Pd-1 antibodies and uses thereof

Abstract

This disclosure provides a PD-1 antibody and its application. The PD-1 antibody or antigen-binding moiety provided in this disclosure has a high affinity for human PD-1 protein and can bind at a rate of 0.9 x 10⁻⁶. ‑8 The Kd value of M binds to human PD-1. Furthermore, the binding specificity is strong, and it does not cross-react with human CTLA-4 positive cells (3T3 cells overexpressing human CTLA-4) or human PD-1 negative cells (such as Raji cells). Based on these characteristics, the PD-1 antibody or antigen-binding moiety provided in this 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 applied in the preparation of drugs for treating tumors, infectious diseases, autoimmune diseases, and anti-immune rejection.

Description

Technical Field

[0001] This disclosure relates to the field of biomedicine, and in particular, to PD-1 antibodies and their applications. Background Technology

[0002] Immunotherapy, which utilizes immunotechnology principles 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 T cell activity. While it can prevent autoimmune diseases, it can also prevent the immune system from killing cancer cells; therefore, PD-1 has become a popular target for developing tumor immunotherapy drugs.

[0003] Currently, three PD-1 monoclonal antibody inhibitors are available globally: Merck's Keytruda, Bristol-Myers Squibb's Opdivo, and Sanofi / Regeneron's Libtayo. In recent years, two domestically developed PD-1 monoclonal antibody drugs—LOQTORZI (Junshi Biosciences) and TEVIMBRA (BeiGene)—have been approved for marketing in the United States. These drugs have indications spanning various solid tumors, including non-small cell lung cancer, melanoma, colorectal cancer, and nasopharyngeal carcinoma, and the scope of indications continues to expand. All of this demonstrates the significant clinical value of PD-1 antibodies.

[0004] Clinical trial data shows that some patients do not respond to PD-1 monoclonal antibody therapy, and each monoclonal antibody drug has varying degrees of side effects. Currently, the mechanism underlying the lack of response in most patients remains unclear, and new anti-human PD-1 antibodies are needed to provide new treatment strategies. Summary of the Invention

[0005] Technical problems to be solved:

[0006] One aspect of this disclosure addresses the lack of ideal anti-human PD-1 antibodies in the prior art by providing a PD-1 antibody. This antibody, or its antigen-binding portion, 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 moiety that specifically binds to human programmed death receptor 1 (PD-1) protein, the antibody or antigen-binding moiety comprising:

[0009] The 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

[0010] The complementarity-determining region (CDRL) of the light chain variable region is 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 include:

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

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

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

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

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

[0017] In this disclosure, having more than 90% identity means having 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 this disclosure, the antibody or antigen-binding portion can prevent PD-1 from binding to its ligands PD-L1 / PD-L2, thereby relieving the inhibition of T cells and restoring or enhancing T cell function.

[0019] In some embodiments, the antibody may be a mammalian-derived antibody, such as that from mice, rabbits, sheep, horses, monkeys, pigs, camels, sharks, chickens, 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 IgG. 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, including N-glycosylation, O-glycosylation, phosphorylation, methylation, acetylation, or labeling.

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

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

[0025] Another aspect of this disclosure is to provide a multivalent antibody comprising the antibody or antigen-binding portion described above.

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

[0027] Another aspect of this disclosure is to provide a multispecific antibody that selectively binds at least to human PD-1 protein, the multispecific antibody comprising the antibody or antigen-binding portion described above; the multispecific antibody is a monovalent antibody or a multivalent antibody.

[0028] Another aspect of this disclosure is to provide an isolated polynucleotide encoding the aforementioned antibody or antigen-binding moiety, or encoding the aforementioned multivalent antibody, or encoding the aforementioned 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 this disclosure is to provide a vector comprising the aforementioned polynucleotide.

[0030] Another aspect of this disclosure is to provide a cell comprising the aforementioned antibody or antigen-binding moiety, the aforementioned multivalent antibody, the aforementioned polynucleotide, or the aforementioned carrier. In some embodiments, the cell may 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 this disclosure is to provide a method for generating an anti-human PD-1 antibody or antigen-binding moiety, wherein the above-mentioned cells are subjected to protein expression to obtain the anti-human PD-1 antibody or antigen-binding moiety.

[0032] Another aspect of this disclosure is to provide a pharmaceutical composition comprising the aforementioned antibody or antigen-binding moiety, the aforementioned multivalent antibody, the aforementioned polynucleotide, the aforementioned carrier or the aforementioned cell, and a pharmaceutically acceptable carrier. In some embodiments, to achieve better therapeutic effects, the pharmaceutical composition may further comprise other therapeutic agents.

[0033] Another aspect of this disclosure is to provide an immunoconjugate comprising:

[0034] a) The antibody or antigen-binding moiety described above, or the multivalent antibody described above; and

[0035] b) Therapeutic agents or detectable markers; and

[0036] c) The connecting body between parts a) and b) above;

[0037] The therapeutic agents include drugs, enzymes, toxins, cytokines, or radionuclides.

[0038] Another aspect of this disclosure is the use of the aforementioned antibody or antigen-binding moiety, the aforementioned multivalent antibody, the aforementioned polynucleotide, the aforementioned carrier, the aforementioned cell, the aforementioned pharmaceutical composition, or the aforementioned immunoconjugate in the preparation of a medicament for the treatment or adjuvant treatment of tumors, infectious diseases, or autoimmune diseases. In some embodiments, by administering a certain dose of the aforementioned antibody or antigen-binding moiety, the aforementioned multivalent antibody, the aforementioned polynucleotide, the aforementioned carrier, the aforementioned cell, the aforementioned pharmaceutical composition, or the aforementioned immunoconjugate to a subject or patient, the effect of treating or adjuvant treatment of tumors, infectious diseases, or autoimmune diseases is achieved.

[0039] Furthermore, in some embodiments, the tumor may be breast cancer, neurotumor, 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 hematologic malignancies.

[0040] Another aspect of this disclosure is the use of the aforementioned antibody or antigen-binding moiety, the aforementioned multivalent antibody, and the aforementioned multispecific antibody in the preparation of products for detecting the presence or level of human PD-1 molecules in a sample. The detection of the presence or level of human PD-1 molecules in a sample can be achieved using any suitable method in the prior art, such as enzyme-linked immunosorbent assay (ELISA), Western blotting, etc.

[0041] Another aspect of this disclosure is a method for inhibiting the interaction between PD-1 and PD-L1 / PD-L2 in cells, the method comprising contacting cells with the aforementioned antibody or antigen binding moiety, the aforementioned multivalent antibody, the aforementioned polynucleotide, the aforementioned carrier, the aforementioned cells, the aforementioned pharmaceutical composition, or the aforementioned immunoconjugate. In some embodiments, the cells are human cells. In other embodiments, the cells are non-human cells. In some embodiments, the cells are contacted in vitro. In other embodiments, the cells are contacted in vivo.

[0042] Beneficial effects:

[0043] The mouse anti-human PD-1 antibody or antigen-binding moiety disclosed herein has a high affinity for human PD-1 protein and can bind at a rate of 0.9 x 10⁻⁶. -8The Kd value of M binds to human PD-1. Furthermore, the binding specificity is strong, and it does not cross-react with human CTLA-4 positive cells (3T3 cells overexpressing human CTLA-4) or human PD-1 negative cells (such as Raji cells). Based on these characteristics, the PD-1 antibody or antigen-binding moiety provided in this 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 applied in the preparation of drugs for treating tumors, infectious diseases, autoimmune diseases, and anti-immune rejection. Attached Figure Description

[0044] Figure 1 In the embodiments of this disclosure, antibody 2E7 and SP2 / 0 (hPD-1) + Affinity constant analysis diagram of cells;

[0045] Figure 2 The image shows the results of flow cytometry detection of cross-reactivity between antibody 2E7 and PD-1 family member CTLA-4 in this embodiment of the present disclosure. The cells used for detection are 3T3 cells overexpressing CTLA-4. The left image is the negative control, the middle image is the commercial CTLA-4 antibody and the positive control, and the right image is 2E7.

[0046] Figure 3 The image shows the cross-reactivity results of antibody 2E7 and Raji (human PD-1 negative cells) detected by flow cytometry in the embodiments of this disclosure. The left image is the negative control, the middle image is the commercial PD-1 antibody, and the right image is 2E7.

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

[0048] Figure 5 This figure shows the results of FACS assay in this embodiment 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.

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

[0050] Serial Number illustrate SEQ ID No:1 The amino acid sequence of the CDRH1 variable region of the 2E7 heavy chain in the examples SEQ ID No:2 The amino acid sequence of the CDRH2 variable region of the 2E7 heavy chain in the examples SEQ ID No:3 The amino acid sequence of the CDRH3 variable region of the 2E7 heavy chain in the examples SEQ ID No:4 The amino acid sequence of the CDRL1 variable region of the 2E7 light chain in the examples SEQ ID No:5 The amino acid sequence of the CDRL2 variable region of the 2E7 light chain in the examples SEQ ID No:6 The amino acid sequence of the CDRL3 variable region of the 2E7 light chain in the examples SEQ ID No:7 The amino acid sequence of the 2E7 heavy chain variable region in the examples SEQ ID No:8 The amino acid sequence of the 2E7 light chain variable region in the examples SEQ ID No:9 The nucleotide sequence of the 2E7 heavy chain variable region in the example SEQ ID No:10 The nucleotide sequence of the 2E7 light chain variable region in the example SEQ ID No:11 In the example, the upstream primer P1 for the RT-PCR heavy chain backbone region SEQ ID No:12 In this example, the downstream primer P2 for the RT-PCR heavy chain variable region SEQ ID No:13 In the examples, the upstream primer P3 for the RT-PCR light chain leader peptide was used. SEQ ID No:14 In the example, the downstream primer P4 for the RT-PCR light chain variable region Detailed Implementation

[0051] This disclosure provides a PD-1 antibody and its application. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. Furthermore, those skilled in the art can clearly modify or appropriately change and combine the content described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0052] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprising of," etc., shall be understood to include the stated elements or components without excluding other elements or other components. The term "a," "an," and "the" includes plural indicators. The term "a plurality of" means 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 this disclosure.

[0053] In this disclosure, when a range of values ​​is provided, it should be understood that, unless the context otherwise explicitly indicates otherwise, the range includes endpoints and each intermediate value between the upper and lower limits of the range, as well as any other specified value or intermediate value within the specified range and any value within a smaller range between specified values.

[0054] In this disclosure, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 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] In this disclosure, terms such as "one embodiment," "an example," "some embodiments," "a particular embodiment," "related embodiment," "a certain embodiment," "some embodiments," "additional embodiment," or "further embodiment," "further implementation," or "another embodiment," "some other embodiments," mean that at least one feature or characteristic description is included in relation to the embodiment. Therefore, throughout this disclosure, the above phrases do not necessarily refer to the same embodiment. Furthermore, specific features may be combined in any suitable manner in one or more embodiments.

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

[0057] Unless otherwise specified, the experimental techniques used in this paper employ standard techniques from immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which can be found in standard books such as *Molecular Cloning: A Laboratory Manual* and *Cell Biology: A Laboratory Handbook*.

[0058] definition:

[0059] The term "PD-1" (programmed death receptor-1) used in this disclosure is a key immunosuppressive receptor on the surface of T cells, whose core function is to act as a "brake" on the immune system. When PD-1 binds to its ligand (mainly PD-L1 on the surface of tumor cells or some normal cells), it transmits a strong inhibitory signal to T cells, leading to reduced T cell activity, decreased proliferation, and even apoptosis. Under normal circumstances, this mechanism helps maintain autoimmune tolerance and prevent excessive immune responses from damaging healthy tissues; however, tumor cells often overexpress 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 "separated" in this disclosure refers to a substance or entity that has been removed from its natural environment or the environment in which it existed prior to separation and is separate from other components. For example, a separated protein substantially does not originate from cellular material or other proteins derived from the cell or tissue from which it originates. The separation ratio 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%. Separated substances may have different levels of purity relative to the substances before their separation.

[0061] The term "antibody" in this disclosure refers to an immunoglobulin molecule 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 κ and λ 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 both the light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 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 regions of an antibody mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated 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 in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form the antibody-binding sites. The term "antibody" is not limited to any particular method of antibody production. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be different isotypes of antibody, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), 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 antibody type 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] Antibody preparation:

[0063] In some embodiments, the antibodies are generated using mammalian cells. For example, monoclonal antibodies are generated in mammalian cells using hybridoma technology. The monoclonal antibodies can be prepared using the hybridoma preparation method reported by Kohler et al. in Nature 256:495 (1975). Mice or other suitable host animals are first immunized with an immunogen (with adjuvants added if necessary).

[0064] Immunogens or adjuvants are typically administered via subcutaneous multi-site injection or intraperitoneal injection. Adjuvants can include Freund's adjuvant (complete or incomplete) or MPL-TDM, etc. After immunization, animals produce lymphocytes that secrete antibodies specifically binding to the immunogen. Target lymphocytes are collected and fused with myeloma cells using a suitable fusion agent (such as PEG4000) to obtain hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996).

[0065] The hybridoma cells prepared above are seeded into a suitable culture medium for growth, the medium containing one or more substances that can inhibit the growth of unfused maternal myeloma cells. For example, for maternal myeloma cells lacking hypoxanthine-guanine phosphotransferase (HGPRT or HPRT), the addition of substances such as hypoxanthine, aminopterin, and thymine (HAT medium) to the culture medium can inhibit the growth of HGPRT-deficient cells.

[0066] Preferred myeloma cells should possess high fusion rates, stable antibody secretion capabilities, and sensitivity to HAT culture medium. Among these, murine myeloma cells are preferred, such as the MOP-21 and MC-11 mouse tumor-derived lines (THE Salk Institute Cell Distribution Center, San Diego, Calif.USA), and SP-2 / 0 or X63-Ag8-653 cell lines (American Type Cell Collection, Rockville, Md.USA). Additionally, human monoclonal antibodies can be prepared using human myeloma and human-mouse heterologous myeloma cell lines (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 used for hybridoma cell growth is used to detect the production of monoclonal antibodies against specific antigens. The binding specificity of monoclonal antibodies produced by hybridoma cells can be determined using methods such as immunoprecipitation or in vitro binding assays, including radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). For example, the affinity of monoclonal antibodies can be determined using the Scatchard assay described by Munson et al. in Anal. Biochem. 107:220 (1980).

[0068] After determining the specificity, affinity, and reactivity of the antibodies produced by hybridomas, the target 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. Additionally, hybridoma cells can also grow in animals in the form of ascites tumors.

[0069] Traditional immunoglobulin purification methods, such as protein A agarose gel chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography, can be used to separate monoclonal antibodies secreted by subclonal cells from cell culture medium, ascites fluid, or serum, thereby obtaining the monoclonal antibodies.

[0070] In other embodiments, antibodies against human CD180 can also be generated by known recombinant methods, such as selecting a recombinant antibody library in a phage or similar vector, see, for example, the description in Smith GP. Filamentous fusionphage: novel expression vectors that display cloned antigens on the virionsurface. Science. 1985; 228:1315–17.

[0071] Antibody modification and alteration:

[0072] In some embodiments, the isolated antibody may be a humanized antibody. Humanization of the antibody may improve its affinity or other characteristics. A description and method for humanized 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 (crystallizable region fragment, Fc) is modified to enhance its effector functions triggered 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 modifications may include: 1) modifying glycosylation, for example, modifying the aspartic acid at position 297 (N297) in the Fc region with N-acetylglucosamine. Mutations of N297 to alanine (A), glutamine (Q), or glycine (G) all inhibit antibody glycosylation, thereby reducing Fc-mediated effector functions. Antibody deglycosylation reduces its ability to induce ADCC or CDC activity; sialic acid modification reduces its binding affinity to FcγRIIIa, thus leading to a decrease in CDC and ADCC activity.

[0074] In addition to the functions mentioned above, glycosylation modification of antibodies can also affect their conformation and stability. For example, the glycans in glycosylation can maintain the antibody's conformation, preventing aggregation or unfolding. For instance, the sugars on the a1-3 arm do not contact the antibody surface but are embedded deep within the space formed by the Fc segments of the two heavy chains. The mannose on the a1-3 arms of the two glycans interacts with each other, which is crucial for maintaining the antibody's conformation. Without the presence of glycans, the CH2 domain of the Fc segment would be slightly enlarged, leading to earlier elution times in size exclusion chromatography and increased sensitivity and aggregation in thermally accelerated stability experiments. Simultaneously, glycosylation modification can also affect the binding of antibodies to receptors on cell membranes, forming complexes that play 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, LALA mutations (L234A / L235A) can lead to changes in the antibody's affinity for FcγR (eliminating binding to low-affinity FcγR and reducing binding to FcγRI), thereby significantly reducing its ADCC and CDC activities. Additionally, combining trans-subtype antibodies can also modulate the antibody's effector function.

[0076] In some embodiments, the point mutation results in the substitution of some conserved amino acids, thereby obtaining a “conservative amino acid substitution variant”. The change results in some amino acids being substituted by others with similar chemical properties and / or functions. Providing conserved substitutions of amino acids with similar chemical properties and / or functions is well known in the art. Typical examples of mutually conserved 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 above modifications can also include: 3) Phosphorylation modification. Phosphorylation modification refers to the process of adding phosphate groups to amino acids of proteins within cells. Phosphorylation antibodies can specifically recognize specific phosphorylation sites, thereby detecting the increase or decrease in the phosphorylation level of proteins when cells are stimulated. These antibodies play an important role in life science research fields such as cell signaling, apoptosis, and cancer. 4) Methylation modification: Methylation modification is an important dynamic modification and biological phenomenon catalyzed by methyltransferases acting on specific residues of proteins. Methylation antibodies can specifically recognize specific methylated amino acid sites, used to distinguish between methylated and unmethylated forms of proteins. They have wide applications in research fields such as epigenetics, cancer, Alzheimer's disease, and aging. 5) Acetylation modification. Acetylation is one of the most common types of acylation modification. Acetylation antibodies can specifically recognize the acetylated form of target proteins and specific acetylated amino acid sites, detecting the activity level of the protein. These antibodies are widely used in research on cell cycle regulation, signal transduction, neurodegenerative diseases, metabolic diseases, and the occurrence and development of cancer.

[0078] The above modifications can also include: 6) Labeling. Antibodies can be cross-linked with different chemical reagents to attach to substances such as enzymes, fluorescent dyes, biotin, or colloidal gold, thereby altering their detection or analytical performance. For example, enzyme labeling: Antibodies can be cross-linked to enzymes such as horseradish peroxidase (HRP) and alkaline phosphatase. This is commonly used in immunohistochemistry, ELISA, and other experiments, where a color reaction is produced through enzyme catalysis, thus detecting the presence of the antibody. For example, HRP-labeled antibodies can produce a color precipitate after binding to an antigen by adding a substrate, facilitating observation and quantification. Fluorescent dye labeling: Antibodies can also be bound to fluorescent dyes (such as FTC, PE, APC, etc.) for use in flow cytometry, fluorescence microscopy, and other detection methods. Fluorescently labeled antibodies can locate specific antigens in cells or tissue sections, and the intensity of the fluorescence signal can be used to determine the antigen expression level. Biotin labeling: Biotin is a small molecule compound that can bind to antibodies without affecting their antigen-binding ability. Biotin-labeled antibodies can amplify and detect signals by binding to avidin (such as streptavidin). It is commonly used in multiplex immunolabeling assays to detect multiple antigens simultaneously.

[0079] In this disclosure, modifications and alterations to the antibody typically occur in the Fc region and the framework regions (FRs) of the antibody variable region, but not in the complementarity-determining regions (CDRs) of the antibody variable region. The framework regions of the antibody variable region have relatively conserved amino acid sequences, providing stable support for the hypervariable structure and participating in maintaining the three-dimensional conformation of the antigen-binding groove. Therefore, modifications and alterations to these regions do not affect the antibody's binding ability.

[0080] Separated antigen-binding portion:

[0081] The term "antigen-binding moiety" in this disclosure refers to a polypeptide fragment containing 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; it is also referred to as an "antigen-binding fragment." See Fundamental Immunology, Ch.7 (Paul, W., et al., 2nd ed.). Antigen-binding fragments of antibodies can be generated through recombinant DNA technology or through enzymatic or chemical fragmentation of intact antibodies. In some cases, antigen-binding fragments include Fab, Fab′, F(ab′)2, Fd, Fv, etc.

[0082] The term "Fab fragment" refers to an antibody fragment composed of VL, VH, CL, and CH1 domains; the term "F(ab′)2 fragment" refers to an antibody fragment containing two Fab fragments connected by a disulfide bridge on the hinge region; the term "Fd fragment" refers to an antibody fragment composed of VH and CH1 domains; and the term "Fv fragment" refers to an antibody fragment composed of the VL and VH domains of a single arm of the antibody.

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

[0084] Humanized antibodies:

[0085] Humanized antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof composed primarily of human sequences (e.g., Fab, Fab', F(ab')2, Fd, FCL, dAb, or single-chain antibodies scFv). Humanization of the antibody or antigen-binding portion can be performed according to methods in the prior art (see Samuel Ken-En Gan, et al. Sagacity in antibody humanization for therapeutics, diagnosis and research purposes: considerations of antibody elements and their roles, Antibody Therapeutics, DOI: 10.1093 / abt / tbaa004). Generally, humanized antibodies will contain substantially all or at least two variable domains, wherein all or substantially all CDR regions correspond to CDR regions of non-human immunoglobulins, and all or substantially all frame regions are regions of human immunoglobulin common sequence. Humanized antibodies preferably also contain at least a portion of the immunoglobulin constant region (Fc), typically the constant region of human immunoglobulins. For example, in some embodiments of this disclosure, a murine variable region (Vc) may be utilized. H (SEQ ID No: 7) / V LHumanized antibodies can be formed by using (SEQ ID No: 8) + human constant region. Humanized antibodies can also be formed by retaining only the mouse complementarity-determining region (CDR) and replacing the remaining frame regions (FR) with human sequences. Humanized antibodies can also be formed by replacing only the exposed residues on the surface of the mouse antibody while retaining the internal structural stability. Humanized antibodies can also be formed by directly obtaining the full 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 antibody or antigen-binding moieties as described in this disclosure, capable of competitively binding to human PD-1 molecules and producing 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 affinity and stability. This enhanced binding affinity helps improve the affinity of the anti-human PD-1 antibody for the antigen and enhances the interaction between the antigen and cell surface receptors or other molecules. The multivalent antibody can be obtained, for example, by protein fusion, the addition of linkers, covalent bonds, or non-covalent bonds.

[0088] The aforementioned multivalent and multispecific antibodies can be prepared using conventional techniques in the art. For the preparation of engineered antibodies, please refer to, 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 may also be used interchangeably with "nucleic acid," referring to a chain of nucleotides of any length, including DNA or RNA. It may include any known nucleotide analogs or modified nucleotides or bases.

[0090] The term "vehicle / vector" in this disclosure refers to a polynucleotide molecule capable of delivering and / or expressing one or more target genes. Examples of vectors include viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as generating cells.

[0091] Pharmaceutical composition:

[0092] The term "pharmaceutical composition" as used in this disclosure refers to a composition that, in addition to containing the antibody, the antigen-binding moiety, the multivalent antibody, the polynucleotide, the carrier, the cell, or the multispecific antibody described in this disclosure, also contains at least one other substance. In some embodiments, this other substance may be, for example, a pharmaceutically acceptable carrier (a substance 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), excipients, stabilizers, surfactants, preservatives, isotonic agents, etc. It may also be other therapeutic agents, such as chemotherapeutic drugs: melphalan, doxorubicin, cyclophosphamide, vincristine, etc.; glucocorticoids: 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, 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 above-described pharmaceutical composition may further include other PD-1 immunotherapeutic agents, such as pembrolizumab (…). Merck, nivolumab ( Bristol-Myers Squibb, toripalimab ( Junshi Biosciences), Camrelizumab (Hengrui Medicine), Tislelizumab (BeiGene), Sintilimab ( Innovent Biologics, Peanpulimab ( Sino Biopharmaceutical, cepalimab (Yuhang Biotechnology), Rosnilimab (AnaptysBio), GenSci120 (Jinsai Pharmaceutical), etc.

[0094] In some embodiments, the above pharmaceutical compositions can be prepared into any suitable formulation. For example, pills, tablets, creams, gelatin capsules, capsules, suppositories, soft gelatin capsules, gels, films, tubes, solutions, or suspensions. In some embodiments, the above pharmaceutical compositions can be administered via any suitable route of administration, such as intranasal, intrapulmonary, intrabronchial, intravenous, oral, intra-facial, intra-articular, intracranial, intradermal, intralesional, intramuscular, intraperitoneal, intrapleural, intracystic, local, mucosal, parenteral, intra-intestinal, subcutaneous, sublingual, local, buccal, transdermal, by inhalation, by injection, cream, lipid composition, via catheter, by irrigation, by continuous infusion, by infusion via local delivery or by local perfusion, etc.

[0095] Immunoconjugates:

[0096] In some embodiments, the immunoconjugates provided in this disclosure may be in any suitable form, such as antibody-drug conjugates (ADCs), radionuclide drug conjugates (RDCs), antibody fusion proteins, etc.

[0097] The aforementioned antibody-drug conjugates comprise the antibody, antigen-binding moiety, multivalent or multispecific antibody, linker, and payload described in this disclosure. Known linkers include, for example, N-succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylate (SMCC), hydrazone linkers, Val-Cit dipeptide, tetrapeptide Gly-Gly-Phe-Gly, glucuronic acid-containing linkers, β-galactosidase-containing linkers, etc. Known payloads include, for example, calendula extract, maytansin derivatives, tubulysins, cryptomycins (CR), pyrrolo[2,1-c][1,4]benzodiazepines (PBD), ducamycin, camptothecin (CPT), chachiin, apoptosis inducers, thailanstatin A, amatoxins, nicotinamide phosphoribosyltransferase, carbamycin, etc.

[0098] Indications:

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

[0100] Exemplary examples of tumors include breast cancer, neurotumor, melanoma, lung cancer, head and neck cancer, colorectal cancer, pancreatic cancer, stomach 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] Exemplary examples of autoimmune diseases include, for example, autoimmune hematologic disorders (including, for example, hemolytic anemia, aplastic anemia, simple erythrocytic anemia, and idiopathic thrombocytopenic purpura), 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 ophthalmopathy, Graves' disease, sarcoidosis, multiple sclerosis, primary biliary cirrhosis, type I diabetes, uveitis, keratoconjunctivitis sicca and vernal keratoconjunctivitis, interstitial pulmonary fibrosis, psoriatic arthritis, and glomerulonephritis.

[0102] Example:

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

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

[0105] Balb / c mice were immunized intraperitoneally using human PD-1 protein as an immunogen. Booster immunizations were administered at weeks 3 and 5 following the initial immunization. On day 8 post-booster immunization, tail blood was collected from the mice. After standing at room temperature for 1 hour, the serum was centrifuged at 12,000 rpm for 10 minutes at 4°C. The serum was then 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 counted. Each sample was treated with 1×10⁶ ppm. 6For each cell type, 100 μL of serum at different dilutions was added to the cells. The negative control group used serum from unimmunized mice instead of antiserum. Cells were incubated at 4°C for 1 hour and washed twice with PBS. 2 μL of PE-labeled rat anti-mouse IgG antibody was added, and the cells were 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 cells and fluorescence intensity in the serum were detected by flow cytometry. An effective titer was defined as an average fluorescence intensity more than twice that of the negative control, and fusion was only performed when the titer was higher than 6400. Three days before fusion, immunized mice were given a pulse immunization via tail vein injection of the immunogen. Spleen cells from successfully immunized mice were fused with myeloma SP2 / 0 cells at a ratio of 10:1. During fusion, 50% PEG was added to the mixed spleen and myeloma cell clusters (after discarding the supernatant) within 1 minute in a 37°C water bath. The mixture was shaken at 37°C for 1 minute, and then 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 96-well plates (2.5 x 10⁻⁶). 7 Cells / plate). Culture cells at 37°C and 5% CO2. When the colonies in the fusion plate are large enough, take 100 μL of supernatant from each well and mix with 2×10⁻⁶ cells / plate. 5Jurkat cells were co-incubated for detection using the same method and titer assay. Wells with an average immunofluorescence intensity more than twice that of negative wells were considered positive and proceeded to the next step of clonal culture. The selected positive hybridoma clones were expanded from 96-well plates to 24-well plates and cultured for 3-5 days. The culture supernatant was screened again, and positive clones were further subjected to subclonal culture. The remaining cells were cryopreserved. Hybridoma cells were collected from the 24-well plates, counted, and the cell density was adjusted to 10 cells / mL. Cells were seeded into 96-well plates at 100 μL per well and incubated at 37°C in a 5% CO2 incubator. After approximately 10 days of culture, clone formation was observed. Wells containing only a single clone were selected, and the culture supernatant was aspirated. The detection method was the same as before. Positive clones were selected, expanded to 24-well plates, and the supernatant was detected again. Positive clones were then selected for a second round of subclonal culture. Multiple rounds of subclonal culture were generally performed until all test wells were positive, thus obtaining a stable hybridoma cell line. The supernatant from positive hybridoma cultures was selected, and the antibody subtype was detected using antibody subtype detection strips. The monoclonal antibody in this invention is numbered 2E7, is a mouse IgG1 subtype, and has a κ light chain. Protein sequencing revealed the following amino acid sequences: CDRH1-3 as shown in SEQ ID No: 1-3, CDRL1-3 as shown in SEQ ID No: 4-6, the heavy chain variable region amino acid sequence as shown in SEQ ID No: 7, and the light chain variable region amino acid sequence as shown in SEQ ID No: 8. Nucleotide sequencing revealed the following polynucleotide sequences: the heavy chain variable region polynucleotide sequence as shown in SEQ ID No: 9, and the light chain variable region polynucleotide sequence as shown in SEQ ID No: 10.

[0106] Example 2: Ascites preparation and purification.

[0107] Hybridoma cells were washed with sterile PBS solution at 5 x 10⁻⁶ ppm. 6 0.5 mL of cells per mouse was injected intraperitoneally into Balb / c mice pre-sensitized with liquid paraffin. Ascites fluid was collected 7-10 days later. The supernatant was collected by centrifugation at 3000 rpm for 10 min at room temperature. The antibody was crudely purified with 33% saturated ammonium sulfate. The method involved adding one part PBS to one part ascites fluid, then adding one part saturated ammonium sulfate dropwise while stirring. The mixture was incubated overnight at 4°C, centrifuged at 10000 rpm for 10 min to remove the supernatant, and the precipitate was dissolved in a small amount of PBS. The precipitate was then dialyzed against PBS at 4°C for 24 h to remove salts, with three medium changes during this period. The crudely purified antibody was further purified using the AKTA protein purification system with a 1 mL Protein G pre-packed column, according to the purification manual provided by GE. The obtained purified antibody was used for subsequent antibody detection and functional experiments.

[0108] Example 3: Monoclonal antibody titer detection.

[0109] PE was directly labeled with 2E7. The labeled antibody was then mixed with 2.5 × 10⁻⁶ antibodies at final concentrations of 400 nM, 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.2 nM, 1.6 nM, 0.8 nM, 0.4 nM, 0.2 nM, 0.1 nM, 0.05 nM, 0.025 nM, 0.0125 nM, 0.0061 nM, and 0.003 nM. 5 SP2 / 0 cells (transfected with hPD-1) were incubated at room temperature for 30 min, protected from light. Centrifuged at 1800 rpm for 10 min, the supernatant was discarded, and cells were washed with PBS. This process was repeated three times. Cells were resuspended in 400 μL of PBS, and fluorescence intensity was measured using FACS. The mean value was calculated. The Kd value of the antibody was calculated using the data analysis software GraphPad Prism 5. Figure 1 For antibody 2E7 and SP2 / 0 (hPD-1) + Affinity constant analysis of cells; the Kd value of antibody 2E7 is 0.9 x 10⁻⁶. -8 M.

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

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

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

[0113] The PCR reaction mixture (50 μL) was prepared 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: bring to 50 μL. Reaction conditions: 95℃ pre-denaturation for 5 min; repeat the following cycles 35 times: 95℃ for 30 s, 58℃ for 30 s, 72℃ for 1 min; finally, 72℃ extension 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) as follows: 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 solution; ddH2O to 10 μL, ligation overnight at 4℃. The ligation product was transformed into *E. coli* DH5α competent cells and cultured overnight at 37°C. Single colonies were picked, and after shaking at 37°C for 2 hours, bacterial culture was performed for identification by PCR. cDNA containing the corresponding antibody was used as a positive control. The reaction mixture (25 μL) was prepared as follows: bacterial culture: 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 are the same as before. Select PCR-positive clones for amplification culture, extract plasmids from positive clones using a plasmid extraction kit (Takara), and send for sequencing. For each antibody, at least 5 clone samples should be sent for testing, and sequencing results should be obtained from at least three samples. Successful cloning yielded the heavy and light chain variable region sequences of antibody 2E7, which conform to the typical variable region sequence characteristics of antibodies.

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

[0116] The antibody obtained in this example was mixed with 2x10 at a final concentration of 100 nM. 5 3T3 cells overexpressing human CTLA-4 were incubated, with a commercial CTLA-4 antibody (purchased from BD) as a positive control. Cells were incubated at 4°C for 1 hour, washed twice with PBS, resuspended in 100 μL of APC-labeled anti-mouse IgG antibody, and incubated at 4°C in the dark for 40 minutes. Cells were then resuspended in 500 μL of PBS buffer and analyzed by FACS. Results are shown below. Figure 2 It can be seen that antibody 2E7 does not cross-react with human CTLA-4.

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

[0118] The antibody obtained in this example was mixed with 2x10 at a final concentration of 100 nM. 5 Raji cells (PD-1 negative) were incubated with a commercial PD-1 antibody (purchased from BD) as a control. Cells were incubated at 4°C for 1 hour, washed twice with PBS, resuspended in 100 μL of PBS, and incubated at 4°C in the dark for 40 minutes. Cells were then resuspended in 500 μL of PBS buffer and analyzed by FACS. Results are shown below. Figure 3 It is evident that antibody 2E7 does not cross-react with Raji cells.

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

[0120] FACS detection of the binding of the antibody obtained in this example to the PD-1 protein on the surface of Jurkat: the antibody was used at final concentrations of 100 nM, 10 nM, 1 nM, and 0.1 nM with 1 × 10 6 Jurkat cells were incubated at room temperature for 40 min, followed by two washes with PBS. Cells were resuspended in 100 μL of the solution, and 2 μL of PE-labeled rat anti-mouse IgG1 antibody was added. The cells were incubated at room temperature in the dark for 40 min. Cells were then resuspended in 500 μL of PBS buffer and analyzed by FACS. Results are shown below. Figure 4 It is evident that antibody 2E7 can effectively bind to Jurkat cells, demonstrating strong affinity.

[0121] Example 8: FACS method for detecting the competitive relationship between 2E7 and commercial anti-hPD-1 antibody.

[0122] The antibody 2E7 obtained in this example was diluted with PBS to final concentrations of 100 nM, 10 nM, and 1 nM. Simultaneously, 2.5 μL of commercial PD-1 antibody (PE direct label, purchased from BD) was added to each well. After mixing the antibody 2E7 at different dilutions with the commercial antibody, the solution was added to 2 × 10⁻⁶ ppm. 5 Jurkat cells were incubated at room temperature in the dark for 30 min. After centrifugation at 1800 rpm for 10 min, the supernatant was discarded, and the cells were washed with PBS. This process was repeated three times. The cells were then resuspended in 400 μL of PBS and analyzed by FACS. Results are shown below. Figure 5 It is evident that antibody 2E7 is in complete competition with commercial anti-hPD-1 antibodies.

[0123] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A separated antibody or antigen-binding moiety, characterized in that, The antibody or antigen-binding moiety specifically binds to human programmed death receptor 1 (PD-1) protein, and the antibody or antigen-binding moiety comprises: Complementary determinant regions (CDRHs) of heavy chain variable regions: heavy chain variable region CDRH1 as shown in SEQ ID No:1, heavy chain variable region CDRH2 as shown in SEQ ID No:2, and heavy chain variable region CDRH3 as shown in SEQ ID No:3; and Complementary determining regions (CDRLs) of light chain variable regions: light chain variable region CDRL1 as shown in SEQ ID No:4, light chain variable region CDRL2 as shown in SEQ ID No:5, and light chain variable region CDRL3 as shown in SEQ ID No:

6.

2. The isolated antibody or antigen-binding moiety according to claim 1, 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 more than 90% identity with the heavy chain variable region; and / or Such as 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.

3. The antibody or antigen-binding moiety according to claim 1 or 2, characterized in that, The antibody is a chimeric antibody or a humanized antibody.

4. The isolated antibody or antigen-binding moiety according to claim 1 or 2, characterized in that, The antibody is selected from one or more of IgG1, IgG2, IgG3 or IgG4.

5. The isolated antibody or antigen-binding moiety according to claim 4, characterized in that, The antibody is IgG1.

6. The antibody or antigen-binding moiety according to claim 1 or 2, characterized in that, The antigen-binding portion is Fab, Fab', F(ab')2, Fd, dAb, or a single-chain antibody scFv.

7. An isolated polynucleotide, characterized in that, The polynucleotide encodes the antibody or antigen-binding portion as described in any one of claims 1 to 6.

8. The polynucleotide according to claim 7, characterized in that, 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.

9. A carrier, characterized in that, The vector comprises the polynucleotide as described in claim 7 or 8.

10. A cell, characterized in that, The cells include an antibody or antigen-binding portion as described in any one of claims 1 to 6, a polynucleotide as described in claim 7 or 8, or a carrier as described in claim 9.

11. The use of the antibody or antigen-binding moiety as described in any one of claims 1 to 6 in the preparation of a product for detecting the presence or level of human PD-1 molecules in a sample.

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