Anti-PD-1 monoclonal antibodies, their derivatives and uses

Abstract

The present invention relates to the field of biomedical or biopharmaceutical technology, more specifically to anti-PD-1 monoclonal antibodies, their derivatives and uses thereof.

Description

[Technical field]

[0001] The present invention relates to the field of biomedical or biopharmaceutical technology, more specifically to anti-PD-1 monoclonal antibodies and their derivatives and uses. [Background technology]

[0002] Programmed cell death protein-1 (PD-1, also known as CD279) is normally expressed on activated lymphocytes and is upregulated on tumor-infiltrating lymphocytes. PD-1 inhibits the hyperactivation and proliferation of T cells by binding to its ligands PD-L1 and PD-L2, which are highly expressed on tumor cells and antigen-presenting cells, and can exert a negative immunoregulatory effect and mediate tumor immune escape. At present, several anti-PD-1 monoclonal antibodies have been approved for the treatment of various tumors (such as classical Hodgkin's lymphoma, squamous or non-squamous NSCLC, hepatocellular carcinoma, nasopharyngeal carcinoma, urothelial carcinoma, esophageal squamous cell carcinoma, melanoma, etc.). However, the approved indications of anti-PD-1 monoclonal antibodies are mostly focused on certain tumors that are sensitive to immunotherapy, and the objective response rate of monotherapy for most tumors is low (usually less than 30%). One of the important reasons may be that there are few immune cells infiltrating into tumor tissue and / or there is a high proportion of immune suppressive cells (such as Treg, MDSC), as well as the participation of other immune suppressive pathways. Therefore, there is an urgent need to develop anti-PD-1 antibodies or their derivatives with better clinical efficacy that will provide additional drug therapy options for patients with diseases such as cancer and infectious diseases. Summary of the Invention

[0003] After intensive research and creative studies, the present inventors have obtained new anti-PD-1 antibodies and their derivatives. The present inventors have surprisingly found that the anti-PD-1 antibodies and their derivatives of the present invention have good affinity and biological activity, and have anti-tumor potential. The following invention is therefore provided:

[0004] Antibodies of the Invention Accordingly, in one aspect, the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to PD-1, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): a VH CDR1 having the sequence set forth in SEQ ID NO: 7 or 13, a VH CDR2 having the sequence set forth in SEQ ID NO: 8, and a VH CDR3 having the sequence set forth in SEQ ID NO: 9; and / or A light chain variable region (VL) comprising three complementarity determining regions (CDRs): a VL CDR1 having the sequence set forth in SEQ ID NO: 10, a VL CDR2 having the sequence set forth in SEQ ID NO: 11 or 14, and a VL CDR3 having the sequence set forth in SEQ ID NO: 12. The present invention provides an antibody or antigen-binding fragment thereof comprising: In certain embodiments, the antibody or antigen-binding fragment thereof comprises the three CDRs contained in the VH set forth in SEQ ID NO: 1, 3 or 5, and / or the three CDRs contained in the VL set forth in SEQ ID NO: 2, 4 or 6. In certain embodiments, the CDRs are identified according to the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system. In certain embodiments, the antibody or antigen-binding fragment thereof comprises: (1) three CDRs contained in the VH set forth in SEQ ID NO:1 and / or three CDRs contained in the VL set forth in SEQ ID NO:2; (2) three CDRs contained in the VH set forth in SEQ ID NO: 3 and / or three CDRs contained in the VL set forth in SEQ ID NO: 4; or (3) Three CDRs contained in the VH set forth in SEQ ID NO: 5 and / or three CDRs contained in the VL set forth in SEQ ID NO: 6 Includes.

[0005] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH comprising the following three complementarity determining regions (CDRs): a heavy chain variable region (VH) CDR1 having the sequence set forth in SEQ ID NO:7, a VH CDR2 having the sequence set forth in SEQ ID NO:8, and a VH CDR3 having the sequence set forth in SEQ ID NO:9; and / or a light chain variable region (VL) CDR1 having the sequence set forth in SEQ ID NO:10, a VL CDR2 having the sequence set forth in SEQ ID NO:11, and a VL CDR3 having the sequence set forth in SEQ ID NO:12. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH comprising the following three complementarity determining regions (CDRs): a heavy chain variable region (VH) CDR1 having the sequence set forth in SEQ ID NO: 13, a VH CDR2 having the sequence set forth in SEQ ID NO: 8, and a VH CDR3 having the sequence set forth in SEQ ID NO: 9; and / or a light chain variable region (VL) CDR1 having the sequence set forth in SEQ ID NO: 10, a VL CDR2 having the sequence set forth in SEQ ID NO: 14, and a VL CDR3 having the sequence set forth in SEQ ID NO: 12.

[0006] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising the sequence set forth in SEQ ID NO: 1, 3, or 5, or a variant thereof; and / or a light chain variable region (VL) comprising the sequence set forth in SEQ ID NO: 2, 4, or 6, or a variant thereof. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising the sequence set forth in SEQ ID NO: 1 or 3, or a variant thereof; and / or a light chain variable region (VL) comprising the sequence set forth in SEQ ID NO: 2 or 4, or a variant thereof. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising the sequence set forth in SEQ ID NO:1 or a variant thereof; and / or a light chain variable region (VL) comprising the sequence set forth in SEQ ID NO:2 or a variant thereof.

[0007] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising the sequence set forth in SEQ ID NO:3 or a variant thereof; and / or a light chain variable region (VL) comprising the sequence set forth in SEQ ID NO:4 or a variant thereof. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising the sequence set forth in SEQ ID NO:5 or a variant thereof; and / or a light chain variable region (VL) comprising the sequence set forth in SEQ ID NO:6 or a variant thereof. A variant described in any of the above embodiments has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) or a sequence having 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 compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0008] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: (1) a VH comprising the sequence set forth in SEQ ID NO:1 and a VL comprising the sequence set forth in SEQ ID NO:2; (2) a VH comprising the sequence set forth in SEQ ID NO: 3 and a VL comprising the sequence set forth in SEQ ID NO: 4; or (3) VH comprising the sequence set forth in SEQ ID NO:5 and VL comprising the sequence set forth in SEQ ID NO:6 Includes.

[0009] In certain embodiments, the antibody or antigen-binding fragment thereof in any of the above embodiments may further comprise a constant region sequence derived from a mammalian (e.g., murine or human) immunoglobulin. In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof of any of the above embodiments comprises a heavy chain constant region derived from a mouse or human immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4). In certain embodiments, the light chain of the antibody or antigen-binding fragment thereof of any of the above embodiments comprises a light chain constant region derived from a mouse or human immunoglobulin (e.g., κ or λ). In certain embodiments, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region.

[0010] In certain embodiments, the heavy chain constant region (CH) has the same or substantially the same effector function as a wild-type heavy chain constant region sequence. In certain embodiments, the heavy chain constant region (CH) may comprise one or more amino acid mutations or chemical modifications to alter one or more of the following properties of the antibodies of the invention: Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function. A functional alteration can be produced by replacing at least one amino acid residue in the constant region of the antibody with a different residue or chemical modification, for example, that alters the affinity of the antibody for an effector ligand (e.g., FcR or complement C1q), thereby altering (e.g., reducing or enhancing) the effector function. The Fc region of an antibody mediates several important effector functions, such as ADCC, phagocytosis, CDC, etc.

[0011] In certain embodiments, the heavy chain constant region (CH) has reduced or abolished ADCC activity, for example, comprises LALA mutations (L234A, L235A). In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) set forth in SEQ ID NO:15 and / or a light chain constant region (CL) set forth in SEQ ID NO:16. In certain embodiments, the antigen-binding fragment in any of the above embodiments may be selected from a Fab, a Fab', a (Fab')2, an Fv, a disulfide-linked Fv, an scFv, and a diabody. In certain embodiments, the antibody in any of the above embodiments is a chimeric antibody, a humanized antibody, a bispecific antibody, or a multispecific antibody.

[0012] scFv In certain embodiments, the antigen-binding fragment in any of the above embodiments is an scFv. It is known in the art that the stability of the scFv structure can be enhanced by forming interchain disulfide bonds. Thus, in certain embodiments, the scFv comprises disulfide bonds. Methods for introducing disulfide bonds into scFvs are well known to those skilled in the art, for example, by introducing cysteine ​​(C) into the VH and VL of the scFv, respectively. In certain embodiments, the VH and VL of the first antigen-binding domain each comprise a residue in the FR region that is mutated to cysteine ​​(C), so that the scFv formed by the VH and VL may comprise a disulfide bond. In certain embodiments, the residue in the FR2 region of the VH and the residue in the FR4 region of the VL of the first antigen-binding domain are mutated to cysteine ​​(C).

[0013] In certain embodiments, the scFv preferably comprises a VH set forth in SEQ ID NO: 3 and a VL set forth in SEQ ID NO: 4, or a VH set forth in SEQ ID NO: 5 and a VL set forth in SEQ ID NO: 6. In such embodiments, the scFv comprises an interchain disulfide bond. In certain embodiments, the scFv has a structure shown as VH-L-VL or VL-L-VH, where L is a peptide linker. In certain embodiments, L is one or more glycines (G) and / or one or more serines (S) and / or one or more alanines (A), e.g., (G4S). n Or (G4A) n (n=1, 2, 3 or 4). In certain exemplary embodiments, L is (G4S)4. In certain embodiments, the scFv may further comprise an additional biologically active polypeptide in its N-segment and / or C-segment to extend its half-life to form a polypeptide construct, in certain embodiments, the additional biologically active polypeptide is an immunoglobulin Fc domain.

[0014] In certain embodiments, the immunoglobulin Fc domain may be connected to the N-terminus or C-terminus (e.g., the N-terminus) of the scFv via a peptide linker. In certain embodiments, the peptide linker is one or more glycines (G) and / or one or more serines (S) and / or one or more alanines (A), such as (G4S). n Or (G4A) n (wherein n=1, 2, 3, or 4). In certain exemplary embodiments, the peptide linker is (G4S)2 or (G4A)2. In certain embodiments, the immunoglobulin Fc domain is an IgG Fc domain (eg, an IgG1 Fc domain). In certain embodiments, the immunoglobulin Fc domain comprises the sequence set forth in SEQ ID NO:22. In certain exemplary embodiments, the scFv comprises the sequence set forth in SEQ ID NO:23 or 24.

[0015] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention have the following biological functions: (1) Specifically recognizing / binding to PD-1 (especially human PD-1); (2) blocking the binding of PD-1 to PD-L1, or inhibiting and / or blocking intracellular signaling mediated by the binding of PD-1 to PD-L1; (3) Improving the activity of immune cells (e.g., T cells) in vitro and in vivo, for example, increasing the secretion levels of effector cytokines (e.g., IL-2, IFN-γ, etc.), proliferation activity, expression levels of activation markers (e.g., CD25, CD69, etc.), and / or cell killing activity; (4) enhancing immune responses (e.g., T cell-mediated immune responses) in vitro and in vivo; (5) Preventing and / or treating a tumor or an infectious disease in a subject. It has at least one of the following.

[0016] As used herein, an antibody or antigen-binding fragment thereof of the present invention may include variants that differ from the antibody or antigen-binding fragment thereof from which it is derived only in conservative substitutions of one or more amino acid residues (e.g., conservative substitutions of up to 20, up to 15, up to 10, or up to 5 amino acids) or have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the antibody or antigen-binding fragment thereof from which it is derived, and that substantially retain the above-mentioned biological functions of the antibody or antigen-binding fragment thereof from which it is derived.

[0017] Derivatized antibodies The antibodies or antigen-binding fragments thereof of the invention may be derivatized, e.g., linked to another molecule (e.g., another polypeptide or protein). In general, derivatization (e.g., labeling) of an antibody or antigen-binding fragment thereof does not adversely affect its binding to PD-1 (particularly human PD-1). Thus, the antibodies or antigen-binding fragments thereof of the invention are also intended to include such derivatized forms. For example, the antibodies or antigen-binding fragments thereof of the invention may be functionally linked (by chemical coupling, genetic fusion, non-covalent bonding, or other methods) to one or more other molecular groups, e.g., another antibody (e.g., to form a bispecific antibody), a detection agent, a pharmaceutical agent, and / or a protein or polypeptide that can mediate binding of the antibody or antigen-binding fragment to another molecule (e.g., an avidin or polyhistidine tag). Additionally, the antibodies or antigen-binding fragments thereof of the invention may be derivatized with chemical groups, e.g., polyethylene glycol (PEG), methyl or ethyl, or glycosyl. These groups may be used to improve the biological properties of the antibody, e.g., to increase serum half-life.

[0018] Thus, in certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention are labeled. In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention are labeled with a detectable label, such as an enzyme, a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin. A 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 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 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), luminescent materials (e.g., chemiluminescent materials, such as acridinium ester compounds), magnetic beads (e.g., Dynabeads®), calorimetric labels, such as gold colloids 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 labels. In certain embodiments, such labels may be suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.). In certain embodiments, the detectable labels described above can be attached to the antibodies or antigen-binding fragments thereof of the present invention through linkers of different lengths to reduce potential steric hindrance.

[0019] In another aspect, the present invention also provides a conjugate comprising an antibody, or antigen-binding fragment thereof, of the present invention and a therapeutic agent attached to the antibody or antigen-binding fragment thereof.

[0020] In certain embodiments, the conjugate is an antibody-drug conjugate (ADC). In certain preferred embodiments, the therapeutic agent is selected from the group consisting of a cytotoxin or a radioactive isotope. In the present invention, non-limiting examples of suitable therapeutic agents include antimetabolites, alkylating agents, DNA minor groove binders, DNA intercalators, DNA cross-linking agents, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and antimitotic agents. In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention may be conjugated to a therapeutic agent through a linker. In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention are conjugated to a therapeutic agent through a cleavable linker (e.g., a peptide linker, a disulfide or a hydrazone linker).

[0021] Fusion proteins As mentioned above, the objective response rate of anti-PD-1 antibody monotherapy for some tumor types is low due to the low number of immune cells infiltrating into tumor tissue and / or the presence of a high proportion of immunosuppressive cells (e.g., Tregs, MDSCs), as well as the participation of other immunosuppressive pathways. To solve the above problems, the inventors of the present application further provide a PD-1 antibody-based fusion protein with superior performance to provide better anti-tumor activity. In another aspect, the present invention also provides a fusion protein comprising an antibody of the present invention, or an antigen-binding fragment thereof, and an additional biologically active polypeptide fused thereto.

[0022] In certain embodiments, the additional biologically active polypeptide is connected to the antibody or antigen-binding fragment thereof via a peptide linker. In certain embodiments, the peptide linker is one or more glycines (G) and / or one or more serines (S) and / or one or more alanines (A), such as (G4S). n Or (G4A) n (where n=1, 2, 3, or 4). In certain exemplary embodiments, the peptide linker is (G4A)4G. In certain embodiments, the additional biologically active polypeptide is an immunomodulatory agent. In certain embodiments, the immunomodulatory agent is a TGF-β / TGF-βR pathway inhibitor.

[0023] Transforming growth factor-β (TGF-β) is a class of cytokines with multiple biological activities secreted by tumor cells or immune cells, mainly including TGF-β1, TGF-β2 and TGF-β3. TGF-β signaling pathway is a key factor in initiating an immunosuppressive tumor microenvironment. The concentration of TGF-β in serum is negatively correlated with clinical outcomes. The main mechanisms include (1) inhibition of CD8+ T cell proliferation and activation; (2) inhibition of NK cell killing activity; (3) induction of the production of immunosuppressive cells, such as M2 macrophages, MDSCs and Tregs. Additionally, TGF-β can also promote tumor cell invasion by inducing fibrosis and angiogenesis. Neutralization of TGF-β in the tumor microenvironment helps reduce tumor invasion, inhibits tumor growth and restores vascular normalization. At the same time, blocking TGF-β signals can enhance the clinical efficacy of immune checkpoint inhibitors. Based on this, the inventors of the present application have developed a fusion protein comprising the PD-1 antibody of the present invention and a TGF-β / TGF-βR pathway inhibitor, which specifically binds to PD-1 and TGF-β, blocks the PD-L1 / PD-1 and TGF-β / TGF-βR pathway, alleviates T cell immunosuppression, better exerts anti-tumor activity, improves the objective response rate of tumor patients, and prolongs the survival of tumor patients.

[0024] In certain embodiments, the immunomodulatory agent is a TGF-βRII extracellular domain. In certain embodiments, the TGF-βRII extracellular domain comprises the sequence set forth in SEQ ID NO:19. In certain embodiments, the fusion protein comprises a first peptide chain comprising the light chain of an antibody or antigen-binding fragment thereof, and a second peptide chain comprising the heavy chain of an antibody or antigen-binding fragment thereof, and an additional biologically active polypeptide. In certain embodiments, the first peptide chain comprises the sequence set forth in SEQ ID NO:17 and / or the second peptide chain comprises the sequence set forth in SEQ ID NO:18.

[0025] Polypeptide Constructs When the antigen-binding fragment of the present invention is used, in order to increase its half-life, it can be linked to a biologically active polypeptide capable of increasing half-life to form a construct. Thus, the present invention also provides such a polypeptide construct. In another aspect, the present invention also provides a polypeptide construct comprising an antibody or antigen-binding fragment thereof of the present invention and an immunoglobulin Fc domain. In certain embodiments, the antibody or antigen-binding fragment thereof is an antigen-binding fragment, hi certain embodiments, the antigen-binding fragment is a Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv, or diabody. Herein, the Fc domain is also referred to as the Fc region and refers to the portion of the heavy chain constant region that includes CH2 and CH3. In some embodiments, the Fc domain includes a hinge, CH2 and CH3. When the Fc domain includes a hinge, the hinge regulates dimerization between two Fc-containing polypeptides. The Fc domain can be any antibody heavy chain constant region isotype. In some embodiments, the Fc domain is IgG1, IgG2, IgG3 or IgG4.

[0026] In certain embodiments, the Fc domain contained in the polypeptide construct of the present invention is a native Fc region that comprises an amino acid sequence that matches the amino acid sequence of an Fc region found in nature. For example, the Fc domain can be a human IgG1 Fc region with native sequence, a human IgG2 Fc region with native sequence, a human IgG3 Fc region with native sequence, or a human IgG4 Fc region with native sequence. The native Fc region can have an effector function. Exemplary "effector functions" include Fc receptor binding; Clq binding and complement-dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation, etc. A functional alteration can be produced by replacing at least one amino acid residue in the native Fc region with a different residue or chemical modification, e.g., a different residue or chemical modification that alters the affinity of the antibody for an effector ligand (e.g., FcR or complement C1q) and thereby alters (e.g., reduces or enhances) effector function.

[0027] Thus, in certain embodiments, the Fc domain contained in the polypeptide construct of the invention may also be a variant Fc region, which may comprise one or more (e.g., 1-10, e.g., 1-5) amino acid mutations or chemical modifications compared to a native Fc region so as to alter one or more of the following properties of an antibody of the invention: Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function, etc. In certain embodiments, the Fc domain has reduced or abolished ADCC activity, for example, comprises a LALA mutation (L234A, L235A). In certain embodiments, the immunoglobulin Fc domain is an IgG Fc domain (eg, an IgG1 Fc domain). In certain exemplary embodiments, the immunoglobulin Fc domain comprises the sequence set forth in SEQ ID NO:22.

[0028] In certain embodiments, the immunoglobulin Fc domain may be connected to the antibody or antigen-binding fragment thereof (e.g., at the N-terminus and / or C-terminus) via a peptide linker. In certain embodiments, the peptide linker may include one or more glycines (G) and / or one or more serines (S) and / or one or more alanines (A), such as (G4S). n Or (G4A) n (wherein n=1, 2, 3, or 4). In certain exemplary embodiments, the peptide linker is (G4S)2 or (G4A)2. In certain embodiments, the polypeptide construct comprises an scFv as described above and an immunoglobulin Fc domain, in certain embodiments, the immunoglobulin Fc domain is linked to the N-terminus of the scFv. In certain exemplary embodiments, the polypeptide construct comprises the sequence set forth in SEQ ID NO:23 or 24.

[0029] Preparation of Antibodies, Fusion Proteins and Polypeptide Constructs The antibody of the present invention can be prepared by various methods known in the art, for example, by recombinant gene engineering. For example, DNA molecules encoding the heavy and light chain genes of the antibody of the present invention can be obtained by chemical synthesis or PCR amplification. The obtained DNA molecules are inserted into an expression vector and then transfected into a host cell. The transfected host cell is then cultured under specific conditions, and the antibody of the present invention is expressed.

[0030] The antigen-binding fragments of the present invention can be obtained by hydrolysis of intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). Additionally, these antigen-binding fragments can also be produced directly 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 obtained directly from host cells; Fab' fragments can be chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology, 10: 163-167 (1992)). Additionally, Fv, Fab or F(ab')2 fragments can also be directly isolated from the culture medium of recombinant host cells. Those skilled in the art are fully aware of other techniques for preparing these antigen-binding fragments. Thus, in another aspect, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof, or a heavy and / or light chain variable region thereof of the invention. In certain embodiments, the isolated nucleic acid molecule encodes an antibody or antigen-binding fragment thereof, or a heavy and / or light chain variable region thereof of the invention.

[0031] In another aspect, the invention provides a vector (eg, a cloning vector or an expression vector) comprising the isolated nucleic acid molecule. In another aspect, the invention provides a host cell comprising the isolated nucleic acid molecule or vector. Such host cells include, but are not limited to, prokaryotic cells, such as Escherichia coli cells, and eukaryotic cells, such as yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells, such as mouse cells, human cells, etc.). In another aspect, a method for preparing an antibody or antigen-binding fragment thereof of the present invention is provided, comprising culturing host cells under conditions that allow for expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the culture of the cultured host cells.

[0032] In another aspect, the present invention also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a fusion protein of the present invention. In certain embodiments, the isolated nucleic acid molecule encodes a fusion protein of the present invention. The present invention also provides a vector (e.g., a cloning vector or an expression vector) comprising the isolated nucleic acid molecule. The present invention also provides a host cell comprising the isolated nucleic acid molecule or vector. The present invention also provides a method for preparing a fusion protein of the present invention, comprising culturing a host cell under conditions allowing expression of the fusion protein, and recovering the fusion protein from the culture of the cultured host cell.

[0033] In another aspect, the present invention also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide construct of the present invention. In certain embodiments, the isolated nucleic acid molecule encodes a polypeptide construct of the present invention. The present invention also provides a vector (e.g., a cloning vector or an expression vector) comprising the isolated nucleic acid molecule. The present invention also provides a host cell comprising the isolated nucleic acid molecule or vector. The present invention also provides a method for preparing a polypeptide construct of the present invention, comprising culturing a host cell under conditions allowing expression of the polypeptide construct, and recovering the polypeptide construct from the culture of the cultured host cell.

[0034] composition In another aspect, the present invention also provides a composition comprising: (1) an antibody or antigen-binding fragment thereof, or a polypeptide construct of the present invention; and (2) an immunomodulatory agent. In certain embodiments, the immunomodulatory agent is a TGF-β / TGF-βR pathway inhibitor, such as a TGF-βRII extracellular domain. In certain embodiments, the composition comprises an antibody of the present invention or an antigen-binding fragment thereof (e.g., a VH comprising the sequence set forth in SEQ ID NO:1, and a VL comprising the sequence set forth in SEQ ID NO:2), and a TGF-βRII extracellular domain. In certain embodiments, the agents described in (1) and (2) are provided as separate components or as components of the same composition. Thus, the agents described in (1) and (2) can be administered simultaneously, separately or sequentially.

[0035] Pharmaceutical Compositions and Therapeutic Uses The antibodies or antigen-binding fragments thereof of the present invention may be used in vitro or in vivo in a subject to inhibit and / or block intracellular signaling mediated by binding of PD-1 to PD-L1, to increase immune cell activity, to enhance immune responses, and to prevent and / or treat tumors or infectious diseases. Thus, in another aspect, the present invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof, fusion protein, polypeptide construct or conjugate, or composition of the invention and a pharma- ceutically acceptable carrier and / or excipient. In certain embodiments, the pharmaceutical composition comprises a fusion protein comprising an antibody or antigen-binding fragment thereof of the present invention and a TGF-β / TGF-βR pathway inhibitor (e.g., a TGF-βRII extracellular domain), where the fusion protein simultaneously specifically binds to PD-1 and TGF-β, blocks PD-L1 / PD-1 and the TGF-β / TGF-βR pathway, relieves T cell immunosuppression, better exerts anti-tumor activity, improves the objective response rate of tumor patients, and prolongs the survival of tumor patients.

[0036] In certain embodiments, the pharmaceutical composition may also include an additional pharma- ceutical active agent. In certain embodiments, the additional pharma- ceutical active agent is a pharmaceutical agent with anti-tumor activity, such as an additional immune checkpoint inhibitor, an oncolytic virus, a chemotherapeutic agent, an anti-angiogenic drug, an antimetabolite, a tumor-targeting drug, an immunostimulant, etc. In certain embodiments, the additional pharma- ceutical active agent is a medication for treating an infectious disease, such as an antiviral agent, an antifungal agent, an antibacterial agent, an immunostimulant, and the like.

[0037] In certain embodiments, in the pharmaceutical composition, the antibody or antigen-binding fragment thereof, fusion protein, polypeptide construct or conjugate, or composition of the present invention and the additional pharma- ceutical active agent are provided as separate components or as components of the same composition. Thus, the antibody or antigen-binding fragment thereof, fusion protein, polypeptide construct or conjugate, or composition of the present invention and the additional pharma-ceutical active agent may be administered simultaneously, separately or sequentially. In certain exemplary embodiments, the medicament comprises a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such a sterile injectable liquid is selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a surfactant-containing solution (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0038] In another aspect, the present invention provides the use of an antibody or antigen-binding fragment thereof, a fusion protein, a polypeptide construct or conjugate, or a composition of the present invention in the preparation of a medicament, comprising: (1) increasing the activity of immune cells (e.g., T cells) in vitro or in a subject (e.g., a human); (2) enhancing an immune response (e.g., a T cell-mediated immune response) in a subject (e.g., a human); (3) treating a tumor in a subject (e.g., a human); or (4) Treating infectious diseases in a subject (e.g., a human) Used for, relating to use.

[0039] In certain embodiments, the medicine comprises a fusion protein comprising an antibody or antigen-binding fragment thereof of the present invention and a TGF-β / TGF-βR pathway inhibitor (e.g., the extracellular domain of TGF-βRII), which simultaneously specifically binds to PD-1 and TGF-β to block the PD-L1 / PD-1 and TGF-β / TGF-βR pathway, alleviating T cell immunosuppression, better exerting anti-tumor activity, improving the objective response rate of tumor patients, and prolonging the survival of tumor patients. In another aspect, the present invention provides a method for increasing immune cell activity and / or enhancing an immune response in a subject, comprising administering to a subject in need thereof an effective amount of an antibody (or antigen-binding fragment thereof), fusion protein, polypeptide construct, conjugate, composition or pharmaceutical composition of the present invention.

[0040] In certain embodiments, the immune response is a T cell-mediated immune response. In certain embodiments, the methods are used to prevent and / or treat a tumor. In such embodiments, the subject is afflicted with a tumor. In certain embodiments, the methods are used to prevent and / or treat an infectious disease. In such embodiments, the subject is suffering from an infectious disease. In certain embodiments, the immune cell is a T cell, e.g., a cytotoxic T cell (CTL), an antigen-specific T cell, or a tumor-infiltrating T cell (TIL-T). In certain exemplary embodiments, the immune cell is a tumor-infiltrating lymphocyte, e.g., a tumor-infiltrating T cell. In certain embodiments, a fusion protein comprising an antibody or antigen-binding fragment thereof of the present invention and a TGF-β / TGF-βR pathway inhibitor (e.g., a TGF-βRII extracellular domain) is administered to a subject, and the fusion protein simultaneously specifically binds to PD-1 and TGF-β to block the PD-L1 / PD-1 and TGF-β / TGF-βR pathway, alleviating T cell immunosuppression, better exerting anti-tumor activity, improving the objective response rate of tumor patients, and prolonging the survival of tumor patients. In another aspect, the present invention provides a method for preventing and / or treating a tumor in a subject (e.g., a human), comprising administering to a subject in need thereof an effective amount of an antibody (or antigen-binding fragment thereof), fusion protein, polypeptide construct, conjugate, composition or pharmaceutical composition of the present invention.

[0041] In certain embodiments, a fusion protein comprising an antibody or antigen-binding fragment thereof of the present invention and a TGF-β / TGF-βR pathway inhibitor (e.g., a TGF-βRII extracellular domain) is administered to a subject, and the fusion protein simultaneously specifically binds to PD-1 and TGF-β to block the PD-L1 / PD-1 and TGF-β / TGF-βR pathway, alleviating T cell immunosuppression, better exerting anti-tumor activity, improving the objective response rate of tumor patients, and prolonging the survival of tumor patients. In certain embodiments, the antibody (or antigen-binding fragment thereof), fusion protein, polypeptide construct or conjugate, or composition of the present invention is used in combination with an additional drug having anti-tumor activity. The additional drug having anti-tumor activity may be administered before, simultaneously with, or after administration of the antibody (or antigen-binding fragment thereof), fusion protein, polypeptide construct or conjugate, or composition.

[0042] In certain embodiments, the antibody (or antigen-binding fragment thereof), fusion protein, polypeptide construct, conjugate, composition, or pharmaceutical composition of the present invention is administered in combination with an additional treatment. This additional treatment can be any known treatment for tumors, such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormonal therapy, gene therapy, or palliative care. The additional treatment can be administered before, simultaneously, or after the administration of the antibody (or antigen-binding fragment thereof), fusion protein, polypeptide construct, conjugate, composition, or pharmaceutical composition of the present invention. In another aspect, the present invention provides a method for preventing and / or treating an infectious disease in a subject (e.g., a human), comprising administering to a subject in need thereof an effective amount of an antibody (or antigen-binding fragment thereof), fusion protein, polypeptide construct, conjugate, composition or pharmaceutical composition of the present invention.

[0043] In certain embodiments, the antibody (or antigen-binding fragment thereof), fusion protein, polypeptide construct or conjugate, or composition of the invention is used in combination with an additional drug for treating an infectious disease. The additional drug for treating an infectious disease may be administered before, simultaneously with, or after administration of the antibody (or antigen-binding fragment thereof), fusion protein, polypeptide construct or conjugate, or composition. In certain embodiments of the uses and methods described above, the tumor is a tumor with microsatellite instability high (MSI-H) and / or mismatch repair deficient (dMMR).

[0044] According to the MSI detection classification criteria established by the National Cancer Institute (NCI) (Boland CR, et al. Cancer Res. 1998 Nov 15;58(22):5248-57.), for five standard microsatellite loci, BAT26, BAT25, D2S123, D5S346, and D17S250, if the tumor tissue contains two or more unstable loci compared with the normal tissue, it is judged as microsatellite instability high (MSI-H), i.e., mismatch repair dysfunctional (dMMR); if there are less than two or no unstable loci, it is judged as microsatellite instability low (MSI-L) or microsatellite stable (MSS), i.e., the mismatch repair function is intact (pMMR). Methods for detecting high frequency microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR) are known to those skilled in the art. For example, by detecting the length change of the above five standard microsatellite loci in tumor tissue by PCR or second generation sequencing, if two or more loci are unstable, it is judged as MSI-H. Additionally, the expression of MLH1, MSH2, MSH6 and PSM2 in tumor tissue can also be detected by immunohistochemistry (ICH). If all four proteins are expressed positively, it is determined to be pMMR, and if any one of the proteins is expressed negatively, it is determined to be dMMR.

[0045] In certain embodiments of the above uses and methods, the tumor is a solid tumor, such as melanoma (e.g., metastatic malignant melanoma), breast cancer, renal cancer (e.g., clear cell carcinoma), prostate cancer, bladder cancer, pancreatic cancer, lung cancer (e.g., non-small cell lung cancer), colon cancer, esophageal cancer, squamous cell carcinoma of the head and neck, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, and glioma. In certain embodiments of the above uses and methods, the tumor is a hematological tumor, such as lymphoma or leukemia. In certain embodiments, the lymphoma is Hodgkin's lymphoma or non-Hodgkin's lymphoma. In certain embodiments, the non-Hodgkin's lymphoma is one or more of peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma, NK / T-cell lymphoma with Epstein-Barr virus positivity (nasal type), and B-cell non-Hodgkin's lymphoma. In certain embodiments of the above uses and methods, infectious disease refers to any infectious disease caused by any pathogenic microorganism, such as a virus, a bacterium, a fungus, a parasite, etc. In certain embodiments, the infectious disease is selected from the group consisting of a viral infection, a bacterial infection, a fungal infection, and a parasitic infection. In certain embodiments of the above uses and methods, the subject is a mammal, such as a human.

[0046] The antibody or antigen-binding fragment thereof, fusion protein, polypeptide construct, conjugate, composition, and pharmaceutical composition of the present invention can be formulated into any dosage form known in the medical art, such as tablets, pills, suspensions, emulsions, liquids, 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 pharmaceutical composition of the present invention should be sterile and stable under the conditions of production and storage. The 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 required dose of the recombinant protein of the present invention in a suitable solvent, and optionally incorporating other desired ingredients (including but not limited to pH adjusting agents, surfactants, adjuvants, ionic strength enhancing agents, isotonicity agents, preservatives, diluents, or any combination thereof), followed by filtration sterilization. Additionally, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), surfactant-containing solution (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof, prior to use.

[0047] Additionally, the antibodies or antigen-binding fragments thereof, fusion proteins, polypeptide constructs, and conjugates of the present invention may be presented in pharmaceutical compositions in unit dosage form for ease of administration. The antibodies or antigen-binding fragments thereof, fusion proteins, polypeptide constructs, conjugates, compositions, and pharmaceutical compositions of the present invention may be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic, inguinal, intravesical, topical (e.g., powders, ointments, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / mode of administration is parenteral administration (e.g., intravenous injection or bolus, subcutaneous injection, intraperitoneal injection, intramuscular injection). Those skilled in the art will appreciate that the route and / or mode of administration will vary depending on the intended purpose. In a preferred embodiment, the antibodies or antigen-binding fragments thereof, fusion proteins, polypeptide constructs, conjugates, compositions, or pharmaceutical compositions of the present invention are administered by intravenous injection or bolus.

[0048] The pharmaceutical composition of the present invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of the antibody or antigen-binding fragment thereof, fusion protein, polypeptide construct, conjugate, or composition of the present invention. A "prophylactically effective amount" refers to an amount sufficient to prevent, stop, or delay the onset of the 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 of the antibody or antigen-binding fragment thereof, fusion protein, polypeptide construct or conjugate, or composition of the present invention may vary depending on the severity of the disease to be treated, the overall state of the patient's own immune system, the general condition of the patient, such as age, weight, and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0049] Kits and detection uses The antibodies, or antigen-binding fragments thereof, of the present invention are capable of specifically binding to PD-1 and can therefore be used to detect the presence or level of PD-1 in a sample. Thus, in another aspect, the present invention provides a kit comprising the antibody of the present invention or an antigen-binding fragment thereof. In a preferred embodiment, the antibody of the present invention or an antigen-binding fragment thereof has a detectable label. In another preferred embodiment, the kit further comprises a second antibody that specifically recognizes the antibody of the present invention or an antigen-binding fragment thereof. Preferably, the second antibody further comprises a detectable label.

[0050] In the present invention, a detectable label may be any substance that can be detected by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Particularly preferably, such a label may be suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.). Such labels are well known in the art and include 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), phycoerythrin (PE), Texas Red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), luminescent substances (e.g., chemiluminescent substances, such as acridinium ester compounds), magnetic beads (e.g., Dynabeads®), calorimetric labels, such as gold colloids 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-mentioned labels. In certain embodiments, the detectable labels described above may be linked to the antibody or antigen-binding fragment thereof of the present invention via linkers of various lengths to reduce potential steric hindrance.

[0051] In another aspect, the present invention provides a method for detecting the presence or level of PD-1 in a sample, comprising using an antibody or antigen-binding fragment thereof of the present invention. In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention also bears a detectable label. In another preferred embodiment, the method further comprises detecting the antibody or antigen-binding fragment thereof of the present invention using a reagent bearing a detectable label. The method may be used for diagnostic or non-diagnostic purposes (e.g., the sample is a cell sample rather than a sample from a patient). In certain embodiments, the PD-1 is human PD-1.

[0052] In another aspect, there is provided the use of an antibody or antigen-binding fragment thereof of the invention in the manufacture of a kit for detecting the presence or level of PD-1 in a sample. In certain embodiments, the PD-1 is human PD-1. In certain embodiments, the sample is a cell sample (eg, tumor cells) or a tissue sample (eg, tumor tissue) from a subject (eg, a mammal, such as a human).

[0053] Definition of Terms In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, biochemistry, nucleic acid chemistry, immunology laboratory and other operation steps used herein are all routine steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

[0054] As used herein, the term "antibody" refers to an immunoglobulin molecule that is generally composed of two pairs of polypeptide chains, each pair having 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 antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and heavy chains also contain 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 domains are not directly involved in binding of the antibody to an antigen, but exhibit various effector functions, for example, 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 of the classical complement system (C1q). The VH and VL regions can also be further divided into highly variable regions (called complementarity determining regions (CDRs)) interspersed with more conserved 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 / light chain pair form the respective antigen-binding site. The distribution of amino acids in the various regions or domains can follow the definitions 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.

[0055] 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. The precise boundaries of these amino acid residues 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, one skilled in the art can easily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those of skill in the art (see, e.g., Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0056] In the present invention, the CDRs contained in the antibody or antigen-binding fragment thereof of the present invention can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibody or antigen-binding fragment thereof of the present invention are determined by the numbering method described in the "Sequence analysis" section of Lu X, Nobrega RP, Lynaugh H, et al. Deamidation and isomerization liability analysis of 131 clinical-stage antibodies. MAbs. 2019 Jan;11(1):45-57. doi: 10.1080 / 19420862.2018.1548233. Page 11, the entire contents of which are incorporated herein by reference.

[0057] As used herein, the term "framework region" or "FR" residues refers to amino acid residues in an antibody variable region other than the CDR residues defined above. 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 of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0058] 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 bound by the full-length antibody and / or competes with the full-length antibody for specific binding to an antigen, 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 techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementarity determining region (CDR) fragments, single chain antibodies (e.g., scFv), chimeric antibodies, diabodies, linear antibodies, nanobodies (technology from Domantis), domain antibodies (technology from Ablynx), probodies, and polypeptides containing at least a sufficient portion of an antibody to confer specific antigen-binding capability to the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0059] As used herein, the term "full-length antibody" refers to an antibody consisting of two "full-length heavy chains" and two "full-length light chains", and "full-length heavy chain" refers to a polypeptide chain consisting of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the N-terminal to C-terminal direction; if the full-length antibody is an IgE isotype, it may further comprise a heavy chain constant region CH4 domain. Preferably, the "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2, and CH3 in the N-terminal to C-terminal direction. The "full-length light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction. The two pairs of full-length antibody chains are linked together by disulfide bonds between CL and CH1 and between the HRs of the two full-length heavy chains. The full-length antibody of the present invention may be from a single species, e.g., human; it may also be a chimeric or humanized antibody. The full-length antibody of the present invention comprises two antigen-binding sites formed by a VH and VL pair, respectively, that specifically recognize / bind to the same antigen.

[0060] As used herein, the term "Fd fragment" refers to an antibody fragment consisting of the VH and CH1 domains; the term "dAb fragment" refers to an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544 546 (1989)); the term "Fab fragment" refers to an antibody fragment consisting of the VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" refers to an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab' fragment" refers to a fragment obtained after reduction of the disulfide bonds linking the two heavy chain fragments in the F(ab')2 fragment, consisting of the complete light chain and the Fd fragment of the heavy chain (consisting of the VH and CH1 domains).

[0061] As used herein, the term "Fv fragment" refers to 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 capable of forming a complete antigen-binding site. The six CDRs are generally considered to confer antigen-binding specificity to an antibody. However, even a single variable region (e.g., an Fd fragment containing only three CDRs specific for an antigen) can recognize and bind to an antigen, although the affinity may be lower than that of the complete binding site. As used herein, the term "Fc fragment" refers to an antibody fragment formed by disulfide bonding of the second and third constant regions of a first heavy chain to the second and third constant regions of a second heavy chain of the antibody. The Fc fragment of an antibody has a variety of different functions but does not participate in antigen binding.

[0062] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and a VH domain, where the VL and VH are connected by a linker. Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. A suitable prior art linker consists of a repeated GGGGS amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 may be used, but variants thereof may also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). In some cases, a disulfide bond may also be present between the VH and VL of the scFv. As used herein, the term "di-scFv" refers to an antibody fragment formed by linking two scFvs.

[0063] As used herein, the term "diabody" means that its VH and VL domains are expressed on a single polypeptide chain, but the linker 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 to 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)). Each of the above antibody fragments retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specific binding to antigen. Antigen-binding fragments of an antibody (e.g., the antibody fragments described above) can be obtained from a given antibody (e.g., an antibody provided by the present invention) using conventional techniques known to those of skill in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and antigen-binding fragments of an antibody can be screened for specificity in the same manner as for intact antibodies. As used herein, unless the context clearly dictates otherwise, when the term "antibody" is referred to it includes not only intact antibodies but also antigen-binding fragments of antibodies.

[0064] As used herein, the terms "monoclonal antibody", "McAb" and "mAb" have the same meaning and are used interchangeably to refer to an antibody or antibody fragment from a group of highly homologous antibody molecules, i.e., completely identical except for naturally occurring mutations that may occur naturally. A monoclonal antibody has high specificity for a single epitope on an antigen. A polyclonal antibody is the opposite of a monoclonal antibody and usually contains at least two or more different antibodies that usually recognize different epitopes on an antigen. Additionally, the modifier "monoclonal" merely indicates that the antibody is characterized by being obtained from a group of highly homologous antibodies and cannot be understood to require that the antibody be prepared by any particular method.

[0065] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and an antigen to which it is directed. The strength or affinity of a specific binding interaction is determined by the equilibrium dissociation constant (K D In the present invention, the term "K D " refers to the dissociation equilibrium constant of a specific antibody-antigen interaction and is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. In certain embodiments, an antibody that specifically binds to an antigen (or an antibody specific for an antigen) has an affinity of about 10 -9 Less than M, e.g., about 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 Affinity (K D ) refers to an antibody that binds to an antigen. The specific binding properties between two molecules can be determined using methods known in the art, for example, using surface plasmon resonance (SPR) in a BIACORE instrument.

[0066] As used herein, the term "vector" refers to a nucleic acid carrier into which a polynucleotide can be inserted. If the vector is capable of expressing a 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 element it carries is 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 (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs); 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 (e.g., herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, papova viruses (e.g., SV40). The vector may contain various elements for controlling expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may also contain an origin of replication.

[0067] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell, such as E. coli or Bacillus subtilis, a fungal cell, such as a yeast cell or Aspergillus, an insect cell, such as an S2 Drosophila cell or Sf9, or an animal cell, such as a fibroblast cell, a CHO cell, a COS cell, an NSO cell, a HeLa cell, a BHK cell, a HEK 293 cell or a human cell. As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. If a position in both compared sequences is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences, divided by the number of positions compared, multiplied by 100. For example, if 6 out of 10 positions of the two sequences are matched, the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (3 out of a total of 6 positions are matched). Typically, two sequences are compared when aligned to produce maximum identity. Such alignments can be accomplished, for example, by using the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program, such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J MoI Biol. 48:444-453 (1970)) as incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix, gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0068] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or adversely change the expected properties of a protein / polypeptide containing 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 the replacement of an amino acid residue with an amino acid residue having a similar side chain, such as a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., has similar size, shape, charge, chemical properties including the ability to form covalent 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). Therefore, it is preferred to replace the 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).

[0069] The twenty conventional amino acids referred to herein are written according to 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. Also in the present invention, amino acids are generally represented by one-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0070] As used herein, the term "pharmaceutical acceptable carriers and / or excipients" refers to carriers and / or excipients that are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) that are pharmacologically and / or physiologically compatible with the subject and active ingredient, and include, but are not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancing agents, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, and preservatives. For example, pH adjusting agents 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 enhancing agents include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as, but not limited to, parabens, chlorobutanol, phenol, and sorbic acid. Agents for maintaining osmotic pressure include, but are not limited to, sugars and NaCl. Agents for delaying absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (e.g., buffered saline), alcohols and polyols (e.g., glycerol), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. Stabilizers have the meanings commonly understood by those skilled in the art and can stabilize the desired activity of the active ingredient in the drug, and include, but are not limited to, sodium glutamate, gelatin, SPGA, sugars (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin, or casein) or their decomposition products (e.g., lactalbumin hydrolyzates), and the like.In certain exemplary embodiments, the pharma- ceutically acceptable carrier or excipient comprises a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such a sterile injectable liquid is selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a surfactant-containing solution (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0071] As used herein, the term "prevention" refers to a method performed to prevent or delay the occurrence of a disease or condition or symptom (e.g., a tumor or an infectious disease) in a subject. As used herein, the term "treatment" refers to a method performed to obtain beneficial or desired clinical results. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of the disease, stabilization of the disease state (i.e., no further worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and alleviation of symptoms (whether partial or complete). Additionally, "treatment" may also refer to prolonging survival compared to expected survival in the absence of treatment.

[0072] As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the subject (e.g., a human) is suffering from or at risk of suffering from a tumor or an infection.

[0073] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain a desired effect. For example, an effective amount for preventing a disease (e.g., a tumor or an infection) refers to an amount sufficient to prevent, stop, or delay the onset of the disease (e.g., a tumor or an infection); 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 already suffering from the disease. Determination of such an effective amount is entirely within the capabilities of a person skilled in the art. For example, an effective amount for therapeutic use depends on the severity of the disease to be treated, the overall state of the patient's own immune system, the general condition of the patient, such as age, weight, and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0074] Beneficial Effects of the Invention The antibody of the present invention can not only specifically recognize / bind to PD-1 and block the binding of PD-1 to PD-L1, but also enhance immune cell activity and stimulate immune response in vitro / in vivo. Therefore, the antibody of the present invention has the potential to be used for the prevention and / or treatment of tumors or infectious diseases. In addition, the present invention also provides a fusion protein comprising the antibody of the present invention and a TGF-β / TGF-βR pathway inhibitor, which simultaneously specifically binds to PD-1 and TGF-β and blocks the PD-L1 / PD-1 and TGF-β / TGF-βR pathway, alleviating T cell immunosuppression, better exerting anti-tumor activity, improving the objective response rate of tumor patients, and prolonging the survival of tumor patients.

[0075] The embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but those skilled in the art will understand that the following drawings and examples are used only to illustrate the practical aspects of the present invention, and not to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art in accordance with the accompanying drawings and the following detailed description of the preferred embodiments. [Brief description of the drawings]

[0076] [Figure 1A] 1 is a graph showing the binding curves of anti-PD-1 antibodies / scFv to human PD-1 overexpressed on CHO cells. [Figure 1B] Graph showing binding curves of anti-PD-1 antibodies / scFv to cynomolgus PD-1 overexpressed on CHO cells. [Diagram 2] Figure 13 is a graph showing the curve of anti-PD-1 antibodies / scFv blocking the binding of human PD-L1 to human PD-1 overexpressed on CHO cells. [Diagram 3] Graph showing binding curves of anti-PD-1 antibodies / scFv to PD-1 on activated human primary T cells. [Figure 4] Graph showing the curves of anti-PD-1 antibodies / scFv blocking the PD-L1 / PD-1 luciferase reporter gene. [Diagram 5] 1 is a bar graph of stimulation of IL-2 secretion by anti-PD-1 antibodies / scFv in a mixed lymphocyte assay. [Figure 6] Pharmacodynamics graphs of anti-PD-1 antibodies in h-PD-1 KI mice bearing the h-PD-L1 KI MC38 tumor model. [Figure 7] Pharmacodynamic graph of anti-PD-1 antibody in B-NDG B2M KO plus mice incubated with A375 tumor cell model. [Figure 8] Schematic diagram of the molecular structure of the anti-PD-1 / TGF-β fusion protein 54872-TGF-βRII. [Figure 9A] 1 is a graph showing the binding curve of the anti-PD-1 / TGF-β fusion protein 54872-TGF-βRII to human PD-1 overexpressed on CHO cells. [Figure 9B] 1 is a graph showing the binding curve of anti-PD-1 / TGF-β fusion protein 54872-TGF-βRII to cynomolgus PD-1 overexpressed on CHO cells. [Figure 10]Figure 13 is a graph showing the curve of anti-PD-1 / TGF-β fusion protein 54872-TGF-βRII blocking the binding of human PD-L1 to human PD-1 overexpressed on CHO cells. [Figure 11] Graph showing the curve of anti-PD-1 / TGF-β fusion protein 54872-TGF-βRII blocking the PD-L1 / PD-1 luciferase reporter gene. [Figure 12A] 1 is a graph showing the binding curves of anti-PD-1 / TGF-β fusion protein 54872-TGF-βRII to human TGF-β1, TGF-β2, and TGF-β3. [Figure 12B] 1 is a graph showing the binding curves of anti-PD-1 / TGF-β fusion protein 54872-TGF-βRII to human TGF-β1, TGF-β2, and TGF-β3. [Figure 12C] 1 is a graph showing the binding curves of anti-PD-1 / TGF-β fusion protein 54872-TGF-βRII to human TGF-β1, TGF-β2, and TGF-β3. [Figure 13] Graph showing the curve of anti-PD-1 / TGF-β fusion protein 54872-TGF-βRII blocking the TGF-β1 / SMAD signaling pathway. [Figure 14A] FIG. 1 is a graph showing the ELISA level co-binding curve of 54872-TGF-βRII molecule to human PD-1 protein and anti-TGF-βRII antibody (human PD-1 protein coated on plate, biotin-labeled anti-TGF-βRII antibody as detection signal). [Figure 14B] FIG. 1 is a graph showing ELISA level co-binding curves of 54872-TGF-βRII molecule to TGF-β1 and human PD-1 protein (TGF-β1 coated on plate, biotin-labeled PD-1 protein as detection signal). [Figure 15] 13 is a graph showing the binding curve of 54872-TGF-βRII molecule to human PD-1 overexpressed on CHO cells, where binding is not affected by the presence of TGF-β1. [Figure 16]Figure 13 is a graph showing the curve of 54872-TGF-βRII molecule blocking the binding of human PD-L1 to human PD-1 overexpressed on CHO cells, regardless of the presence or absence of TGF-β1 binding. [Figure 17A] 1 is a bar graph of IL-2 and IFN-γ release stimulated by 54872-TGF-βRII molecule in a mixed lymphocyte assay. [Figure 17B] 1 is a bar graph of IL-2 and IFN-γ release stimulated by 54872-TGF-βRII molecule in a mixed lymphocyte assay. [Figure 18] 13 is a graph showing the anti-tumor efficacy of 54872-TGF-βRII molecule in B-NDG B2M KO mice incubated with A375 tumor cell model. [Figure 19] 1 is a graph showing serum concentration curves of 54872-TGF-βRII molecule over time in mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0077] Sequence information Sequence information relevant to the present invention is provided in Table 1 below. [Table 1-1] [Table 1-2] [Table 1-3]

[0078] Specific Models for Carrying Out the Invention The present invention will now be described with reference to the following examples, which are intended to illustrate (but not limit) the present invention. Unless otherwise specified, the molecular biological experimental methods and immunoassays used in the present invention were essentially based on the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., Compiled Molecular Biology Laboratory Manual, 3rd edition, John Wiley & Sons, Inc., 1995; the use of restriction endonucleases was in accordance with the conditions recommended by the product manufacturers. Those skilled in the art will understand that the examples describe the present invention by way of example and are not intended to limit the scope of protection claimed by the present invention. The control antibody involved in this invention, pembrolizumab: an anti-PD-1 monoclonal antibody, trade name Keytruda, was the original drug produced by Merck.

[0079] Example 1 Antibody screening, sequence design, expression and purification 1. Animal Immunization and Single Cell Sorting Mouse IgG1 Fc fusion protein of PD-1 extracellular segment (Genbank sequence number: AY238517) was mixed with Ribi adjuvant (sigma) and immunized subcutaneously at a dose of 10 μg / mouse in 6-8 week old BALB / c mice. PD-1 extracellular segment-his fusion protein was harvested and injected intraperitoneally at a dose of 10 μg / mouse for booster immunization. Mice were euthanized, spleens and lymph nodes were harvested, subjected to cell screening, gently crushed, and rinsed with PBS to obtain single cell suspensions. Cells were co-incubated with biotin-labeled PD-1 protein and biotin-conjugated fluorescent secondary antibody, washed, resuspended in FACS buffer, and sorted by flow cytometry. Single cells were sorted into 96-well PCR plates (BioRad). To each well was added 20 μL of lysis buffer (5 μL of 5× single-stranded cDNA buffer (Invitrogen), 0.625 μL of NP-40 (New England Biolabs), 0.25 μL of RNaseOUT (Invitrogen), 1.25 μL of dithiothreitol (Invitrogen), and 12.6 μL of double distilled water). After sorting, plates were immediately transferred to −80° C. for storage.

[0080] 2. Cloning and Amplification of Single B Cell Antibody Variable Region Sequences According to a previous report (Tiller et al. 2009 J Immunol Methods 350:183-93), cDNA sequences encoding antibody heavy and light chain variable regions were amplified from B cell lysates by reverse transcription PCR and nested PCR using specific IgG and IgM specific mixed primers. 10 μL of PCR amplified heavy and light chain variable region sequences and 200 ng of enzyme digested yeast expression plasmid were co-transfected into yeast cells. A single transfected yeast cell was used for subsequent enrichment, amplification, antibody expression, screening, sequencing, etc.

[0081] 3. Humanization of anti-PD-1 antibodies For example, to reduce the potential immunogenicity of mouse antibodies, antibodies have been humanized.Generally speaking, humanized antibodies contain the CDR regions of non-human antibodies and the framework and constant regions of human antibodies.In some cases, some amino acids in the framework regions of human antibodies are also back-mutated to better maintain the activity of antibodies. Humanization was performed as previously reported (Imagro et al. Front. Biosci. 13:1619-1633 (2008); Riechmann et al. Nature 332:323-329 (1988); Queen et al. Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al. Methods 36:25-34 (2005); Padlan, Mol. Immunol. 28:489-498 (1991); Dall'Acqua et al. Methods 36:43-60 (2005)). Methods for selecting human antibody framework regions include, but are not limited to, the "optimal selection method" (Sims et al. J. Immunol. 151:2296 (1993)). The antibody framework regions were derived from the sequence comparison method of heavy and light chain variable region libraries (Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285). (1992) and the like. The antibody humanization modification of the present invention was based on computational biology selection of a humanized antibody sequence library, and the most suitable human antibody framework region of a mouse antibody and the mouse antibody CDR region were grafted onto the human framework region. For each parent antibody to be humanized, 3 to 4 most suitable human antibody heavy chain variable regions and 3 to 4 most suitable human antibody light chain variable regions were selected and constructed according to the above method. The antibodies were expressed in yeast by transfection and subjected to human / monkey PD-1 protein binding screening.

[0082] 4. Antibody Engineering Optimization Further engineering optimization of the humanized monoclonal antibody / scFv was carried out as follows: random point mutation was carried out in antibody heavy chain variable region CDR 1, 2, 3, paired with the original light chain, transfected into yeast, constructed and screened a library to obtain a yeast population with improved affinity; a plasmid library with improved heavy chain affinity was obtained by extraction. Random point mutation was carried out in antibody light chain variable region CDR 1, 2, 3, paired with the original heavy chain, transfected into yeast, constructed and screened a library to obtain a yeast population with improved affinity; a plasmid library with improved light chain affinity was obtained by extraction. The plasmid library with improved heavy chain affinity and the plasmid library with improved light chain affinity were randomly mixed, transfected into yeast, constructed and screened a library to obtain a final yeast monoclonal clone with improved affinity, which was sequenced to obtain an antibody sequence with improved affinity.

[0083] Anti-PD-1 antibody ADI-54872 was finally obtained through screening and modification according to the above method, and its derivative named ADI-54872-1 (anti-PD-1 scFv molecule) was further obtained based on the variant region sequence, and ADI-54872-1 was further engineered to obtain its derivative ADI-63628. The full-length sequences of ADI-54872-1 and ADI-63628 are set forth in SEQ ID NO:23 and 24, respectively, which contained the IgG1 CH2 / CH3 constant region sequence (SEQ ID NO:22) and scFv sequence (VH-linker-VL). The CDR and variable region sequences of the above anti-PD-1 antibodies and their scFv derivatives are shown in the following table.

[0084] [Table 2]

[0085] The amino acid sequence of the heavy chain variable region and the amino acid sequence of the heavy chain constant region of ADI-54872 (SEQ ID NO: 15) were connected, and the amino acid sequence of the light chain variable region and the amino acid sequence of the light chain constant region of ADI-54872 (SEQ ID NO: 16) were connected, and then they were cloned into the pcDNA3.1 expression vector, respectively, and the antibody ADI-54872 was transiently expressed and purified through the HEK293 expression system. The amino acid sequence of ADI-54872-1 (SEQ ID NO: 23) and the amino acid sequence of ADI-63628 (SEQ ID NO: 24) were cloned into the pcDNA3.1 expression vector, respectively, and the corresponding scFv derivatives ADI-54872-1 and ADI-63628 were transiently expressed and purified through the HEK293 expression system. The specific operation was as follows: the pcDNA3.1 vector carrying the antibody heavy chain and / or light chain was transferred into HEK293 cells by chemical transfection, and cultured at 37°C and 8% CO2 for 7 days. The cell clearing was collected and centrifuged at 13000 rpm for 20 min. The supernatant was collected and purified by Protein A while controlling the endotoxin content, and the antibody purity was detected by SEC.

[0086] Example 2 Antibody affinity detection The binding dissociation constants (K D ) values ​​were determined by biofilm optical interference technology (ForteBio). Fortebio affinity determination was performed according to a previous method (Este, P et al. High throughput solution-based measurement of antibody-antigen affinity and epitope binning. Mabs, 2013.5(2):p.270-8), and the extracellular amino acid sequences of human and cynomolgus PD-1 are listed in SEQ ID NOs: 20 and 21, respectively.

[0087] Measurement of monovalent affinity of intact antibody (full length IgG) or anti-PD-1 Fc-scFv protein to human and cynomolgus PD-1-his protein: Sensors were equilibrated offline for 20 min in assay buffer, then tested online for 120 s to establish a baseline, and intact PD-1 antibody or Fc-scFv was loaded onto AHQ sensors to a thickness of 1 nm for affinity detection. Antibody-loaded sensors were exposed to 100 nM PD-1-his antigen until the plateau phase, and then the sensors were transferred to assay buffer for at least 2 min for dissociation rate measurements. Kinetic analysis was performed using a 1:1 binding model. The results are shown in the table below. ADI-54872, ADI-54872-1, and ADI-63628 had better monovalent affinity for binding to human and cynomolgus monkey PD-1 proteins than the control antibody pembrolizumab.

[0088] [Table 3]

[0089] Example 3 Binding activity of anti-PD-1 antibodies / scFv to CHO cells overexpressing human / cynomolgus PD-1 Human PD-1 (Uniprot: Q15116), cynomolgus monkey PD-1 (Uniprot: B0LAJ3) cDNA pCHO1.0 vector (purchased from Invitrogen) was transfected into CHO-S cells, and subjected to pressure screening to produce CHO-S cells overexpressing human PD-1 (CHO-huPD-1 cells) and CHO-S cells overexpressing cynomolgus monkey PD-1 (CHO-cynoPD-1 cells). The overexpressing cells after expansion culture were adjusted to an appropriate cell density, added to a 96-well flow plate, centrifuged, and then gradient diluted samples were added and incubated at 4°C for 30 minutes. Washing was performed twice with PBS, fluorescent secondary antibodies diluted correspondingly to appropriate concentrations were added, incubated at 4°C for 30 minutes, and washed twice with PBS. The cells were resuspended in PBS, and detection was performed on a CytoFlex flow cytometer, and the corresponding MFI was calculated. EC was measured using Graphpad software for graph analysis. 50 The results are shown in Table 4 and Figures 1A and 1B: ADI-54872, ADI-54872-1, and ADI-63628 had good binding activity to CHO cells overexpressing human PD-1 and cynomolgus PD-1.

[0090] [Table 4]

[0091] Example 4 Activity of anti-PD-1 antibodies / scFv in blocking the binding of PD-L1 to CHO cells overexpressing human PD-1 After expansion, CHO-huPD-1 cells were cultured at 2 × 10 6The purified monoclonal antibodies were adjusted to reach a cell density of 0.5μg / mL, added to a 96-well flow plate at 100μL / well, and centrifuged for further use. The purified monoclonal antibodies were diluted in PBS by 3-fold dilutions starting from 400nM for a total of 12 points. The diluted samples were added to the above 96-well flow plate containing cells at 60μL / well and incubated at 4℃ for 30 minutes. Then bio-human PD-L1-Fc protein was added at 60μL / well to reach a final concentration of 0.5μg / mL, incubated at 4℃ for 30 minutes, and washed twice with PBS. SA-PE diluted 100-fold in PBS was added at 100μL / well, incubated at 4℃ for 30 minutes, and washed twice with PBS. PBS was added at 100μL / well to resuspend the cells, and detection was performed on a CytoFlex flow cytometer, and the corresponding MFI was calculated. The results are shown in Table 4 and FIG. 2: ADI-54872, ADI-54872-1, and ADI-63628 had good blocking activity.

[0092] Example 5 Binding of anti-PD-1 antibodies / scFv to surface PD-1 on primary T cells The binding activity of the anti-PD-1 antibodies / scFv of the present invention to surface PD-1 on activated T cells was detected based on flow cytometry. Specifically, human PBMCs were selected to obtain human total T cells according to the experimental protocol provided by STEMCELL (STEMCELL, Catalog No.: #17951C). T cell concentration was adjusted to 1.0 × 10 using X-VIVO15 culture medium (purchased from lonza, Catalog No.: 04-418Q). 6The cells were adjusted to 100 / mL, 1 μL of IL-2 stock solution (1 million IU) was added, CD3 / CD28 Dynabeads (purchased from Gibco, Catalog No.: 11132D) were added at 1:1 (beads to cells), and cultured for 48 hours in a 5% CO2 incubator at 37°C. The activated T cells were adjusted to the appropriate cell density and added to a 96-well flow plate. After centrifugation, the gradient diluted samples to be tested were added and incubated at 4°C for 30 minutes. Washing was performed twice with PBS, and the corresponding fluorescent secondary antibodies diluted to the appropriate concentration were added and incubated at 4°C for 30 minutes, and washing was performed twice with PBS. The cells were resuspended in PBS, detection was performed in a CytoFlex flow cytometer, and the corresponding MFI was calculated. The results are shown in Figure 3. The anti-PD-1 antibodies / scFv ADI-54872, ADI-54872-1 and ADI-63628 of the present invention were able to bind to PD-1 molecules on the surface of activated T cells, with ADI-54872 being superior to the control antibody pembrolizumab.

[0093] Example 6 Experiments with anti-PD-1 antibodies / scFv blocking the PD-L1 / PD-1 luciferase reporter gene To further detect the blocking activity of PD-1 antibody / scFv at the cellular level, a luciferase reporter gene system was constructed in this example. Briefly, cells were transfected with lentivirus to construct a CHO-K1 cell line (CHO-K1-PD-L1) overexpressing human PD-L1 and OKT-3 scFv, and a Jurkat cell line (Jurkat-PD-1-luc) overexpressing human PD-1 and NF-AT luciferase reporter gene (purchased from Promega), and the reporter gene system was subsequently used to carry out related experiments.

[0094] Specifically, CHO-K1-PD-L1 functional cells were obtained by digestion, adjusted to an appropriate cell density, and added to a 96-well white-bottom plate at 100μL / well and cultured overnight. The next day, Jurkat-PD-1-luc effector cell suspension was prepared, and the samples to be tested were gradient diluted with reaction medium (RPMI1640+10% FBS). The white-bottom plate was removed, the culture supernatant was removed by pipetting, and the diluted samples were added to the white-bottom plate at 40μL / well, and Jurkat-PD-1-luc effector cell suspension was added at the same time at 40μL / well. The culture was performed in a 37℃ 5% CO2 incubator for 6 hours, during which Bio-Glo™ reagent (purchased from Promega, Catalog No.: G7940) was allowed to warm to room temperature. After the incubation was completed, the cells were removed and equilibrated at room temperature for 5 minutes, Bio-Glo™ reagent was added at 80μL / well, and the fluorescent signal values ​​were read using a multi-function microplate reader.As shown in Figure 4, the anti-PD-1 antibodies / scFv ADI-54872, ADI-54872-1, and ADI-63628 of the present invention could block PD-L1-mediated PD-1 downstream signaling pathway and up-regulate the reporter gene luciferase expression.

[0095] Example 7 Mixed lymphocyte reaction experiment In this example, the activity of PD-1 antibody / scFv to activate T cells was detected by a mixed lymphocyte reaction (MLR) experiment. The specific experimental method was as follows. PBMC cells (purchased from SAILY BIO, SLB-HPB) were revived and centrifuged. PBMC were resuspended in 10 ml of X-VIVO-15 medium (purchased from LONZA) and cultured at 37°C for 2 hours in a cell culture incubator, and non-adherent cells were removed by pipetting. Add 10ml of DC culture medium, add 10ng / ml GM-CSF (purchased from R&D) and 20ng / ml IL-4 to X-VIVO-15 culture medium, culture for 3 days, add 5ml of DC culture medium, and culture continuously until day 6; add DC maturation culture medium, add 1000U / ml TNF-α (purchased from R&D), 10ng / ml IL-6 (purchased from R&D), 5ng / ml IL-1β (purchased from R&D), and 1μM PGE2 (purchased from Tocris) to X-VIVO-15 culture medium, culture for 2 days, collect mature DC cells, and then collect 2×10 5 The cells were cultured in X-VIVO-15 medium to reach a cell density of 100 / ml.

[0096] PBMC cells from another donor (purchased from SAILY BIO, SLB-HPB) were revived and centrifuged. PBMC were resuspended in 10ml of X-VIVO-15 culture medium. Total T cells were enriched with a total T cell sorting kit (purchased from Stemcell), and total T cells were resuspended in X-VIVO-15 to a cell density of 2 × 10 6 The T cells were mixed with the mature DC cells collected above at a 1:1 ratio and added to a 96-well U-bottom plate at 100 μl / well. PD-1 antibodies / scFv were diluted with X-VIVO-15 medium by 5-fold dilution starting from 200nM for a total of 5 points, and added to the above mixed cell wells at 100μl / well, cultured for 3 days, and the supernatants were collected. ELISA (purchased from eBioscience) method was used to detect the expression level of IL2. The results are shown in Figure 5. The anti-PD-1 antibodies / scFv ADI-54872, ADI-54872-1, and ADI-63628 of the present invention could activate T cells to secrete IL-2 in MLR experiments, and the activation activity of ADI-54872 was better than that of the control antibody pembrolizumab.

[0097] Example 8 In vivo pharmacodynamic study of anti-PD-1 antibodies in human PD-1 transgenic mice In this experiment, human PD-L1 KI MC38 tumor cells (MC38-huPD-L1 cells) were inoculated subcutaneously into human PD-1 transgenic C57 mice (huPD-1 KI mice) to determine the anti-tumor effect of the PD-1 antibodies of the invention. Specifically, a MC38-huPD-L1 cell tumor-bearing mouse model was first established by subcutaneous inoculation. The mean tumor volume was approximately 173 mm 3 When the mice reached 100 mm Hg, they were divided into groups and intraperitoneally injected with PBS, 5 mg / kg ADI-54872, and 5 mg / kg pembrolizumab for treatment. The tumor volume and body weight changes of the mice in each group were monitored. The monitoring frequency was once every 2-4 days, and the monitoring was carried out for 3 consecutive weeks. The dosage and administration method are shown in Table 5. The results are shown in Figure 6. The anti-PD-1 antibody ADI-54872 of the present invention had significant anti-tumor activity.

[0098] [Table 5]

[0099] Example 9 In vivo pharmacodynamic study of anti-PD-1 antibodies in B-NDG mice inoculated with A375 tumor cells and human PBMCs In this experiment, the anti-tumor activity of the anti-PD-1 antibodies of the present invention in a tumor model in which B-NDG mice were inoculated with A375 tumor cells and human PBMCs. Specifically, an A375 tumor-bearing mouse model was first established by subcutaneous co-inoculation of A375 tumor cells + human PBMCs. The mean tumor volume was approximately 147 mm. 3When the mice reached 100 mm Hg, they were divided into groups and treated with PBS, 2.1 mg / kg ADI-54872, and 2.1 mg / kg pembrolizumab by intraperitoneal injection. The changes in tumor volume and body weight of the mice in each group were monitored. The monitoring frequency was once every 2 days, and the monitoring continued for 2 weeks. The dosage and administration method are shown in Table 6. As shown in Figure 7, the anti-PD-1 antibody ADI-54872 of the present invention had significant anti-tumor activity. [Table 6]

[0100] Example 10 Cloning and expression of anti-PD-1 / TGF-β trap fusion protein In this example, the TGF-βR2 extracellular domain (SEQ ID NO: 19) was used as the immunomodulatory molecule portion of the fusion protein, and a PD-1 antibody was used as the targeting portion of the fusion protein to form a PD-1 antibody / TGF-βRII extracellular domain fusion protein designated 54872-TGF-βRII. The heavy chain C-terminal amino acids of ADI-54872 were linked to the extracellular domain of TGF-βRII via (G4A)4G by molecular cloning technology, and conventional expression was performed by Expi-CHO expression system. The expression and purification methods were the same as those in Example 1, and the fusion protein 54872-TGF-βRII with the structure shown in Figure 8 was obtained. The full-length heavy chain sequence and light chain sequence are listed in SEQ ID NO: 17 and SEQ ID NO: 18, respectively.

[0101] Example 11 Binding activity of 54872-TGF-βRII molecule to CHO cells overexpressing human / cynomolgus PD-1 The methods for detecting the binding activity of purified PD-1 antibody ADI-54872, 54872-TGF-βRII molecule, and TGF-βR2-Fc fusion protein to cell surface PD-1 were the same as those in Example 3. In the detection experiments of the above methods, the experimental results are shown in Figures 9A-9B, and the 54872-TGF-βRII molecule of the present invention had binding activity to CHO cells overexpressing human PD-1 and cynomolgus PD-1.

[0102] Example 12 The activity of the 54872-TGF-βRII molecule in blocking PD-L1 binding to CHO cells overexpressing human PD-1 The methods for detecting the activity of purified PD-1 antibody ADI-54872, 54872-TGF-βRII molecule, and TGF-βR2-Fc fusion protein in blocking the binding of PD-L1 to CHO cells overexpressing human PD-1 were the same as those in Example 4. In the detection experiment of the above method, the experimental results are shown in Figure 10, and the 54872-TGF-βRII molecule of the present invention could block the binding of PD-L1 to CHO cells overexpressing human PD-1.

[0103] (Example 13) Experiments with 54872-TGF-βRII molecules blocking the PD-L1 / PD-1 luciferase reporter gene The methods for detecting purified PD-1 antibody ADI-54872, 54872-TGF-βRII molecule, TGF-βR2-Fc fusion protein and negative control, which block PD-L1-mediated PD-1 downstream signaling pathway at the cellular level, were the same as those in Example 6. In the above detection experiment, the experimental results were shown in Figure 11. The 54872-TGF-βRII molecule of the present invention could block PD-L1-mediated PD-1 downstream signaling pathway and up-regulate reporter gene luciferase expression.

[0104] Example 14 Experiments of 54872-TGF-βRII molecules binding to human TGF-β family proteins at the ELISA level Human TGF-β1 (Acrobiosystems, TG1-H421), TGF-β2 (PeproTech, 100-35B), and TGF-β3 (PeproTech, 100-36E) proteins were diluted in ELISA coating solution, added to the ELISA plate, and coated overnight at 4°C. The coating solution was discarded, and the plate was washed three times by adding 250μL / well of PBST, and blocked with 5% BSA at room temperature for 1 hour for later use. Purified PD-1 antibody ADI-54872, 54872-TGF-βRII molecule, TGF-βR2-Fc fusion protein, and negative control were gradient diluted in 1% BSA, added to the blocked ELISA plate, and incubated at room temperature for 2 hours. The plate was washed three times by adding PBST, mouse anti-human Fc-HRP (SouthernBiotech, 9040-05) was added to the wells, incubated at room temperature for 1 hour, washed three times by adding PBST, then ELISA developing solution was added, left at room temperature for 3 minutes, ELISA stop solution was added, and the absorbance value at 450 nm was read. In the detection experiment of the above method, the experimental results are shown in Figures 12A to 12C. The 54872-TGF-βRII molecule of the present invention has good binding activity to human TGF-β1 and TGF-β3 proteins at the ELISA level, and weak binding activity to human TGF-β2 protein.

[0105] Example 15 Experiments with 54872-TGF-βRII molecule blocking the TGF-β / SMAD signaling pathway An appropriate amount of 293-TGF-β / SMAD effector cells was harvested and inoculated into a 96-well cell culture white-bottom plate, which was cultured overnight in a 5% CO2 incubator at 37°C. The purified PD-1 antibody ADI-54872, 54872-TGF-βRII molecule, TGF-βR2-Fc fusion protein, and negative control were gradient diluted and mixed with TGF-β1 (Acrobiosystems, TG1-H421) and incubated at room temperature for 30 minutes. The above mixture was added to the white-bottom plate containing cells and cultured continuously overnight. Bio-Glo™ reagent (Promega) was added to each well, and the fluorescent signal value was read using a multifunction microplate reader. In the detection experiment of the above method, the experimental results are shown in Figure 13. The 54872-TGF-βRII molecule of the present invention could block the TGF-β / SMAD signaling pathway in vitro.

[0106] Example 16 Experiment of 54872-TGF-βRII molecule co-binding to human TGF-β1 / PD-1 protein at ELISA level Human TGF-β1 (Acrobiosystems, TG1-H421) or human PD-1 protein was diluted in ELISA coating solution, then added to the ELISA plate and coated overnight at 4℃. The coating solution was discarded, and the plate was washed 3 times by adding 250μL / well of PBST, and blocked with 5% BSA at room temperature for 1 hour for later use. Purified PD-1 antibody ADI-54872, 54872-TGF-βRII molecule, TGF-βR2-Fc fusion protein, and negative control were gradient diluted in 1% BSA, then added to the blocked ELISA plate and incubated at room temperature for 2 hours. Washed 3 times with PBST, and biotin-PD-1 or biotin-anti-TGFβR2 was added to the wells and incubated at room temperature for 1 hour. Washed 3 times with PBST, and streptavidin-HRP (abcam, ab7403) was added to the wells, incubated at room temperature for half an hour, and washed 3 times with PBST. ELISA developing solution was added and left at room temperature for 3 minutes, then ELISA stop solution was added and absorbance values ​​were read at 450 nm.

[0107] In the detection experiment of the above method, the experimental results are shown in Figures 14A to 14B. The 54872-TGF-βRII molecule of the present invention can co-bind with human TGF-β1 and PD-1 proteins at the ELISA level.

[0108] (Example 17) Binding activity of 54872-TGF-βRII molecule to CHO cells overexpressing human PD-1 in the presence of human TGF-β1 protein After gradient dilution of 54872-TGF-βRII molecule, an equal volume of human TGF-β1 protein was added and incubated at room temperature for 30 minutes. The CHO cells overexpressing human PD-1 after expansion culture were adjusted to an appropriate cell density and added to a 96-well flow plate. After centrifugation, the above incubated sample was added and incubated at 4°C for 30 minutes. The subsequent detection method was the same as that in Example 3. In the determination experiment of the above method, the experimental results were shown in Figure 15. The presence of human TGF-β1 protein did not affect the binding activity of the 54872-TGF-βRII molecule of the present invention to CHO cells overexpressing human PD-1.

[0109] (Example 18) The activity of the 54872-TGF-βRII molecule to block the binding of PD-L1 to CHO cells overexpressing human PD-1 in the presence of human TGF-β1 protein After gradient dilution of 54872-TGF-βRII molecule, an equal volume of human TGF-β1 protein was added and incubated at room temperature for 30 minutes. The CHO cells overexpressing human PD-1 after expansion culture were adjusted to an appropriate cell density and added to a 96-well flow plate. After centrifugation, the above incubated sample was added and incubated at 4°C for 30 minutes. The subsequent detection method was the same as that in Example 4. In the determination experiment of the above method, the experimental results were shown in Figure 16. The presence of human TGF-β1 protein did not affect the activity of the 54872-TGF-βRII molecule of the present invention in blocking the binding of PD-L1 to CHO cells overexpressing human PD-1.

[0110] (Example 19) Mixed lymphocyte reaction experiment The methods of purified PD-1 antibody ADI-54872, 54872-TGF-βRII molecule, TGF-βR2-Fc fusion protein, and negative control mixed lymphocyte detection for activating human T lymphocytes were the same as those in Example 7. The results are shown in Figures 17A-17B. The 54872-TGF-βRII molecule of the present invention could activate T cells to secrete IL-2 and IFN-γ in MLR experiments.

[0111] (Example 20) In vivo pharmacodynamic study of 54872-TGF-βRII molecule in B-NDG mice inoculated with A375 tumor cells and human PBMCs In this experiment, the anti-tumor activity of anti-PD-1 antibodies of the invention in a tumor model in which B-NDG mice were inoculated with A375 tumor cells and human PBMCs. Specifically, an A375 tumor-bearing mouse model was first established by subcutaneous co-inoculation of A375 tumor cells + human PBMCs. The mean tumor volume was approximately 150 mm 3 When the mice reached the age of 18, they were divided into groups and treated by intraperitoneal injection with PBS, 5 mg / kg Keytruda (pembrolizumab), 5 mg / kg ADI-54872 in combination with 2.5 mg / kg TGF-βRII-Fc, and 6 mg / kg 54872-TGF-βRII (equivalent molar dose to 5 mg / kg Keytruda). The tumor volume and body weight changes of the mice in each group were monitored. The monitoring frequency was once every 2-3 days, and monitoring was performed continuously for 2-3 weeks. The dosage and method of administration are shown in Table 7. The results are shown in Figure 18. The antitumor activity of the 54872-TGF-βRII molecule of the present invention was better than that of Keytruda at an equivalent molar dose. [Table 7]

[0112] Example 21 Pharmacokinetic evaluation of 54872-TGF-βRII molecule in mice Eighteen BALB / C mice, half male and half female, were used in the experiment and were subjected to a 12 / 12 h light / dark regime at a temperature of 24±2°C and humidity of 40-70%, with free access to water and food. Mice were purchased from Zhejiang Weitong Lihua Experimental Technology Co., Ltd. On the day of the experiment, mice were injected with 54872-TGF-βRII molecule once through the tail vein at an injection dose of 10 mg / kg.

[0113] Serum collection time points: 5 min, 0.5 h, 2 h, 6 h, 24 h, 48 h, 96 h, 168 h, and 336 h after administration, blood was collected from the mouse orbital vein. The whole blood sample was placed at 2-8°C for 30 min, centrifuged at 12000 rpm for 5 min to collect serum, and the resulting serum was centrifuged at 2-8°C and 12000 rpm for 5 min, stored at -80°C, and the molecular weight of free 54872-TGF-βRII in serum was detected using ELISA. The results are shown in Figure 19 and Table 8. The half-life of the free state molecule of 54872-TGF-βRII in BALB / C mice was about 160 hours.

[0114] [Table 8]

[0115] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings disclosed, and these modifications are within the scope of protection of the present invention. The whole of the present invention is represented by the appended claims and any equivalents thereof.

Claims

1. An antibody or its antigen-binding fragment that has the ability to specifically bind to PD-1, wherein the antibody or its antigen-binding fragment is (i) The following three complementarity-determining regions (CDRs): a heavy chain variable region (VH) having the sequence described in SEQ ID NO: 7 or 13 CDR1, a VH having the sequence described in SEQ ID NO: 8 CDR2, and a VH having the sequence described in SEQ ID NO: 9 CDR3, and / or The following three complementarity-determining regions (CDRs): a light chain variable region (VL) having the sequence described in SEQ ID NO: 10; a VL having the sequence described in SEQ ID NO: 11 or 14; a VL including CDR2 and a VL having the sequence described in SEQ ID NO: 12; and a VL including CDR3. or (ii) Three CDRs contained in VH as described in Sequence ID No. 1, 3, or 5, and / or three CDRs contained in VL as described in Sequence ID No. 2, 4, or 6 The antibody or antigen-binding fragment thereof comprises; preferably, the CDR is determined by the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system.

2. An antibody or its antigen-binding fragment, (a) The following three complementarity-determining regions (CDRs): a heavy chain variable region (VH) CDR1 having the sequence described in SEQ ID NO: 7, a VH CDR2 having the sequence described in SEQ ID NO: 8, and a VH CDR3 having the sequence described in SEQ ID NO: 9; and / or the following three complementarity-determining regions (CDRs): a light chain variable region (VL) CDR1 having the sequence described in SEQ ID NO: 10, a VL CDR2 having the sequence described in SEQ ID NO: 11, and a VL CDR3 having the sequence described in SEQ ID NO: 12; or (b) The following three complementarity-determining regions (CDRs): a heavy chain variable region (VH) having the sequence described in SEQ ID NO: 13 CDR1, a VH having the sequence described in SEQ ID NO: 8 CDR2, and a VH having the sequence described in SEQ ID NO: 9 CDR3; and / or the following three complementarity-determining regions (CDRs): a light chain variable region (VL) having the sequence described in SEQ ID NO: 10 CDR1, a VL having the sequence described in SEQ ID NO: 14 CDR2, and a VL having the sequence described in SEQ ID NO: 12 CDR3 The antibody or antigen-binding fragment thereof according to claim 1, comprising:

3. The antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) containing the sequence or variant thereof described in SEQ ID NO: 1, 3, or 5; and / or a light chain variable region (VL) containing the sequence or variant thereof described in SEQ ID NO: 2, 4, or 6; The antibody or antigen-binding fragment thereof according to claim 1, 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 a sequence having 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.

4. (a) a heavy chain variable region (VH) containing the sequence or variant thereof described in SEQ ID NO: 1 or 3; and / or a light chain variable region (VL) containing the sequence or variant thereof described in SEQ ID NO: 2 or 4; or (b) A heavy chain variable region (VH) containing the sequence or a variant thereof described in SEQ ID NO: 5; and / or a light chain variable region (VL) containing the sequence or a variant thereof described in SEQ ID NO:

6. Including; The antibody or antigen-binding fragment thereof according to claim 1, 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 a sequence having 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.

5. (1) VH containing the sequence described in SEQ ID NO: 1 and VL containing the sequence described in SEQ ID NO: 2; (2) VH containing the sequence described in SEQ ID NO: 3 and VL containing the sequence described in SEQ ID NO: 4; or, (3) VH containing the sequence described in Sequence ID No. 5 and VL containing the sequence described in Sequence ID No. 6 The antibody or antigen-binding fragment thereof according to claim 1, comprising:

6. It further includes a constant region derived from human immunoglobulin; Preferably, the heavy chain of the antibody or its antigen-binding fragment includes a heavy chain constant region derived from human immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4), and the light chain of the antibody or its antigen-binding fragment includes a light chain constant region derived from human immunoglobulin (e.g., κ or λ); Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) described in SEQ ID NO: 15 and / or a light chain constant region (CL) described in SEQ ID NO: 16, according to claim 1.

7. The antigen-binding fragments are Fab, Fab', (Fab') 2 An antibody or antigen-binding fragment thereof according to claim 1, selected from the group consisting of Fv, disulfide-linked Fv, scFv, diabody, chimeric antibody, humanized antibody, bispecific antibody, or polyspecific antibody.

8. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is an IgG antibody (for example, an IgG1, IgG2, IgG3, or IgG4 antibody).

9. The antibody or antigen-binding fragment thereof is labeled; preferably, the antibody or antigen-binding fragment thereof has 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, according to claim 1.

10. A fusion protein comprising the antibody or antigen-binding fragment thereof described in claim 1 and an additional biologically active polypeptide.

11. The fusion protein according to claim 10, wherein the additional biologically active polypeptide is an immunomodulator.

12. The fusion protein according to claim 11, wherein the additional biologically active polypeptide is a TGF-β / TGF-βR pathway inhibitor, such as a TGF-βRII extracellular domain.

13. A first peptide chain comprising an antibody or its antigen-binding fragment's light chain: and A second peptide chain comprising the heavy chain of an antibody or its antigen-binding fragment and an additional biologically active polypeptide. The fusion protein according to claim 10, comprising:

14. The fusion protein according to claim 13, wherein the first peptide chain comprises the sequence described in SEQ ID NO: 17, and / or the second peptide chain comprises the sequence described in SEQ ID NO:

18.

15. A polypeptide construct comprising the antibody or antigen-binding fragment thereof described in claim 1, and an immunoglobulin Fc domain; Preferably, the antibody or its antigen-binding fragment is an antigen-binding fragment, for example, Fab, Fab', (Fab') 2 A polypeptide construct comprising Fv, disulfide bond Fv, scFv, or diabody.

16. The antibody or its antigen-binding fragment is scFv; Preferably, scFv contains a disulfide bond; Preferably, an additional biologically active polypeptide may be linked to the N-terminus of the scFv via a peptide linker. The polypeptide construct according to claim 15.

17. A polypeptide construct according to claim 16, comprising the sequence shown in sequence number 23 or 24.

18. An isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or its heavy chain variable region and / or light chain variable region, or encoding a fusion protein according to any one of claims 10 to 14, or encoding a polypeptide construct according to any one of claims 15 to 17.

19. A vector comprising the nucleic acid molecule described in claim 18, preferably a cloning vector or an expression vector.

20. A host cell comprising the nucleic acid molecule described in claim 18.

21. A method for preparing an antibody or an antigen-binding fragment thereof, or a fusion protein or polypeptide construct, comprising culturing the host cells described in claim 20 under conditions that enable protein expression, and recovering the antibody or an antigen-binding fragment thereof, or a fusion protein or polypeptide construct from the cultured host cells.

22. A conjugate comprising the antibody or its antigen-binding fragment as described in claim 1 and a therapeutic agent linked to the antibody or its antigen-binding fragment; Preferably, the therapeutic agent is a conjugate selected from the group consisting of cytotoxins or radioactive isotopes.

23. (1) an antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 or a polypeptide construct according to any one of claims 15 to 17; and (2) a composition comprising an immunomodulator.

24. The composition according to claim 23, wherein the immunomodulator is a TGF-β / TGF-βR pathway inhibitor, for example, a TGF-βRII extracellular domain.

25. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, a fusion protein according to any one of claims 10 to 14, a polypeptide construct according to any one of claims 15 to 17, or a conjugate according to claim 22, 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 a drug having antitumor activity, such as an additional immune checkpoint inhibitor, oncolytic virus, chemotherapeutic agent, anti-angiogenic drug, antimetabolite, tumor-targeting drug, or immunostimulant; Preferably, the additional pharmaceutically active agent is a drug for treating an infection, such as an antiviral agent, antifungal agent, antibacterial agent, or immunostimulant; Preferably, a pharmaceutical composition comprising an antibody or its antigen-binding fragment, fusion protein or conjugate and an additional pharmaceutically active agent, provided as separate components or as components of the same composition.

26. (1) To increase immune cell activity in vitro or in vivo in a subject (e.g., human); (2) To enhance the immune response in the target (e.g., humans); (3) To prevent and / or treat tumors in subjects (e.g., humans); (4) To prevent and / or treat infectious diseases in subjects (e.g., humans) The use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, a fusion protein according to any one of claims 10 to 14, a polypeptide construct according to any one of claims 15 to 17, or a conjugate according to claim 22 in the manufacture of a pharmaceutical product; Preferably, the tumor is a tumor with high-frequency microsatellite instability (MSI-H) and / or mismatch repair failure (dMMR); Preferably, the tumor is a solid tumor, such as melanoma (e.g., metastatic malignant melanoma), breast cancer, kidney cancer (e.g., clear cell carcinoma), prostate cancer, bladder cancer, pancreatic cancer, lung cancer (e.g., non-small cell lung cancer), colon cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, or glioma; Preferably, the tumor is a hematological malignancy, such as lymphoma or leukemia; preferably, the lymphoma is Hodgkin lymphoma or non-Hodgkin lymphoma; preferably, the non-Hodgkin lymphoma is one or more of peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma, Epstein-Barr virus-positive NK / T-cell lymphoma (nasal type), and B-cell non-Hodgkin lymphoma; Preferably, the infectious disease is selected from the group consisting of viral infections, bacterial infections, fungal infections, and parasitic infections; Preferably, the subject is a mammal, such as a human.

27. A pharmaceutical composition according to claim 25 for enhancing the immune response and / or preventing and / or treating tumors or infections in a subject, Preferably, the tumor is a tumor with high-frequency microsatellite instability (MSI-H) and / or mismatch repair failure (dMMR); Preferably, the tumor is a solid tumor, such as melanoma (e.g., metastatic malignant melanoma), breast cancer, kidney cancer (e.g., clear cell carcinoma), prostate cancer, bladder cancer, pancreatic cancer, lung cancer (e.g., non-small cell lung cancer), colon cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, or glioma; Preferably, the tumor is a hematological malignancy, such as lymphoma or leukemia; preferably, the lymphoma is Hodgkin lymphoma or non-Hodgkin lymphoma; preferably, the non-Hodgkin lymphoma is one or more of peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma, Epstein-Barr virus-positive NK / T-cell lymphoma (nasal type), and B-cell non-Hodgkin lymphoma; Preferably, the infectious disease is selected from the group consisting of viral infections, bacterial infections, fungal infections, and parasitic infections; Preferably, the target is a mammal, such as a human, and is a pharmaceutical composition.

28. A kit comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 9; Preferably, the antibody described in claim 9 or its antigen-binding fragment; Preferably, the kit comprises an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8, and a second antibody having the ability to specifically recognize the antibody or the antigen-binding fragment thereof; the second antibody may further comprise 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.

29. A method for detecting the presence or level of PD-1 in a sample, comprising using the antibody or antigen-binding fragment thereof described in claim 1; Preferably, the method is an immunological assay, such as immunoblotting, enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescence immunoassay, or radioimmunoassay.

30. The method comprises using the antibody or antigen-binding fragment thereof described in Claim 9; A method comprising using an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 8, further comprising detecting the antibody or antigen-binding fragment thereof using a second antibody having a detectable label (e.g., 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); or The method according to claim 29, wherein the sample is a cell sample (e.g., tumor cells) or a tissue sample (e.g., tumor tissue) from a subject (e.g., a mammal, e.g., a human).

31. The use of an antibody or its antigen-binding fragment according to any one of claims 1 to 9 in the manufacture of a detection reagent for detecting the presence or level of PD-1 in a sample; Preferably, the detection reagent detects the presence or level of PD-1 in the sample by the method described in claim 29; Preferably, the sample used is a cell sample (e.g., tumor cells) from a subject (e.g., a mammal, e.g., a human).

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