PD-1 antibody and application thereof

Abstract

The invention provides a PD-1 antibody and application thereof. The PD-1 antibody or antigen binding part provided by the invention has high affinity with human PD-1 protein, and can be bound with human PD-1 at a Kd value of 0.9 * 10 <-8 > M. Moreover, the binding specificity is high, and cross reaction with human CTLA-4 positive cells (3T3 cells overexpressing human CTLA-4) and human PD-1 negative cells (such as Raji cells) is avoided. Based on the characteristics, the PD-1 antibody or the antigen binding part provided by the invention can be used for detecting human PD-1 protein, and can also be independently applied to tumor immunotherapy or applied to tumor immunotherapy in combination with other methods, namely, the PD-1 antibody or the antigen binding part can be effectively applied to preparation of medicines for treating tumors, infectious diseases, autoimmune diseases, immunological rejection and the like.

Description

Technical Field

[0001] The present disclosure relates to the field of biomedicine, and in particular, to PD-1 antibodies and applications thereof. Background Art

[0002] Immunotherapy, which utilizes immune technology to treat cancer, is rapidly becoming a recognized cancer treatment method after surgery, chemotherapy, and radiotherapy. Monoclonal antibodies targeting immune checkpoints, such as cytotoxic T lymphocyte-associated protein 4 (CTLA-4), programmed death receptor 1 (PD-1), or programmed death ligand 1 (PD-L1), have achieved significant breakthroughs in cancer immunotherapy. PD-1, also known as CD279, is an important immunosuppressive molecule that regulates the immune system and promotes immune tolerance by inhibiting the activity of T cells. It can prevent autoimmune diseases, but it can also prevent the immune system from killing cancer cells. Therefore, PD-1 has become a popular target for the development of tumor immunotherapy drugs.

[0003] Currently, three PD-1 monoclonal antibody inhibitors are marketed worldwide: Merck's Keytruda, Bristol-Myers Squibb's Opdivo, and Sanofi / Regeneron's Libtayo. In recent years, two domestically produced PD-1 monoclonal antibody drugs—LOQTORZI (Junshi Biosciences) and TEVIMBRA (BeiGene)—have been approved for marketing in the United States. These drugs are indicated for a wide range of solid tumors, including non-small cell lung cancer, melanoma, colorectal cancer, and nasopharyngeal carcinoma, and their scope of indications is constantly expanding. This demonstrates the excellent clinical value of PD-1 antibodies.

[0004] Clinical trial data show that some patients do not respond to PD-1 monoclonal antibody therapy, and each monoclonal antibody has varying degrees of side effects. Currently, the mechanism by which most patients do not respond to treatment is unclear, and new anti-human PD-1 antibodies are needed to provide new treatment ideas. Summary of the Invention

[0005] Technical issues solved:

[0006] One aspect of the present disclosure addresses the lack of an ideal anti-human PD-1 antibody in the prior art and provides a PD-1 antibody. This antibody, or an antigen-binding portion thereof, is capable of competitively binding to human PD-1 protein molecules and blocking the binding of PD-L1 / PD-L2 to PD-1.

[0007] Technical solution:

[0008] An isolated antibody or antigen-binding portion that specifically binds to human programmed death receptor 1 (PD-1) protein, comprising:

[0009] Complementarity determining region (CDRH) of the heavy chain variable region: at least one selected from the amino acid sequences shown in SEQ ID No: 1, SEQ ID No: 2 or SEQ ID No: 3; and / or

[0010] Complementarity determining region (CDRL) of the light chain variable region: selected from at least one of the amino acid sequences shown in SEQ ID No: 4, SEQ ID No: 5 or SEQ ID No: 6.

[0011] In some embodiments, the antibody or antigen binding portion may comprise:

[0012] The heavy chain variable region CDRH1 shown in SEQ ID No: 1, the heavy chain variable region CDRH2 shown in SEQ ID No: 2, and the heavy chain variable region CDRH3 shown in SEQ ID No: 3; and / or

[0013] The light chain variable region CDRL1 is shown in SEQ ID No: 4, the light chain variable region CDRL2 is shown in SEQ ID No: 5, and the light chain variable region CDRL3 is shown in SEQ ID No: 6.

[0014] In some embodiments, the antibody or antigen binding portion may comprise:

[0015] The heavy chain variable region as shown in SEQ ID No: 7, or an amino acid sequence having 90% or more identity thereto; and / or

[0016] The light chain variable region shown in SEQ ID No: 8 or an amino acid sequence having more than 90% identity with the light chain variable region.

[0017] In the present disclosure, the above-mentioned identity of more than 90% refers to a sequence that has at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity with the heavy chain variable region / light chain variable region.

[0018] In the present disclosure, the above-mentioned antibodies or antigen-binding portions can prevent the binding of PD-1 to its ligands PD-L1 / PD-L2, relieve the inhibition of T cells, and thus restore or enhance the function of T cells.

[0019] In some embodiments, the antibody may be an antibody of mammalian origin, for example, mouse, rabbit, sheep, horse, monkey, pig, camel, shark, chicken, etc. In other embodiments, the antibody may be a chimeric antibody or a humanized antibody.

[0020] In some embodiments, the antibody may be IgG, IgA, IgM, IgD, or IgE. Preferably, in some embodiments, the antibody may be of IgG type. Further, in some embodiments, the antibody may be one or more selected from IgG1, IgG2, IgG3, or IgG4. Preferably, the antibody may be IgG1.

[0021] In some embodiments, the antibody is a monoclonal antibody.

[0022] In some embodiments, the antibody or antigen-binding portion is modified, and the modification includes N-glycosylation modification, O-glycosylation modification, phosphorylation modification, methylation modification, acetylation modification or label modification.

[0023] In some embodiments, the antibody comprises an Fc portion. Preferably, in some embodiments, the Fc portion of the antibody is modified or engineered to enhance its ADCC activity, CDC activity, or ADCP activity.

[0024] In some embodiments, the antigen-binding portion is Fab, Fab', F(ab')2, Fd, FCL, dAb or single-chain antibody scFv. Preferably, in some embodiments, the antigen-binding portion is a single-chain antibody scFv.

[0025] Another aspect of the present disclosure provides a multivalent antibody comprising the above-mentioned antibody or antigen-binding portion.

[0026] The multivalent antibody can be, for example, bivalent, trivalent, tetravalent, hexavalent, nonavalent, etc. The multivalent antibody can be prepared using a suitable method in the prior art. Preferably, in some embodiments, the multivalent antibody is a bispecific antibody or a trispecific antibody.

[0027] Another aspect of the present disclosure provides a multispecific antibody that selectively binds to at least human PD-1 protein, and the multispecific antibody comprises the above-mentioned antibody or antigen-binding portion; the multispecific antibody is a monovalent antibody or a multivalent antibody.

[0028] Another aspect of the present disclosure is to provide an isolated polynucleotide encoding the above-mentioned antibody or antigen-binding portion, or encoding the above-mentioned multivalent antibody, or encoding the above-mentioned multispecific antibody. Further, in some embodiments, the polynucleotide sequence encoding the heavy chain variable region is shown in SEQ ID No: 9, and the polynucleotide sequence encoding the light chain variable region is shown in SEQ ID No: 10.

[0029] Another aspect of the present disclosure provides a vector comprising the above-mentioned polynucleotide.

[0030] Another aspect of the present disclosure provides a cell comprising the above-mentioned antibody or antigen-binding portion, the above-mentioned multivalent antibody, the above-mentioned polynucleotide, or the above-mentioned vector. In some embodiments, the cell can be any suitable host cell used as a tool for producing the target protein. For example, SP2 / 0, YB2 / 0, IR983F, human myeloma Namalwa, PERC6 or CHO cell lines, insect cells, or Escherichia coli cells.

[0031] Another aspect of the present disclosure provides a method for producing an anti-human PD-1 antibody or antigen-binding portion thereof, wherein the anti-human PD-1 antibody or antigen-binding portion thereof is obtained by expressing the protein in the above-mentioned cells.

[0032] Another aspect of the present disclosure is to provide a pharmaceutical composition comprising the above-mentioned antibody or antigen-binding portion, the above-mentioned multivalent antibody, the above-mentioned polynucleotide, the above-mentioned vector or the above-mentioned cell, and a pharmaceutically acceptable carrier. To achieve better therapeutic effects, in some embodiments, the pharmaceutical composition may also contain other therapeutic drugs.

[0033] Another aspect of the present disclosure is to provide an immunoconjugate, comprising:

[0034] a) an antibody or antigen-binding portion as described above, or a multivalent antibody as described above; and

[0035] b) a therapeutic agent or a detectable marker; and

[0036] c) a connector of parts a) and b) above;

[0037] Wherein, the therapeutic agent includes a drug, an enzyme, a toxin, a cytokine or a radionuclide.

[0038] Another aspect of the present disclosure is to provide the use of the above-mentioned antibodies or antigen-binding portions, the above-mentioned multivalent antibodies, the above-mentioned polynucleotides, the above-mentioned vectors, the above-mentioned cells, the above-mentioned pharmaceutical compositions or the above-mentioned immunoconjugates in the preparation of medicaments for treating or assisting in the treatment of tumors, infectious diseases, and autoimmune diseases. In some embodiments, a certain dose of the above-mentioned antibodies or antigen-binding portions, the above-mentioned multivalent antibodies, the above-mentioned polynucleotides, the above-mentioned vectors, the above-mentioned cells, the above-mentioned pharmaceutical compositions or the above-mentioned immunoconjugates is administered to a subject or patient to achieve the effect of treating or assisting in the treatment of tumors, infectious diseases, and autoimmune diseases.

[0039] Furthermore, in some embodiments, the tumor can be breast cancer, neural tumor, melanoma, lung cancer, head and neck cancer, colorectal cancer, pancreatic cancer, gastric cancer, kidney cancer, bladder cancer, prostate cancer, ovarian cancer, cervical cancer, glioblastoma, esophageal cancer, renal cell carcinoma, endometrial cancer, skin cancer, testicular cancer, thyroid cancer or a hematological tumor.

[0040] Another aspect of the present disclosure provides the use of the aforementioned antibodies or antigen-binding portions, the aforementioned multivalent antibodies, or the aforementioned multispecific antibodies in the preparation of products for detecting the presence or level of human PD-1 molecules in a sample. Detecting the presence or level of human PD-1 molecules in a sample can be accomplished using any suitable method known in the art, such as an enzyme-linked immunosorbent assay (ELISA), Western Blot, or the like.

[0041] Another aspect of the present disclosure provides a method for inhibiting the interaction between PD-1 and PD-L1 / PD-L2 in a cell, the method comprising contacting the cell with the above-mentioned antibody or antigen-binding portion, the above-mentioned multivalent antibody, the above-mentioned polynucleotide, the above-mentioned vector, the above-mentioned cell, the above-mentioned pharmaceutical composition, or the above-mentioned immunoconjugate. In some embodiments, the cell is a human cell. In other embodiments, the cell is a non-human cell. In some embodiments, the cell is contacted in vitro. In other embodiments, the cell is contacted in vivo.

[0042] Beneficial effects:

[0043] The mouse anti-human PD-1 antibody or antigen-binding portion provided by the present disclosure has high affinity to human PD-1 protein and can bind to 0.9x10 -8The Kd value of M binds to human PD-1. The binding specificity is strong and does not cross-react with human CTLA-4 positive cells (3T3 cells overexpressing human CTLA-4) and human PD-1 negative cells (such as Raji cells). Based on these characteristics, the PD-1 antibody or antigen-binding portion provided by the present disclosure can be used for the detection of human PD-1 protein, and can also be used alone or in combination with other methods in tumor immunotherapy, that is, it can be effectively used in the preparation of drugs for treating tumors, infectious diseases, autoimmune diseases and anti-immune rejection. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The antibody 2E7 and SP2 / 0 (hPD-1 + ) Affinity constant analysis diagram of cells;

[0045] Figure 2 Figure 2 is a graph showing the cross-reaction results of the antibody 2E7 and the PD-1 family member CTLA-4 detected by flow cytometry in the embodiments of the present disclosure. The detected cells are 3T3 cells overexpressing CTLA-4. The left graph is a negative control, the middle graph is a commercial CTLA-4 antibody, the positive control, and the right graph is 2E7.

[0046] Figure 3 Graphs showing the cross-reaction results of antibody 2E7 and Raji (human PD-1 negative cells) detected by flow cytometry in the disclosed embodiments, wherein the left graph is a negative control, the middle graph is a commercial PD-1 antibody, and the right graph is 2E7;

[0047] Figure 4 This is a diagram showing the results of FACS detection of the binding of antibody 2F6 to Jurkat cells (human PD-1 positive cells) in the embodiments of the present disclosure;

[0048] Figure 5 This figure shows the results of FACS detection of the competitive ability of antibody 2E7 and commercial anti-hPD-1 antibody to bind to hPD-1 protein on the surface of Jurkat cells in the examples of the present disclosure.

[0049] Sequence Description: This disclosure contains a Sequence Listing which has been submitted electronically in .XML format. The Sequence Listing contained in this .XML file is part of the specification and is hereby incorporated by reference in its entirety.

[0050] Sequence number illustrate SEQ ID No: 1 Amino acid sequence of the heavy chain variable region CDRH1 of 2E7 in the example SEQ ID No: 2 The amino acid sequence of the 2E7 heavy chain variable region CDRH2 in the example SEQ ID No: 3 The amino acid sequence of the 2E7 heavy chain variable region CDRH3 in the example SEQ ID No:4 The amino acid sequence of the light chain variable region CDRL1 of 2E7 in the example SEQ ID No:5 The amino acid sequence of the light chain variable region CDRL2 of 2E7 in the example SEQ ID No:6 The amino acid sequence of the light chain variable region CDRL3 of 2E7 in the example SEQ ID No:7 Amino acid sequence of the heavy chain variable region of 2E7 in the example SEQ ID No:8 Amino acid sequence of the light chain variable region of 2E7 in the example SEQ ID No:9 Nucleotide sequence of the heavy chain variable region of 2E7 in the example SEQ ID No: 10 Nucleotide sequence of the light chain variable region of 2E7 in the example SEQ ID No: 11 RT-PCR heavy chain backbone region upstream primer P1 in the embodiment SEQ ID No:12 RT-PCR heavy chain variable region downstream primer P2 in the embodiment SEQ ID No: 13 RT-PCR light chain leader peptide upstream primer P3 in the embodiment SEQ ID No:14 RT-PCR light chain variable region downstream primer P4 in the embodiment DETAILED DESCRIPTION

[0051] The present disclosure provides a PD-1 antibody and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. It is obvious that relevant persons can modify or appropriately change and combine the contents described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0052] In the present disclosure, unless otherwise indicated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise expressly indicated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprising" etc. will be understood to include the elements or components stated, without excluding other elements or other components. The terms "a", "an" and "the" include plural indicators. The term "multiple" refers to two or more. The terms "such as", "for example" etc. are intended to refer to exemplary embodiments and are not intended to limit the scope of the present disclosure.

[0053] In this disclosure, when a range of values ​​is provided, it is understood that the endpoints are included in the range and that each intervening value between the upper and lower limits of the range and any other specified value or intervening value in the stated range and any smaller range between the specified values ​​are encompassed unless the context clearly dictates otherwise.

[0054] In this disclosure, the term "about" generally refers to a variation within a range of 0.5%-10% above or below a specified value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0055] Throughout this disclosure, references to "one embodiment," "an example," "some embodiments," "specific embodiments," "related embodiments," "an example," "some examples," "additional embodiments," or "further embodiments," "further implementations," or "another embodiment," "other examples" mean that at least one feature or characteristic description is included in connection with an embodiment. Thus, references to these phrases in various places throughout this disclosure are not necessarily referring to the same embodiment. Furthermore, particular features may be combined in any suitable manner in one or more embodiments.

[0056] In this disclosure, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. For definitions of common terms in molecular biology, see Lewin's Genes, Twelfth Edition, Jocelyn E. Krebs, Elliott S. Goldstein, Stephen T. Kilpatrick, Publisher: Jones & Bartlett Learning. For definitions of common terms in biochemistry, see Lehninger Principles of Biochemistry, Eighth Edition, David L. Nelson, Michael M. Cox, Publisher: WH Freeman. For definitions of common terms in cell biology, see Molecular Biology of the Cell, Sixth Edition, Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter, Publisher: Garland Science. For definitions of common terms in genetics, see Genetics: Analysis of Genes and Genomes, Eighth Edition, Daniel L. Hartl, Maryellen Ruvolo, Publisher: Jones & Bartlett Learning.

[0057] Unless otherwise specified, the laboratory techniques herein utilize conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which can be found in standard texts such as Molecular Cloning: A Laboratory Manual; Cell Biology: A Laboratory Handbook, etc.

[0058] definition:

[0059] The term "PD-1" (programmed death receptor-1) in this disclosure is a key immunosuppressive receptor present on the surface of T cells, and its core function is to act as a "brake" for the immune system. When PD-1 binds to its ligand (mainly PD-L1 on the surface of tumor cells or certain normal cells), it transmits a strong inhibitory signal to T cells, resulting in reduced T cell activity, decreased proliferation, and even apoptosis. Under normal circumstances, this mechanism helps maintain autoimmune tolerance and prevent excessive immune response from damaging healthy tissues; however, tumor cells often highly express PD-L1 to hijack this pathway, binding to PD-1 on T cells, thereby inhibiting the anti-tumor function of T cells and achieving immune escape. Therefore, the PD-1 / PD-L1 pathway is an important target for cancer immunotherapy.

[0060] The term "isolated" as used herein refers to a substance or entity that has been separated from its natural environment or the environment in which it existed prior to separation and from other components. For example, an isolated protein is substantially free of cellular material or other proteins from the cell or tissue source from which it was derived. The separation can be, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. Isolated substances may have varying levels of purity relative to their pre-isolation counterparts.

[0061] The term "antibody" in this disclosure refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains (each pair having one "light" chain and one "heavy" chain). Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains 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 region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be further subdivided into regions with high variability (called complementary determining regions (CDRs)), interspersed with more conservative regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair respectively form the antibody binding site. The term "antibody" is not limited to any particular method for producing antibodies. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be an antibody of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibody. In some embodiments, the antibody can be IgG, IgA, IgM, IgD or IgE. Preferably, in some embodiments, the type of the antibody can be IgG. Further, in some embodiments, the antibody can be one or more selected from IgG1, IgG2, IgG3 or IgG4. Preferably, the antibody can be IgG1.

[0062] Preparation of antibodies:

[0063] In some embodiments, the antibodies are produced using mammalian cells. For example, monoclonal antibodies can be produced in mammalian cells using hybridoma technology. The hybridoma preparation method reported by Kohler et al. in Nature 256:495 (1975) can be used to prepare the monoclonal antibodies. First, mice or other suitable host animals are immunized with the immunogen (with an adjuvant if necessary).

[0064] Immunogens or adjuvants are typically injected subcutaneously at multiple sites or intraperitoneally. Adjuvants can include Freund's adjuvant (complete or incomplete) or MPL-TDM. After immunization, animals produce lymphocytes that secrete antibodies that specifically bind to the immunogen. The target lymphocytes are collected and fused with myeloma cells using a suitable fusion agent (such as PEG 4000) to obtain hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996).

[0065] The hybridoma cells prepared as described above are plated and grown in a suitable culture medium containing one or more substances that inhibit the growth of unfused, parental myeloma cells. For example, for parental myeloma cells that lack the enzyme hypoxanthine guanine phosphotransferase (HGPRT or HPRT), the addition of hypoxanthine, aminopterin, and thymidine to the culture medium (HAT medium) will inhibit the growth of HGPRT-deficient cells.

[0066] Preferred myeloma cells should have a high fusion rate, stable antibody secretion capacity, and sensitivity to HAT culture medium. Among them, myeloma cells are preferably murine myeloma cells, such as MOP-21 and MC-11 mouse tumor-derived strains (THE Salk Institute Cell Distribution Center, San Diego, Calif., USA), and SP-2 / 0 or X63-Ag8-653 cell lines (American Type Culture Collection, Rockville, Md., USA). In addition, human myeloma and human-mouse heteromyeloma cell lines can also be used to prepare human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63, Marcel Dekker, Inc., New York, 1987).

[0067] The culture medium in which the hybridoma cells are grown is used to detect the production of monoclonal antibodies against the specific antigen. The binding specificity of the monoclonal antibodies produced by the hybridoma cells can be determined using the following methods: immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). For example, the affinity of the monoclonal antibodies can be determined using the Scatchard analysis described by Munson et al. in Anal. Biochem. 107:220 (1980).

[0068] After determining the specificity, affinity, and reactivity of the hybridoma-produced antibody, the desired cell line can be subcloned using the limiting dilution method described in Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996. Suitable culture media include DMEM or RPMI-1640. Alternatively, hybridoma cells can be grown in animals as ascites tumors.

[0069] The monoclonal antibody secreted by the subcloned cells can be separated from the cell culture medium, ascites or serum using traditional immunoglobulin purification methods, such as protein A agarose gel, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography, thereby obtaining the monoclonal antibody.

[0070] In other embodiments, anti-human CD180 antibodies can also be produced by known recombinant methods, for example, by selecting recombinant antibody libraries in phage or similar vectors, see, for example, Smith GP. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virionsurface. Science. 1985; 228: 1315–17.

[0071] Antibody modification and adaptation:

[0072] In some embodiments, the isolated antibody can be a humanized antibody. Humanization of an antibody can improve the affinity or other characteristics of the antibody. A description and method of humanizing antibodies can be found in Riechmann, L., Clark, M., Waldmann, H., & Winter, G. (1988). Reshaping human antibodies for therapy. Nature, 332(6162), 323–327.

[0073] In some embodiments, the antibody Fc (fragment crystallizable region, Fc) is modified to enhance its effector functions by binding to Fc receptors or complement. These functions may include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). The above modifications may include: 1) modification of glycosylation, for example, aspartic acid at position 297 (N297) in the Fc region can be modified with N-acetylglucosamine. Mutation of N297 to alanine (A), glutamine (Q), or glycine (G) will hinder the glycosylation of the antibody, thereby reducing Fc-mediated effector functions. After deglycosylation, the ability of antibodies to induce ADCC or CDC activity will be reduced; sialic acid modification will reduce the binding affinity with FcγRIIIa, thereby leading to a decrease in CDC and ADCC activity.

[0074] In addition to the aforementioned functions, glycosylation of antibodies can also influence their conformation and stability. For example, the sugar chains in glycosylation can maintain the antibody's conformation, preventing aggregation or unfolding. For example, the sugars on the al-3 arm do not contact the antibody surface, but instead reside deep within the space formed by the two heavy chain Fc segments. The interaction between the mannose groups on the al-3 arms of the two sugar chains is crucial for maintaining the antibody's conformation. Without the presence of sugar chains, the CH2 domain of the Fc segment will slightly expand, resulting in an earlier elution time in size-exclusion chromatography and increased sensitivity and aggregation in thermally accelerated stability tests. Glycosylation can also influence the binding of antibodies to receptors on the cell membrane, forming complexes and playing an important role in signal transduction. This regulation of signal transduction is crucial for physiological processes such as cell proliferation, differentiation, and apoptosis.

[0075] The above modifications may also include: 2) point mutations, for example, the LALA mutation (L234A / L235A) can alter the antibody's affinity for FcγR (eliminating binding to low-affinity FcγRs and reducing binding to FcγRI), thereby significantly reducing its ADCC and CDC activities. In addition, the combination of cross-subtype antibodies can also modulate the antibody's effector function.

[0076] In some embodiments, the point mutation results in the substitution of some conservative amino acids, thereby obtaining a "conservative amino acid substitution variant". The change results in some amino acids being substituted by other amino acids with similar chemical properties and / or functions. Conservative substitution tables providing amino acids with similar chemical properties and / or functions are well known in the art. Typical examples of mutually conservative substitutions include, for example, (1) alanine (A), glycine (G); (2) aspartic acid (D), glutamic acid (E); (3) asparagine (N), glutamine (Q); (4) arginine (R), lysine (K); (5) isoleucine (I), leucine (L), methionine (M), valine (V); (6) phenylalanine (F), tyrosine (Y), tryptophan (W); (7) serine (S), threonine (T); (8) cysteine ​​(C), methionine (M).

[0077] The aforementioned modifications may also include: 3) Phosphorylation. Phosphorylation refers to the process of adding phosphate groups to amino acids in proteins within cells. Phosphoantibodies can specifically recognize specific phosphorylation sites, allowing detection of increased or decreased phosphorylation levels of proteins upon cellular stimulation. These antibodies play an important role in life science research, including cell signaling, apoptosis, and cancer. 4) Methylation: Methylation is an important dynamic modification and biological phenomenon catalyzed by methyltransferases acting on specific residues in proteins. Methylation antibodies can specifically recognize specific methylated amino acid sites and are used to distinguish between methylated and unmethylated forms of proteins. They are widely used in research on epigenetics, cancer, Alzheimer's disease, and aging. 5) Acetylation. Acetylation is one of the most common types of acylation. Acetylation antibodies can specifically recognize the acetylated form of a target protein and specific acetylated amino acid sites, allowing detection of protein activity. These antibodies are widely used in research on cell cycle regulation, signal transduction, neurodegenerative diseases, metabolic diseases, and the development and progression of cancer.

[0078] The aforementioned modifications may also include: 6) Label modification. Antibodies can be cross-linked using various chemical reagents, allowing them to be linked to substances such as enzymes, fluorescent dyes, biotin, or colloidal gold to alter their detection or analytical properties. For example, enzyme labeling: Antibodies can be cross-linked to enzymes such as horseradish peroxidase (HRP) and alkaline phosphatase. This is commonly used in experiments such as immunohistochemistry and ELISA, where the presence of the antibody is detected by a color reaction catalyzed by the enzyme. For example, after binding to an antigen, an HRP-labeled antibody can produce a color precipitate by adding a substrate, facilitating observation and quantification. Fluorescent dye labeling: Antibodies can also be conjugated with fluorescent dyes (such as FTC, PE, and APC) for use in detection methods such as flow cytometry and fluorescence microscopy. Fluorescently labeled antibodies can be used to locate specific antigens in cells or tissue sections, and the intensity of the fluorescent signal can be used to determine the antigen's expression level. Biotin labeling: Biotin is a small molecule that can bind to antibodies without affecting their antigen-binding ability. Biotin-labeled antibodies can be conjugated to avidins (such as streptavidin) to achieve signal amplification and detection. Commonly used in multiplex immunolabeling experiments to detect multiple antigens simultaneously.

[0079] In the present disclosure, modifications and alterations to antibodies typically occur in the Fc region and the framework regions (FRs) of the antibody variable region, rather than in the complementarity-determining regions (CDRs) of the antibody variable region. The framework region of the antibody variable region has a relatively conserved amino acid sequence, provides stable support for the structure of the hypervariable region, and participates in maintaining the three-dimensional conformation of the antigen-binding groove. Therefore, alterations and modifications to it do not affect the binding ability of the antibody.

[0080] Isolated antigen-binding portion:

[0081] The term "antigen-binding portion" in this disclosure 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 the antigen, and is also referred to as an "antigen-binding fragment". See generally, Fundamental Immunology, Ch. 7 (Paul, W., et al., 2nd edition). Antigen-binding fragments of antibodies can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. In some cases, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, and the like.

[0082] 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 "Fd fragment" refers to an antibody fragment consisting of the VH and CH1 domains; and the term "Fv fragment" refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody.

[0083] In some embodiments, the antigen-binding portion is prepared by protease digestion, using proteases such as papain, pepsin, etc. In other embodiments, the antigen-binding portion is prepared by chemical treatment. In other embodiments, the antigen-binding portion is prepared by genetic engineering. That is, a fragment containing all or part of the gene sequence of the antigen-binding portion is connected to a suitable vector and expressed. Examples of expression vectors include bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0084] Humanized antibodies:

[0085] Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fab, Fab', F(ab')2, Fd, FCL, dAb, or single-chain antibody scFv) that are primarily composed of human sequences. Humanization of antibodies or antigen-binding portions can be performed according to methods in the prior art (see Samuel Ken-En Gan, et al. Sagacity in antibody humanization for therapeutics, diagnostics and research purposes: considerations of antibody elements and their roles, Antibody Therapeutics, DOI: 10.1093 / abt / tbaa004). In general, a humanized antibody will comprise substantially all or at least two variable domains, wherein all or substantially all of the CDR regions correspond to the CDR regions of a non-human immunoglobulin, and all or substantially all of the framework regions are regions of human immunoglobulin consensus sequences. The humanized antibody optimally also comprises at least a portion of an immunoglobulin constant region (Fc), typically a constant region of a human immunoglobulin. For example, in some embodiments of the present disclosure, a murine variable region (V H (SEQ ID No: 7) / V L(SEQ ID No: 8)) + human constant region, or by retaining only the mouse complementary determining region (CDR) and replacing the remaining framework region (FR) with a human sequence. Humanized antibodies can also be formed by replacing only the exposed residues on the surface of the mouse antibody and retaining the stability of the internal structure. Humanized antibodies can also be formed by directly obtaining a fully human sequence through phage display or transgenic mouse technology.

[0086] Multivalent and multispecific antibodies:

[0087] In some embodiments, the multivalent antibody comprises at least two antibodies or antigen-binding portions described herein, which can competitively bind to human PD-1 molecules and produce effects different from those of monovalent antibodies, for example, by increasing the number of antigen-binding sites, a tighter antigen-antibody complex can be formed, thereby enhancing binding and stability. This enhanced binding helps to increase the affinity of the anti-human PD-1 antibody for the antigen and enhance the interaction of the antigen with cell surface receptors or other molecules. The multivalent antibody can be obtained, for example, by protein fusion, adding a linker, covalent bonding or non-covalent bonding.

[0088] The multivalent and multispecific antibodies can be prepared using conventional techniques in the art. For the preparation of engineered antibodies, see, for example, Hantao Lou, Xuetao Cao, Antibody variable region engineering for improving cancer immunotherapy, Cancer Communications. 2022; 42: 804–827.

[0089] The term "polynucleotide" in this disclosure is also used interchangeably as "nucleic acid" and refers to a chain of nucleotides of any length and includes DNA or RNA. It may include any known nucleotide analogs or modified nucleotides or bases.

[0090] The term "vehicle" or "vector" as used herein refers to a polynucleotide molecule capable of transporting and / or expressing one or more target genes. Examples of vectors include viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids, or phage vectors, DNA or RNA expression vectors associated with a cationic condensing agent, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as production cells.

[0091] Pharmaceutical composition:

[0092] The term "pharmaceutical composition" in the present disclosure refers to a composition that contains at least one other substance in addition to the antibody, antigen-binding portion, multivalent antibody, polynucleotide, vector, cell, or multispecific antibody described in the present disclosure. In some embodiments, the other substance can be, for example, a pharmaceutically acceptable carrier (a carrier that does not affect the function of the mesenchymal stem cells and has no effect on the patient's physical condition, such as physiological saline, cell culture medium, glucose, water for injection, glycerol, ethanol, and combinations thereof), an excipient, a stabilizer, a surfactant, a preservative, an isotonic agent, and the like. It can also be other therapeutic agents, such as chemotherapeutic drugs: melphalan, doxorubicin, cyclophosphamide, vincristine, etc.; glucocorticoid drugs: prednisone, dexamethasone, betamethasone, etc.; immunomodulatory drugs: thalidomide, lenalidomide, pomalidomide, etc. In some embodiments, the pharmaceutical composition may further include other immune checkpoint inhibitors, the targets of which include but are not limited to, for example, CTLA-4, TIGIT, TIM-3, B7-H3, CD73, LAG3, CD27, CD70, 4-1BB, GITR, OX40, SIRP-α (CD47), CD39, ILDR2, VISTA, BTLA, and VTCN-1.

[0093] In some embodiments, the pharmaceutical composition may further include other PD-1 immunotherapy agents, for example, pembrolizumab ( Merck), nivolumab ( Bristol-Myers Squibb), Tepliizumab ( Junshi Biosciences), Carrelizumab (Hengrui Medicine), Tislelizumab (BeiGene), Sintilimab ( Innovent Biologics), Penpulimab ( Sino Biopharmaceutical), Sepalimab (Yuheng Biopharm), Rosnilimab (AnaptysBio), GenSci120 (Jinsai Pharmaceutical), etc.

[0094] In some embodiments, the above pharmaceutical composition can be prepared into any suitable preparation.For example, pill, tablet, emulsifier, gelatin capsule, capsule, suppository, soft gelatin capsule, gel, film, tubule, solution or suspension.In some embodiments, the above pharmaceutical composition can adopt any suitable mode of administration, for example, intranasal, intrapulmonary, intrabronchial, intravenous, oral, intrafatty, intraarterial, intraarticular, intracranial, intradermal, intralesional, intramuscular, intrapericardial, intraperitoneal, intrapleural, intracystic, local, mucosal, parenteral, enteral, subcutaneous, sublingual, local, through buccal, transdermal, by inhalation, by injection, emulsifier, lipid composition, by catheter, by lavage, by continuous infusion, by infusion via local delivery or via local perfusion, etc.

[0095] Immunoconjugates:

[0096] In some embodiments, the immunoconjugates provided by the present disclosure can be in any suitable form, for example, antibody-drug conjugates (ADCs), radionuclide drug conjugates (RDCs), antibody fusion proteins, and the like.

[0097] The above-mentioned antibody-drug conjugate comprises the antibody, antigen-binding portion, multivalent antibody or multispecific antibody described in the present disclosure, a linker and a payload. Existing known linkers include, for example, N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), hydrazone linkers, Val-Cit dipeptide, tetrapeptide Gly-Gly-Phe-Gly, glucuronic acid-containing linkers, β-galactosidase-containing linkers, etc. Existing known payloads include, for example, calendulin, maytansine derivatives, tubulysins, cryptomycins (CR), pyrrolo[2,1-c][1,4]benzodiazepine (PBD), dukamycin, camptothecin (CPT), calicheamicin, apoptosis inducers, thailanstatin A, amatoxin, nicotinamide phosphoribosyltransferase, carmazolin, etc.

[0098] Indications:

[0099] In some embodiments, drugs prepared from the antibodies or antigen-binding portions, multivalent antibodies, polynucleotides, vectors, cells, pharmaceutical compositions or immunoconjugates described herein can be used to treat the following diseases: tumors, autoimmune diseases, and infectious diseases.

[0100] Illustrative examples of tumors include, for example, breast cancer, neurological tumors, melanoma, lung cancer, head and neck cancer, colorectal cancer, pancreatic cancer, gastric cancer, kidney cancer, bladder cancer, prostate cancer, ovarian cancer, cervical cancer, glioblastoma, esophageal cancer, bladder cancer, renal cell carcinoma, endometrial cancer, skin cancer, testicular cancer, thyroid cancer, acute myeloid leukemia, chronic lymphocytic leukemia, and B-cell lymphoma.

[0101] Illustrative examples of autoimmune diseases include, for example, autoimmune hematological disorders (including, for example, hemolytic anemia, aplastic anemia, simple red blood cell anemia, and idiopathic thrombocytopenia), systemic lupus erythematosus, polychondritis, scleroderma, Wegener granulomatosis, dermatomyositis, chronic active hepatitis, myasthenia gravis, psoriasis, Steven-Johnson syndrome, idiopathic diarrhea, autoimmune inflammatory bowel disease (including, for example, ulcerative colitis and Crohn's disease, endocrine eye diseases, Graves' disease, sarcoidosis, multiple sclerosis, primary biliary cirrhosis, type I diabetes mellitus, uveitis, keratoconjunctivitis sicca and vernal keratoconjunctivitis, interstitial pulmonary fibrosis, psoriatic arthritis, and glomerulonephritis.

[0102] Example:

[0103] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention is further described in detail below with reference to specific embodiments.

[0104] Example 1: Screening of mouse hybridoma monoclonal antibodies.

[0105] Balb / c mice were immunized intraperitoneally with human PD-1 protein as the immunogen. Boost immunizations were performed at 3 and 5 weeks after the initial immunization. Blood was collected from the tail of the mice on the 8th day after the boost immunization. The blood was allowed to stand at room temperature for 1 hour and then centrifuged at 12000 rpm for 10 minutes at 4°C. The serum was collected and diluted with PBS to different concentrations: 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, and 1:12800. Jurkat cells were collected, washed once with PBS, and the cells were counted. 1×10 6For each cell, 100 μL of serum at various dilutions was added to the cells. For the negative control, serum from unimmunized mice was used instead of antiserum. The cells were incubated at 4°C for 1 hour and washed twice with PBS. 2 μL of PE-conjugated rat anti-mouse IgG antibody was added and incubated at 4°C in the dark for 40 minutes. The cells were resuspended in 500 μL of PBS buffer, and the percentage of antibody binding to the cells and the fluorescence intensity were measured by flow cytometry. A mean fluorescence intensity greater than twice that of the negative control was considered effective, and fusion was performed only when the titer exceeded 6400. Three days before fusion, immunized mice were pulsed with the immunogen via tail vein injection. Spleen cells from successfully immunized mice were fused with myeloma SP2 / 0 cells at a 10:1 ratio. For fusion, 50% PEG was added to the spleen and myeloma cell pellet within 1 minute in a 37°C water bath, and the supernatant was discarded. The mixture was shaken at 37°C in a water bath for 1 minute, and 10 ml of serum-free 1640 medium was added within 2 minutes. Centrifuge at 800 rpm for 6 min, discard the supernatant, resuspend the cells in 1640 medium containing HAT, and pipette into a 96-well plate (2.5 x 10 7 cells / plate). The cells were cultured at 37°C and 5% CO2. When the clones in the fusion plate were large enough, 100 μL of supernatant was taken from each well and mixed with 2×10 5Jurkat cells were co-incubated with the cells and tested using the same method as for titer testing. Wells with an average immunofluorescence intensity greater than twice that of negative wells were designated as positive and proceeded to the next cloning stage. Hybridoma clones that screened positive were expanded from the 96-well plate to a 24-well plate for 3-5 days. The culture supernatant was screened again and tested. Positive clones were then subcloned, and the remaining cells were frozen. Hybridoma cells were harvested from the 24-well plate, counted, and adjusted to a cell density of 10 cells / mL. Cells were plated into a 96-well plate at 100 μL per well and incubated at 37°C in a 5% CO2 incubator. After approximately 10 days of culture, if colonies were visible, wells containing only a single colony were selected, and the supernatant was aspirated and tested as before. Positive clones were selected and expanded to a 24-well plate. After the supernatant was tested again, positive clones were selected for a second round of subcloning. Multiple rounds of subcloning were typically performed until all wells tested were positive, resulting in a stable hybridoma cell line. Positive hybridoma culture supernatants were selected and the antibody subtype was determined using an antibody subtype detection test strip. The monoclonal antibody of the present invention is numbered 2E7, is a murine IgG1 subtype, and has a kappa light chain. Protein sequencing revealed that the CDRH1-3 sequences of the antibody are shown in SEQ ID Nos. 1-3, the CDRL1-3 sequences are shown in SEQ ID Nos. 4-6, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 7, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 8. Nucleotide sequencing revealed that the polynucleotide sequence of the heavy chain variable region of the antibody is shown in SEQ ID No. 9, and the polynucleotide sequence of the light chain variable region is shown in SEQ ID No. 10.

[0106] Example 2: Ascites preparation and purification.

[0107] Wash the hybridoma cells with sterile PBS solution and use 5x10 6 Cells were injected intraperitoneally at a volume of 0.5 mL per mouse into Balb / c mice pre-sensitized with liquid paraffin. Seven to ten days later, ascites were collected and incubated at 3000 rpm for 10 minutes at room temperature, and the supernatant was collected. The antibody was crudely purified using saturated ammonium sulfate at a final concentration of 33%. This was accomplished by adding one part of ascites fluid to one part of PBS, then dropwise adding one part of saturated ammonium sulfate with stirring. The mixture was incubated at 4°C overnight, centrifuged at 10,000 rpm for 10 minutes, and the supernatant removed. The precipitate was dissolved with a small amount of PBS and dialyzed against PBS for 24 hours at 4°C, with three changes of buffer. The crude antibody was further purified using a 1 mL Protein G prepacked column using the AKTA protein purification system according to the GE purification manual. The resulting pure antibody was used for subsequent antibody detection and functional experiments.

[0108] Example 3: Monoclonal antibody titer detection.

[0109] PE directly labeled 2E7. The labeled antibodies were mixed with 2.5×10 5 SP2 / 0 cells (transfected with hPD-1) were incubated at room temperature for 30 minutes, protected from light. The cells were centrifuged at 1800 rpm for 10 minutes, the supernatant discarded, and the cells were washed three times with PBS. The cells were resuspended in 400 μL of PBS, and fluorescence intensity was measured by FACS. Mean values ​​were calculated. The Kd values ​​of the antibodies were calculated using GraphPad Prism 5 software. Figure 1 Antibody 2E7 and SP2 / 0 (hPD-1 + ) cell affinity constant analysis chart, the Kd value of antibody 2E7 is 0.9x10 -8 M.

[0110] Example 4: Cloning of Ig variable region genes by RT-PCR.

[0111] Total RNA extraction and single-stranded cDNA synthesis:

[0112] Total RNA from the 2E7 hybridoma cell line was extracted using the Trizol method (kit purchased from Invitrogen) and reverse transcriptase was used to convert the total RNA into a cDNA library using M-MLV reverse transcriptase (purchased from Invitrogen). The heavy chain backbone region upstream primer P1 was designed as shown in SEQ ID No: 11, the heavy chain variable region downstream primer P2 was designed as shown in SEQ ID No: 12, the light chain leader peptide upstream primer P3 was designed as shown in SEQ ID No: 13, and the light chain variable region downstream primer P4 was designed as shown in SEQ ID No: 12.

[0113] Prepare PCR reaction system (50 μL) as follows:

[0114] cDNA: 2 μL; upstream primer (10 μM): 2 μL; downstream primer (10 μM): 2 μL; dNTP mixture: 2 μL; pfu DNA polymerase (5 U / μL): 1 μL; 10X pfu Buffer II: 5 μL; ddH2O: make up to 50 μL. Reaction conditions: 95°C initial denaturation for 5 min; 35 cycles of 95°C for 30 s, 58°C for 30 s, 72°C for 1 min; and finally, extension at 72°C for 10 min. VL and VH fragments were separated and recovered by agarose gel electrophoresis. The recovered VL and VH fragments were ligated into the pMD19-T(simple) vector (Takara) using the following system: 70 ng each of VL PCR product and VH PCR product, 1 μL of pMD19-T(simple) vector, 5 μL of Solution I ligation reaction, and ddH2O to make up to 10 μL. Ligation was incubated overnight at 4°C. The ligation product was transformed into E. coli DH5α competent bacteria and cultured overnight at 37°C. A single colony was picked and shaken at 37°C for 2 hours before PCR identification of the bacterial solution. The cDNA of the corresponding antibody was used as a positive control. The reaction system (25 μL) was prepared as follows: bacterial solution: 1 μL, upstream primer (10 μM): 1 μL; downstream primer (10 μM): 1 μL; dNTP Mixture (2.5 μM each): 2 μL; Taq DNA polymerase (5 U / μL): 0.5 μL; 10× Taq Buffer (MgCl2) 2+ Plus) 2.5 μL; add water to 25 μL. Reaction conditions were the same as before. Select PCR-positive clones for expansion and culture. Use a plasmid extraction kit (Takara) to extract the plasmids of the positive clones and send them for sequencing. At least five clones of each chain of each antibody were sent for testing, and sequencing results of at least three samples were consistent. The heavy and light chain variable region sequences of antibody 2E7 were successfully cloned and conformed to the sequence characteristics of typical antibody variable regions.

[0115] Example 5: Cross-reactivity with PD-1 family member CTLA-4.

[0116] The antibody obtained in this example was mixed with 2×10 5 3T3 cells overexpressing human CTLA-4 were incubated with a commercial CTLA-4 antibody (purchased from BD) as a positive control. Incubate at 4°C for 1 hour, wash twice with PBS; resuspend the cells in 100 μL, add 1 μL of APC-labeled anti-mouse IgG antibody, and incubate at 4°C in the dark for 40 minutes; resuspend the cells in 500 μL of PBS buffer and analyze with FACS. Figure 2 , it can be seen that antibody 2E7 has no cross-reaction with human CTLA-4.

[0117] Example 6: Cross-reaction with human PD-1 negative cells.

[0118] The antibody obtained in this example was mixed with 2×10 5 Raji cells (PD-1 negative) were incubated with a commercial PD-1 antibody (purchased from BD) as a control. Incubate at 4°C for 1 hour, wash twice with PBS; resuspend the cells in 100 μL, add 2 μL of PE-labeled rat anti-mouse IgG1 antibody, and incubate at 4°C in the dark for 40 minutes; resuspend the cells in 500 μL PBS buffer and analyze with FACS. Figure 3 It can be seen that antibody 2E7 has no cross-reaction with Raji cells.

[0119] Example 7: Specific binding to Jurkat cells that highly express human PD-1.

[0120] FACS assay was used to detect the binding of the antibodies obtained in this example to the PD-1 protein on the surface of Jurkat cells: the antibodies were mixed with 1×10 6 Jurkat cells were incubated at room temperature for 40 minutes, washed twice with PBS, resuspended in 100 μL of cells, added with 2 μL of PE-labeled rat anti-mouse IgG1 antibody, and incubated at room temperature in the dark for 40 minutes; cells were resuspended in 500 μL of PBS buffer and detected by FACS. Figure 4 It can be seen that antibody 2E7 can effectively bind to Jurkat cells with strong affinity.

[0121] Example 8: FACS analysis of the competitive relationship between 2E7 and commercial anti-hPD-1 antibodies.

[0122] The antibody 2E7 obtained in this example was diluted with PBS to a final concentration of 100 nM, 10 nM, and 1 nM. At the same time, 2.5 μL of commercial PD-1 antibody (PE direct label, purchased from BD) was added to each well. After the antibody 2E7 at different dilutions was mixed with the commercial antibody, 2×10 5 Jurkat cells were incubated at room temperature in the dark for 30 minutes. Centrifuged at 1800 rpm for 10 minutes, discarded the supernatant, and washed with PBS. Repeat three times and resuspended in 400 μL of PBS for FACS analysis. Figure 5 It can be seen that antibody 2E7 can completely compete with commercial anti-hPD-1 antibodies.

[0123] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An isolated antibody or antigen-binding portion, characterized in that The antibody or antigen-binding portion specifically binds to human programmed death receptor 1 (PD-1) protein, and the antibody or antigen-binding portion comprises: Complementarity determining region (CDRH) of the heavy chain variable region: selected from at least one of the amino acid sequences shown in SEQ ID No: 1, SEQ ID No: 2 or SEQ ID No: 3; and / or Complementarity determining region (CDRL) of the light chain variable region: selected from at least one of the amino acid sequences shown in SEQ ID No: 4, SEQ ID No: 5 or SEQ ID No:

6.

2. The isolated antibody or antigen-binding portion according to claim 1, wherein The antibody or antigen-binding portion comprises: The heavy chain variable region CDRH1 shown in SEQ ID No: 1, the heavy chain variable region CDRH2 shown in SEQ ID No: 2, and the heavy chain variable region CDRH3 shown in SEQ ID No: 3; and / or The light chain variable region CDRL1 is shown in SEQ ID No: 4, the light chain variable region CDRL2 is shown in SEQ ID No: 5, and the light chain variable region CDRL3 is shown in SEQ ID No:

6.

3. The isolated antibody or antigen-binding portion according to claim 1 or 2, characterized in that The antibody or antigen-binding portion comprises: The heavy chain variable region as shown in SEQ ID No: 7, or an amino acid sequence having 90% or more identity thereto; and / or The light chain variable region shown in SEQ ID No: 8 or an amino acid sequence having more than 90% identity with the light chain variable region.

4. The antibody or antigen-binding portion according to any one of claims 1 to 3, wherein The antibody is a chimeric antibody or a humanized antibody.

5. The isolated antibody or antigen-binding portion according to any one of claims 1 to 3, wherein The antibody is one or more selected from IgG1, IgG2, IgG3 or IgG4; Preferably, the antibody is IgG1.

6. The antibody or antigen-binding portion according to any one of claims 1 to 3, wherein The antibody or antigen-binding portion is modified, The modification includes N-glycosylation modification, O-glycosylation modification, phosphorylation modification, methylation modification, acetylation modification or label modification.

7. The antibody or antigen-binding portion according to any one of claims 1 to 3, wherein The antigen binding portion is Fab, Fab', F(ab')2, Fd, FCL, dAb or single-chain antibody scFv.

8. A multivalent antibody, characterized in that The multivalent antibody comprises the antibody or antigen-binding portion according to any one of claims 1 to 7; Preferably, the multivalent antibody is a bispecific antibody or a trispecific antibody.

9. A multispecific antibody, characterized in that The multispecific antibody selectively binds to at least human PD-1 protein, and the multispecific antibody comprises the antibody or antigen-binding portion according to any one of claims 1 to 7; the multispecific antibody is a monovalent antibody or a multivalent antibody.

10. An isolated polynucleotide, characterized in that The polynucleotide encodes the antibody or antigen-binding portion of any one of claims 1 to 7, or encodes the multivalent antibody of claim 8, or encodes the multispecific antibody of claim 9.

11. The polynucleotide according to claim 10, wherein The polynucleotide sequence encoding the heavy chain variable region is shown in SEQ ID No: 9, and the polynucleotide sequence encoding the light chain variable region is shown in SEQ ID No:

10.

12. A carrier, characterized in that The vector comprises the polynucleotide according to claim 10 or 11.

13. A cell, characterized in that The cell comprises the antibody or antigen-binding portion of any one of claims 1 to 7, the multivalent antibody of claim 8, the multispecific antibody of claim 9, the polynucleotide of claim 10 or 11, or the vector of claim 12.

14. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the antibody or antigen-binding portion of any one of claims 1 to 7, the multivalent antibody of claim 8, the multispecific antibody of claim 9, the polynucleotide of claim 10 or 11, the vector of claim 12 or the cell of claim 13, and a pharmaceutically acceptable carrier; Preferably, the pharmaceutical composition further comprises other therapeutic drugs.

15. An immunoconjugate, characterized in that The immunoconjugate comprises: a) the antibody or antigen-binding portion of any one of claims 1 to 7, or the multivalent antibody of claim 8, or the multispecific antibody of claim 9; b) a therapeutic agent or a detectable marker; and c) a connector of parts a) and b) above; Wherein, the therapeutic agent includes a drug, an enzyme, a toxin, a cytokine or a radionuclide.

16. Use of the antibody or antigen-binding portion of any one of claims 1 to 7, the multivalent antibody of claim 8, the multispecific antibody of claim 9, the polynucleotide of claim 10 or 11, the vector of claim 12, the cell of claim 13, the pharmaceutical composition of claim 14, or the immunoconjugate of claim 15 in the preparation of a medicament for the treatment or adjuvant treatment of tumors, infectious diseases, or autoimmune diseases; Preferably, the tumor is breast cancer, neural tumor, melanoma, lung cancer, head and neck cancer, colorectal cancer, pancreatic cancer, gastric cancer, kidney cancer, bladder cancer, prostate cancer, ovarian cancer, cervical cancer, glioblastoma, esophageal cancer, renal cell carcinoma, endometrial cancer, skin cancer, testicular cancer, thyroid cancer or a hematological tumor.

17. Use of the antibody or antigen-binding portion of any one of claims 1 to 7, the multivalent antibody of claim 8, or the multispecific antibody of claim 9 in the preparation of a product for detecting the presence or level of human PD-1 molecules in a sample.

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