C4-zu1-hIgG1 and 1C4-zu1-hIgG1 DD to cPD-1ECD-Avi-His is comparable.

[0246] Table 5: Affinity determination results of anti-PD-1 humanized antibodies and hPD-1

[0247] Antibody Name Ka(1 / Ms) Kd(1 / s) KD(M) 1B5-zu1-hIgG1 2.04E+05 2.40E-03 1.18E-08 1B5-zu1-hIgG1 DD 2.09E+05 2.31E-03 1.11E-08 1C4-zu1-hIgG1 2.36E+05 1.95E-03 8.26E-09 1C4-zu1-hIgG1 DD 2.40E+05 1.94E-03 8.05E-09

[0248] Table 6: Affinity test results of anti-PD-1 humanized antibodies and cPD-1

[0249] Antibody Name Ka(1 / Ms) Kd(1 / s) KD(M) 1B5-zu1-hIgG1 2.19E+05 8.41E-03 3.85E-08 1B5-zu1-hIgG1 DD 2.43E+05 8.56E-03 3.51E-08 1C4-zu1-hIgG1 2.51E+05 3.92E-03 1.56E-08 1C4-zu1-hIgG1 DD 2.59E+05 3.93E-03 1.52E-08

[0250] Example 9: Determination of affinity between anti-PD-1 humanized antibody and Fc receptor

[0251] The anti-His antibody (Cytiva, 28995056) was coupled to the CM5 biosensor chip (Cytiva, BR-1005-30), hCD32A (167His, His Tag, Sino Biological, 10374-H08C1), hCD32B (ECD, His tag, Sino Biological, 10259-H08C) were used as ligands, and different concentrations of antibodies 1B5-zu1-hIgG1, 1B5-zu1-hIgG1 DD, 1C4-zu1-hIgG1, 1C4-zu1-hIgG1 DD were flowed through the chip at a flow rate of 30 μL / min, and the Fc receptors were bound to the antibodies, with a binding time of 60 s and a dissociation time of 60 s. The affinity constants were obtained by fitting with Biacore8K Evaluation software (Cytiva) as shown in Tables 7 and 8 below. The affinity of hCD32A to 1B5-zu1-hIgG1 DD or 1C4-zu1-hIgG1DD was 3-4 times higher than that of 1B5-zu1-hIgG1 or 1C4-zu1-hIgG1. The affinity of hCD32B to 1B5-zu1-hIgG1DD or 1C4-zu1-hIgG1 DD was 6-9 times higher than that of 1B5-zu1-hIgG1 or 1C4-zu1-hIgG1. The above results indicate that the introduction of DD mutation in the Fc region can further enhance the affinity of the antibody to the Fc receptors hCD32A and hCD32B.

[0252] Table 7: Affinity determination results of anti-PD-1 humanized antibody and hCD32A

[0253] Antibody Name KD(M) 1B5-zu1-hIgG1 5.43E-06 1B5-zu1-hIgG1 DD 1.47E-06 1C4-zu1-hIgG1 4.94E-06 1C4-zu1-hIgG1 DD 1.61E-06

[0254] Table 8: Affinity determination results of anti-PD-1 humanized antibody and hCD32B

[0255] Antibody Name KD(M) 1B5-zu1-hIgG1 1.34E-05 1B5-zu1-hIgG1 DD 1.53E-06 1C4-zu1-hIgG1 1.00E-05 1C4-zu1-hIgG1 DD 1.68E-06

[0256] The anti-His antibody was coupled to the CM5 biosensor chip, and the homemade hCD16A(158F)-ECD-C-his and hCD16A(158V)-ECD-C-his were used as ligands. Different concentrations of antibodies 1B5-zu1-hIgG1, 1B5-zu1-hIgG1 DD, 1C4-zu1-hIgG1, and 1C4-zu1-hIgG1 DD were flowed through the chip at a flow rate of 30μL / min. The Fc receptors bound to the antibodies, and the binding time was 60&90s and the dissociation time was 60&90s. The Biacore 8K Evaluation software (Cytiva) was used for kinetic and steady-state fitting, and the affinity constants were obtained as shown in Tables 9 and 10. The affinity of hCD16A(158F) to 1B5-zu1-hIgG1DD or 1C4-zu1-hIgG1 DD was 3-6 times higher than that of 1B5-zu1-hIgG1 or 1C4-zu1-hIgG1. The affinity of hCD16A(158V) to 1B5-zu1-hIgG1 DD or 1C4-zu1-hIgG1 DD was 3-4 times higher than that of 1B5-zu1-hIgG1 or 1C4-zu1-hIgG1. The above results indicate that the introduction of DD mutation in the Fc region can further enhance the affinity of the antibody to the Fc receptor hCD16A.

[0257] Table 9: Affinity determination results of anti-PD-1 humanized antibodies and hCD16A (158F)

[0258] Antibody Name Ka(1 / Ms) Kd(1 / s) KD(M) 1B5-zu1-hIgG1 9.21E+04 3.00E-02 3.26E-07 1B5-zu1-hIgG1 DD 6.84E+05 3.54E-02 5.18E-08 1C4-zu1-hIgG1 1.12E+05 2.48E-02 2.22E-07 1C4-zu1-hIgG1 DD 6.74E+05 4.23E-02 6.27E-08

[0259] Table 10: Affinity determination results of anti-PD-1 humanized antibodies and hCD16A (158V)

[0260]

[0261]

[0262] Example 10: Binding activity assay of anti-PD-1 humanized antibodies to PD-1 overexpressing cell lines

[0263] CHOS-hPD-1 and CHOS-cPD-1 cells were resuspended in 1% BSA / 1×PBS and then added to a 96-well U-shaped plate at 2×10 cells per well. 5cells. Then, the antibodies to be tested were added at a maximum concentration of 100 nM, 3-fold dilutions, and 10 concentration gradients, 100 μL per well, and incubated at 4°C for 30 minutes. Wash twice with 1% BSA / 1×PBS, add 1:200 diluted Goat Anti-Human IgG (Jackson) secondary antibody, 100 μL per well, and incubate at 4°C for 30 minutes. After washing twice with 1% BSA / 1×PBS, resuspend in 100 μL 1% BSA / 1×PBS. The cells were analyzed on a flow cytometer (BD Canto II). The assay results were analyzed using FlowJo software. The mean fluorescence intensity (MFI) value was used to calculate the relative EC50 based on a 4-parameter logistic regression.

[0264] The results of antibody, CHOS-hPD-1 and CHOS-cPD-1 assays are shown in Figure 2 , Figure 3 And Table 11. 1B5-zu1-hIgG1, 1B5-zu1-hIgG1 DD, 1C4-zu1-hIgG1, and 1C4-zu1-hIgG1 DD can efficiently bind to CHOS-hPD-1, and are significantly stronger than P02-hIgG1, and slightly better than P01-hIgG1. 1B5-zu1-hIgG1, 1B5-zu1-hIgG1DD, 1C4-zu1-hIgG1, and 1C4-zu1-hIgG1 DD can efficiently bind to CHOS-cPD-1, and are significantly stronger than P02-hIgG1, and slightly better than P01-hIgG1.

[0265] Table 11: Binding assay results of anti-PD-1 humanized antibodies to CHOS-hPD-1 and CHOS-cPD-1

[0266]

[0267] Example 11: Activity assay of anti-PD-1 humanized antibody inhibiting activation of Jurkat-PD1-NFAT reporter cells

[0268] Jurkat-PD1-NFAT cells (purchased from China Food and Drug Administration) were cultured in RPMI-1640 (Gibco, C11875500BT) medium containing 1% FBS (Gibco, 10091-148) to adjust the density to 2×10 6 / mL, 25μL / well was inoculated in a 96-well all-white plate (CORNING, 3917), i.e. 5×10 4 The antibody to be tested was diluted in the experimental culture medium and added to a 96-well plate at 25 μL / well. The highest concentration was 75 nM, 4-fold dilution, and 9 gradients. THP-1 cells (purchased from BNCC) were adjusted to a density of 1.6×106 / mL, 25μL / well was inoculated in a 96-well plate, i.e. 4×10 4 CD3 antibody (self-made) was diluted in experimental culture medium and added to the corresponding experimental wells at a final concentration of 0.01 nM. The total experimental system was 100 μL / well and placed at 37°C and 5% CO 2 Incubate in an incubator for 6-24 hours. Add 100 μL / well of Bright-Lite (Novozyme, DD1204-02), protect from light, and stand at room temperature for 3 minutes. Detect the luminescence value with a SpectraMax iD5 multifunctional microplate reader (Molecular Devices). Reporter gene inhibition rate calculation formula: Reporter gene inhibition (%) = 100% × [1-(RLU sample -RLU cell ) / (RLU control -RLU cell )].

[0269] The results are as follows Figure 4 As shown in Table 12, the humanized antibodies 1B5-zu1-hIgG1 and 1C4-zu1-hIgG1 have better inhibition of Jurkat-PD1-NFAT reporter cell activation than the positive controls P01-hIgG1 and P02-hIgG1, as reflected by a 2-3-fold decrease in EC50. 1B5-zu1-hIgG1 DD and 1C4-zu1-hIgG1 DD have better inhibition of Jurkat-PD1-NFAT reporter cell activation than 1B5-zu1-hIgG1 and 1C4-zu1-hIgG1, as reflected by an increase in the maximum inhibition rate of about 10%.

[0270] Table 12: Anti-PD-1 humanized antibody inhibits Jurkat-PD1-NFAT reporter cell activation assay results

[0271] Antibody Name EC50(nM) hIgG1 isotype NA hIgG1 DD isotype NA P01-hIgG1 0.25 P02-hIgG1 0.38 1B5-zu1-hIgG1 0.13 1B5-zu1-hIgG1 DD 0.15 1C4-zu1-hIgG1 0.12 1C4-zu1-hIgG1 DD 0.16

[0272] Example 12: Activity determination of anti-PD-1 humanized antibodies in inhibiting IFN-γ secretion of primary T cells

[0273] Human CD4 + T cells (Rubai Biotechnology) were adjusted to a density of 1×10 61×10 cells / mL, 2 mL / well were inoculated in a 12-well plate (CORNING, 3513) pre-coated with 3 μg / mL Anti-Human CD3 antibody (clone OKT3, ACRO, CDE-M120a) and stimulated for 3 days. THP-1 cells were treated with mitomycin C (INALCO, 1758-9327) to inhibit cell proliferation: 1×10 7 THP-1 cells were treated with 10 μg / mL mitomycin C at 37°C for 2 h. After washing three times with the culture medium, THP-1 cells were resuspended in RPMI-1640 experimental culture medium containing 5% human serum (Hengyuan Biotechnology, HY118-25) and the cell density was adjusted to 5×10 5 / mL, 50 μL / well was inoculated in a 96-well U-shaped plate (NEST, 1224B), i.e. 2.5×10 4 The antibody to be tested was diluted to 0.25 nM with RPMI-1640 experimental medium containing 5% human serum, and 50 μL / well was added to the corresponding experimental well. + T cells were diluted to 1 × 10 6 / mL, 50μL / well was inoculated in a 96-well U-shaped plate, i.e. 5×10 4 cells / well. CD3 antibody (homemade) was diluted in the experimental culture medium and 50 μL / well was added to the corresponding experimental wells, with a final concentration of 0.04 nM. The total system was 200 μL / well and placed at 37°C, 5% CO 2 Incubate in an incubator for 18 hours. After incubation, centrifuge and collect the supernatant. ELISA was used to detect IFN-γ secretion (R&D Systems, SIF50C). The OD value was read using a SpectraMaxiD5 multifunctional microplate reader (Molecular Devices) with a detection wavelength of 450 nm and a reference wavelength of 540 nm, and the IFN-γ content was calculated based on the standard curve. The cytokine secretion inhibition rate was calculated as follows: inhibition (%) = 100% × [1-(sample-untreated) / (treated-untreated)].

[0274] The results are as follows Figure 5 1B5-zu1-hIgG1, 1C4-zu1-hIgG1, 1B5-zu1-hIgG1 DD and 1C4-zu1-hIgG1DD can significantly inhibit the expression of human CD4 + T cells secreted IFN-γ, and the activity was significantly better than the positive controls P01-hIgG1 and P02-hIgG1; among them, 1B5-zu1-hIgG1 DD and 1C4-zu1-hIgG1 DD inhibited human CD4 +T cells secrete IFN-γ with better activity.

[0275] Example 13: Determination of ADCC activity of anti-PD-1 humanized antibodies

[0276] Resuscitator CD56 + NK cells (Shanghai Miaoshun) were cultured overnight, and the cells were collected the next day. NK effector cells were diluted with MEM-α (Gibco, 32561-037) experimental medium containing 1% FBS, and 5×10 4 100 μL / well cells were inoculated in a 96-well plate (CORNING, 3599). The antibody to be tested was diluted with experimental culture medium, the highest concentration was 10 nM, 10-fold dilution, 8 gradients, and 50 μL / well was added to the corresponding experimental well. The target cells CHOS-hPD-1 were diluted with experimental culture medium, 1E4 cells / 50 μL / well were inoculated in a 96-well plate, and the effector-target ratio (E:T) = 5:1. At the same time, the maximum killing group was set: 1% Triton + target cells; the minimum killing group: target cells; E+T group: effector cells + target cells. The total system was 200 μL / well. After the antibody, target cells and effector cells were mixed, the culture plate was placed at 37°C and 5% CO 2 Incubate in an incubator for 24 hours. Non-Radioactive Cytotoxicity Assay Kit (Promega, G1780) was used to detect LDH release. 50 μL of supernatant was collected by centrifugation, 50 μL of LDH substrate was added to each well, and the cells were incubated at room temperature in the dark for 20 min. 50 μL of stop solution was added to each well. The OD value of the LDH reaction was detected using a SpectraMax iD5 multifunctional microplate reader (Molecular Devices). The detection wavelength was 490 nm and the reference wavelength was 620 nm. Calculation formula: % Target celllysis = 100% × (OD Sample -OD E+T ) / (OD Maximum release -OD Minimum release ).

[0277] The results are as follows Figure 6 As shown in Table 13, in the primary human NK cell-mediated ADCC experiment, 1B5-zu1-hIgG1, 1C4-zu1-hIgG1, 1B5-zu1-hIgG1 DD and 1C4-zu1-hIgG1 DD all showed good ADCC activity, among which 1B5-zu1-hIgG1 DD and 1C4-zu1-hIgG1 DD had better ADCC activity, which was reflected in a 5-12-fold decrease in EC50.

[0278] Table 13: ADCC activity assay results of anti-PD-1 humanized antibodies

[0279] Antibody Name EC50(pM) hIgG1 isotype NA hIgG1 DD isotype NA 1B5-zu1-hIgG1 5.12 1B5-zu1-hIgG1 DD 0.44 1C4-zu1-hIgG1 1.90 1C4-zu1-hIgG1 DD 0.33

[0280] Example 14: Activity assay of anti-PD-1 humanized antibodies to block or enhance the binding of PD-1 to PD-L1 or PD-L2

[0281] CHOS-hPD-1 cells were resuspended in 1% BSA / 1×PBS and added to a 96-well U-shaped plate, with 1×10 cells per well. 5 Cells were added with 11 concentration gradients of antibodies to be tested, with a maximum concentration of 600nM, 3-fold dilutions, 50μL per well, and incubated at 4°C for 30 minutes. 600nM hPD-L1-hIgG1Fc-Avi(B) or 26.67nM hPD-L2-hIgG1Fc-Avi(B) were added, 50μL per well, and incubated at 4°C for 30 minutes. After washing twice with 1% BSA / 1×PBS, 1:200 diluted PE-conjugated Streptavidin(BD) secondary antibody was added, 50μL per well, and incubated at 4°C for 30 minutes. After washing twice with 1% BSA / 1×PBS, the cells were resuspended with 50μL 1% BSA / 1×PBS. Cells were analyzed on a flow cytometer (BD Canto II). The results of the assay were analyzed using FlowJo software.

[0282] Figure 7 The results showed that the PD-1 antagonist antibody Keytruda-hIgG1 can significantly block the binding of CHOS-hPD-1 to hPD-L1, while the PD-1 agonist antibodies P01-hIgG1 and P02-hIgG1 do not block the binding of CHOS-hPD-1 to hPD-L1; surprisingly, the PD-1 agonist antibodies 1B5-zu1-hIgG1, 1B5-zu1-hIgG1 DD, 1C4-zu1-hIgG1 and 1C4-zu1-hIgG1 DD not only do not block the binding of CHOS-hPD-1 to hPD-L1, but can even enhance the binding of CHOS-hPD-1 to hPD-L1 to varying degrees. Similarly, Figure 8The test results showed that Keytruda-hIgG1 could significantly block the binding of CHOS-hPD-1 and hPD-L2, while P01-hIgG1, P02-hIgG1, 1B5-zu1-hIgG1 and 1B5-zu1-hIgG1 DD only weakly blocked the binding of CHOS-hPD-1 and hPD-L2; surprisingly, 1C4-zu1-hIgG1 and 1C4-zu1-hIgG1 DD not only did not block the binding of CHOS-hPD-1 and hPD-L2, but even enhanced the binding of CHOS-hPD-1 and hPD-L2.

[0283] Example 15: Efficacy determination of anti-PD-1 humanized antibody in mouse GVHD model

[0284] The mouse graft-versus-host disease (GVHD) model was established using 7-8 week old female immunodeficient mice NOD.CB17-PrkdcscidIl2rgtm1 / Bcgen (B-NDG, purchased from Biocytogen, catalog number 110586). The modeling method was 1.5×10 7 Human peripheral blood mononuclear cells (PBMC) were injected into mice through the tail vein to induce modeling. One day after modeling, the antibody to be tested was administered by intraperitoneal injection at a dose of 10 mg / kg, twice a week, for six weeks. During the modeling and administration period, the animals were weighed and monitored for disease three times a week, and GVHD scores were performed based on the observations. When the animal weight was monitored to drop by more than 20% compared to the initial weight, the mice were euthanized.

[0285] The results are as follows Fig. 9 Compared with the negative control group (Vehicle, hIgG1 isotype, hIgG1 DD isotype), 1B5-zu1-hIgG1, 1C4-zu1-hIgG1, 1B5-zu1-hIgG1 DD, and 1C4-zu1-hIgG1 DD can significantly alleviate the symptoms of GVHD in mice, and are significantly better than the positive control P01-hIgG1 in terms of efficacy; 1B5-zu1-hIgG1DD and 1C4-zu1-hIgG1 DD are better than the positive control P02-hIgG1 in terms of efficacy, and the efficacy levels of 1B5-zu1-hIgG1 and 1C4-zu1-hIgG1 are comparable to those of the positive control P02-hIgG1.

[0286] Example 16: Pharmacokinetics of anti-PD-1 humanized antibodies in human FcRn transgenic mice

[0287] Blood samples were collected from human FcRn transgenic mice (purchased from Biocytogen, catalog number 110001) at 0h, 0.083h, 24h, 48h, 72h, 120h, 168h, 240h, 336h, 408h, 504h, 576h and 672h after a single dose to determine the concentration of antibodies in the serum. The ELISA method was used to detect the antibody blood concentration. There were 6 animals in each group, half male and half female, and the dosage was 10 mg / kg, and the administration route was intravenous injection. Table 14 shows the measured pharmacokinetic properties, where T1 / 2 represents half-life, Cmax represents peak concentration, AUClast represents the area under the curve from the start of administration to the last time point, AUCINF-obs represents the area under the curve from the start of administration to theoretical extrapolation infinity, AUC-%Extrap-obs represents the ratio of the area under the curve from the last point to theoretical extrapolation infinity to AUCINF-obs, Vz-obs represents apparent distribution volume, Cl-obs represents clearance, and MRTlast represents mean residence time.

[0288] The results are as follows Fig.10 and Table 14, 1B5-zu1-hIgG1, 1B5-zu1-hIgG1 DD, 1C4-zu1-hIgG1 and 1C4-zu1-hIgG1 DD all have longer half-lives than P02-hIgG1; 1B5-zu1-hIgG1, 1C4-zu1-hIgG1 and 1C4-zu1-hIgG1 DD have longer half-lives than P01-hIgG1.

[0289] Table 14: Pharmacokinetic results of anti-PD-1 humanized antibodies in human FcRn transgenic mice

[0290]

[0291] Example 17: Efficacy of anti-PD-1 humanized antibody in a SLE mouse model induced by lymphocyte adoptive transfer

[0292] The SLE mouse model induced by adoptive transfer of lymphocytes is characterized by the production of a range of autoantibodies, including anti-histone and anti-double-stranded DNA antibodies, similar to the disease indicators observed in human SLE. 70 male BM12 mice (Jackson Laboratory, strain number 001162) were randomly divided into 5 groups (N=8) according to their body weight at 8 weeks of age. All mice received 5x10 from hPD-1C57BL / 6 donor mice (6-8 weeks old, Jicui Yaokang, strain number T003095) 7The experiment lasted for 25 days with intraperitoneal injection of lymphocytes. Drug administration started on D1 and was given twice a week. Blood was collected on Day 0 / 14 / 25 to separate serum and test anti-dsDNA and anti-histone levels.

[0293] like Fig.11 As shown, on the 14th day after lymphocyte adoptive transfer: 1) compared with the negative control group (Vehicle), 1C4-zu1-hIgG1 20mpk and 40mpk can significantly reduce the level of anti-histone antibodies; 2) compared with the negative control group (Vehicle), 1C4-zu1-hIgG1 20mpk and 40mpk can significantly reduce the level of anti-double-stranded DNA (anti-dsDNA) antibodies. On the 25th day after lymphocyte adoptive transfer: 1) compared with the negative control group (Vehicle), 1C4-zu1-hIgG140mpk can significantly reduce the level of anti-histone antibodies; 2) compared with the negative control group (Vehicle), 1C4-zu1-hIgG1 20mpk and 40mpk can significantly reduce the level of anti-double-stranded DNA (anti-dsDNA) antibodies. CTX (Cyclophosphamide) plays the role of a positive drug in this model.

[0294] Example 18: Efficacy of anti-PD-1 humanized antibody in TNBS-induced IBD mouse model

[0295] Female hPD-1BALB / c mice (6-8 weeks old, Jicui Yaokang, strain number T002726) were randomly divided into 5 groups (N=10) according to body weight: 1 blank control group (Sham group) and 4 TNBS-induced model groups. The immune intervention was performed on day 0: 0.1mL of 1.5% TNBS solution was perfused rectally in the 4 model group mice; 0.1mL of 50% ethanol solution was perfused rectally in the sham group mice. The 1C4-zu1-hIgG1 treatment group was intraperitoneally injected on day-1, day 2, and day 5. The same volume of vehicle excipients was intraperitoneally injected into the sham group and the vehicle control group (Vehicle group) mice (day-1-day 6). The animals were weighed every day during the experiment, the fecal characteristics and occult blood scores of the mice were performed, and the disease activity index (DAI) was calculated. At the end of the experiment (i.e., day 6), the animals were euthanized, and the colon (from anus to cecum) of each group of mice was collected, the fat and connective tissue around the colon were removed, and photos were taken. The length was measured, and the contents and cecum were removed and weighed.

[0296] like Fig.12As shown, in the TNBS-induced acute colitis model in mice, 1C4-zu1-hIgG1 has good pharmacological efficacy and can alleviate the weight loss and increased DAI scores of mice caused by colitis. 1C4-zu1-hIgG1 can significantly alleviate the shortening of the colon caused by colitis.

[0297] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

1. An antibody or an antigen-binding fragment thereof that can specifically bind to PD-1, wherein the antibody or the antigen-binding fragment thereof comprises: (a) VH CDR1 or a variant thereof, VH CDR2 or a variant thereof, and VH CDR3 or a variant thereof contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 7 or 15; and / or VL CDR1 or a variant thereof, VL CDR2 or a variant thereof, and VL CDR3 or a variant thereof contained in the light chain variable region (VL) as shown in SEQ ID NO: 8 or 16; or (b) VH CDR1 or a variant thereof, VH CDR2 or a variant thereof, and VH CDR3 or a variant thereof contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 31 or 39; and / or VL CDR1 or a variant thereof, VL CDR2 or a variant thereof, and VL CDR3 or a variant thereof contained in the light chain variable region (VL) as shown in SEQ ID NO: 32 or 40; in, The variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions, e.g., conservative substitutions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions; Preferably, the antibody or antigen-binding fragment thereof comprises: (a) the three CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 7 or 15; and / or the three CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO: 8 or 16; or, (b) the three CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 31 or 39; and / or the three CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO: 32 or 40.

2. The antibody or antigen-binding fragment thereof according to claim 1, comprising: (a) a heavy chain variable region (VH) comprising the following three complementary determining regions (CDRs): VH CDR1 with a sequence of SEQ ID NO: 1, VH CDR2 with a sequence of SEQ ID NO: 2, and VH CDR3 with a sequence of SEQ ID NO: 3; and / or, a light chain variable region (VL) comprising the following three complementary determining regions (CDRs): VL CDR1 with a sequence of SEQ ID NO: 4, VL CDR2 with a sequence of SEQ ID NO: 5, and VL CDR3 with a sequence of SEQ ID NO: 6; or, (b) a heavy chain variable region (VH) comprising the following three complementary determining regions (CDRs): VH CDR1 of SEQ ID NO: 25, VH CDR2 of SEQ ID NO: 26, and VH CDR3 of SEQ ID NO: 27; and / or a light chain variable region (VL) comprising the following three complementary determining regions (CDRs): VL CDR1 of SEQ ID NO: 28, VL CDR2 of SEQ ID NO: 29, and VL CDR3 of SEQ ID NO: 30; in, The CDRs are defined by the Kabat numbering system.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising: (a) a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 7 or a variant thereof; and / or, a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 8 or a variant thereof; or, (b) a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 31 or a variant thereof; and / or a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 32 or a variant thereof; in, The variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, or has at least 80%, at least 85%, 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; preferably, the substitutions are conservative substitutions; Preferably, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO: 7 and a VL comprising the sequence shown in SEQ ID NO: 8; Preferably, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO:31 and a VL comprising the sequence shown in SEQ ID NO:

32.

4. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein The antibody or antigen-binding fragment thereof comprises a framework region sequence derived from a human immunoglobulin; Preferably, the antibody or antigen-binding fragment thereof comprises: a heavy chain framework region of a human heavy chain germline sequence, and / or a light chain framework region of a human light chain germline sequence.

5. The antibody or antigen-binding fragment thereof according to claim 4, wherein The antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region (VH) comprising a sequence as shown in SEQ ID NO: 15 or a variant thereof; and / or, a light chain variable region (VL) comprising a sequence as shown in SEQ ID NO: 16 or a variant thereof; or, (b) a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 39 or a variant thereof; and / or, a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 40 or a variant thereof; wherein the variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, or has at least 80%, at least 85%, 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; Preferably, the substitution is a conservative substitution; Preferably, the antibody or antigen-binding fragment thereof comprises: (a) comprising a heavy chain variable region (VH) as shown in SEQ ID NO: 15; and / or, comprising a light chain variable region (VL) as shown in SEQ ID NO: 16; or, (b) comprises a heavy chain variable region (VH) as shown in SEQ ID NO:39; and / or, comprises a light chain variable region (VL) as shown in SEQ ID NO:

40.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein The antibody or antigen-binding fragment thereof further comprises a constant region derived from a mammalian (eg, human or mouse) immunoglobulin; Preferably, the heavy chain of the antibody or its antigen-binding fragment comprises a heavy chain constant region derived from a human immunoglobulin (e.g., IgG, such as IgG1, IgG2, IgG3 or IgG4), and / or the light chain of the antibody or its antigen-binding fragment comprises a light chain constant region derived from a human immunoglobulin (e.g., κ or λ).

7. The antibody or antigen-binding fragment thereof according to claim 6, wherein The antibody or antigen-binding fragment thereof comprises a human immunoglobulin heavy chain constant region variant, which has enhanced binding activity to Fc receptors (e.g., CD32A, CD32B and / or CD16A) compared to a wild-type human immunoglobulin heavy chain constant region; Preferably, the antibody or antigen-binding fragment thereof comprises a human IgG1 or IgG4 heavy chain constant region variant, which comprises a substitution in one or more of the following positions according to EU numbering: 239, 268; preferably, the substitution is selected from S239D / H268D or S239D / Q268D.

8. The antibody or antigen-binding fragment thereof according to claim 6 or 7, wherein The antibody or antigen-binding fragment thereof comprises: (1) a heavy chain comprising the sequence shown in SEQ ID NO: 11 and a light chain comprising the sequence shown in SEQ ID NO: 12; (2) a heavy chain comprising the sequence shown in SEQ ID NO: 19 and a light chain comprising the sequence shown in SEQ ID NO: 20; (3) a heavy chain comprising the sequence shown in SEQ ID NO: 23 and a light chain comprising the sequence shown in SEQ ID NO: 20; (4) a heavy chain comprising the sequence shown in SEQ ID NO: 35 and a light chain comprising the sequence shown in SEQ ID NO: 36; (5) a heavy chain comprising the sequence shown in SEQ ID NO:43 and a light chain comprising the sequence shown in SEQ ID NO:44; or, (6) A heavy chain comprising the sequence shown in SEQ ID NO:47 and a light chain comprising the sequence shown in SEQ ID NO:

44.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein The antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, disulfide-linked Fv, scFv, di-scFv, (scFv)2, diabody and single-domain antibody (sdAb); and / or, the antibody is a murine antibody, a humanized antibody, a chimeric antibody, a bispecific antibody or a multispecific antibody.

10. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the heavy chain variable region and / or light chain variable region thereof, or the heavy chain and / or light chain thereof.

11. A vector comprising the isolated nucleic acid molecule of claim 10; preferably, the vector is a cloning vector or an expression vector.

12. A host cell comprising the isolated nucleic acid molecule of claim 10 or the vector of claim 11.

13. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, comprising culturing the host cell according to claim 12 under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

14. A bispecific or multispecific molecule comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9; Preferably, the bispecific or multispecific molecule specifically binds to PD-1 and additionally specifically binds to one or more other targets; Preferably, the bispecific or multispecific molecule further comprises at least one molecule with a second binding specificity for a second target (eg, a second antibody).

15. An immunoconjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 and a therapeutic agent linked to the antibody or antigen-binding fragment thereof; Preferably, the therapeutic agent is a cytotoxic agent.

16. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the isolated nucleic acid molecule according to claim 10, the vector according to claim 11, the host cell according to claim 12, the bispecific or multispecific molecule according to claim 14 or the immunoconjugate according to claim 15, and a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent; Preferably, the additional pharmaceutically active agent is an immunosuppressant; Preferably, the additional pharmaceutically active agent is a drug for the treatment of an autoimmune disease, an allergic disease, graft-versus-host disease, transplant rejection or lymphoma.

17. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the isolated nucleic acid molecule according to claim 10, the vector according to claim 11, the host cell according to claim 12, the bispecific or multispecific molecule according to claim 14, the immunoconjugate according to claim 15, or the pharmaceutical composition according to claim 16 in the preparation of a PD-1 signaling pathway activator.

18. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the isolated nucleic acid molecule according to claim 10, the vector according to claim 11, the host cell according to claim 12, the bispecific or multispecific molecule according to claim 14, the immunoconjugate according to claim 15 or the pharmaceutical composition according to claim 16 in the preparation of an immunosuppressant (e.g., a T cell activity inhibitor).

19. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the isolated nucleic acid molecule according to claim 10, the vector according to claim 11, the host cell according to claim 12, the bispecific or multispecific molecule according to claim 14, the immunoconjugate according to claim 15 or the pharmaceutical composition according to claim 16 in the preparation of a medicament for preventing and / or treating an autoimmune disease, an allergic disease, graft-versus-host disease, transplant rejection or lymphoma in a subject; Preferably, the autoimmune disease is selected from arthritis, rheumatoid arthritis, psoriatic arthritis, lupus, lupus nephritis, systemic lupus erythematosus, psoriasis, vitiligo, alopecia areata, inflammatory bowel disease, ulcerative colitis, Crohn's disease, type I diabetes, multiple sclerosis, autoimmune hepatitis, primary biliary cirrhosis, celiac disease, scleroderma, Graves' disease, Hashimoto's thyroiditis, ankylosing spondylitis, myasthenia gravis, Sjögren's syndrome, IgA nephropathy, IgG4-Related Disease, vasculitis, ANCA-associated vasculitis, uveitis, pemphigus, pemphigoid or autoimmune hemolytic anemia; Preferably, the allergic disease is selected from idiopathic thrombocytopenic purpura, atopic dermatitis, asthma, allergic rhinitis, drug allergy, food allergy, anaphylaxis, allergic conjunctivitis, urticaria, eosinophilic sinusitis, eosinophilic digestive organ disease or allergic bronchopulmonary aspergillosis; Preferably, the lymphoma is T-cell lymphoma; Preferably, the subject is a mammal, such as a human or a non-human primate (such as a cynomolgus monkey); Preferably, the antibody or antigen-binding fragment thereof is used alone or in combination with another pharmaceutically active agent; preferably, the other pharmaceutically active agent is an immunosuppressant; preferably, the other pharmaceutically active agent is a drug for treating autoimmune diseases, allergic diseases, graft-versus-host disease, transplant rejection or lymphoma.

20. A conjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, and a detectable label linked to the antibody or antigen-binding fragment thereof; Preferably, the detectable label is selected from an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (such as acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (such as fluorescein or fluorescent protein), a radionuclide or biotin.

21. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 or the conjugate according to claim 20 in the preparation of a detection reagent for detecting the presence or level of PD-1 in a sample and / or diagnosing a disease associated with PD-1; Preferably, the PD-1-related disease is selected from autoimmune diseases, allergic diseases, graft-versus-host disease, transplant rejection, lymphoma or any combination thereof; Preferably, the disease associated with PD-1 is selected from tumors.

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