WO2023125289A1 - Anti-pd-1 antibody and uses thereof - Google Patents

Abstract

The present invention belongs to the field of biomedicine. Provided are an anti-PD-1 antibody or an antigen-binding fragment thereof, and a biological material, a composition, a conjugate, an antibody derivative and a kit of the antibody or the antigen-binding fragment thereof, as well as the uses thereof. The anti-PD-1 antibody may specifically bind to PD-1, block interactions between PD-1 and PD-L1 or PD-L2, activate an immune response, and has good developability.

Description

Anti-PD-1 antibodies and uses thereof technical field

The invention belongs to the field of biomedicine and relates to anti-PD-1 antibodies.

Background technique

The immune system can recognize and eliminate tumor cells, but immunosuppression in the tumor microenvironment limits the immune system's ability to eliminate tumors. The functions of various immune cells in the tumor microenvironment are inhibited, which mainly leads to the exhaustion of T cells that recognize tumor antigens, which is manifested by the reduction of the ability of T cells to proliferate and secrete cytokines. Studies have found that the high expression of immune checkpoint molecules such as PD-1 in T cells in the tumor microenvironment is related to its function reduction (Ahmadzadeh M, et al. Blood. 2009). PD-1 (programmed death receptor 1), also known as CD279, belongs to the immunoglobulin superfamily. After PD-1 on T cells binds to PD-L1 or PD-L2 on cells such as tumors or macrophages, PD-1 intracellular domain tyrosine residues are phosphorylated by tyrosine phosphokinases to recruit intracellular SHP2 to inhibit T cell activity (Sharpe AH, et al. Nat Rev Immunol. 2018). The interaction between PD-1 and PD-L1 or PD-L2 inhibits T cell activity in tumor immunity, which is an important mechanism for tumors to escape host immunity. Animal models and human clinical studies have found that blocking the interaction between PD-1 and PD-L1 or PD-L2 can activate the host's anti-tumor immune response and inhibit tumor growth. Blocking their interaction with anti-PD-1 or PD-L1 or PD-L2 specific antibodies can restore T cell function, including promoting the secretion of cytokines such as IFN-γ and IL-2 and the proliferation of T cells (Kalbasi A , et al. Nat Rev Immunol. 2020). Anti-PD-1 antibodies, such as nivolumab and pembrolizumab, have been tested in various clinical trials, have shown efficacy against a variety of tumors, and can activate the host's anti-tumor immune response (Sun L, et al. Sci Rep. 2020). Different anti-PD-1 or PD-L1/L2 antibodies used clinically may have different efficacy and toxicity in different types of tumors (Chen J, et al. Medicine (Baltimore). 2021; Liang J, et al. Transl Lung Cancer Res. 2020; Zhang W, et al. Oncologist. 2021). Due to differences in tumor types and individuals, the infiltration of T cells and the expression of PD-1/PD-L1/PD-L2 in the tumor microenvironment are different, the response rate of anti-PD-1 antibodies in tumor patients varies greatly, and the general response rate is lower Low, there is also natural and acquired resistance to anti-PD-1 antibodies (Sharma P, et al. Cell. 2017). Therefore, there is still a need to develop new antibodies that bind to human PD-1 and block its interaction with PD-L1 or PD-L2. Compared with clinically approved anti-PD-1 antibodies, anti-PD-1 antibodies with stronger T cell activation activity may translate into greater drug efficacy or lower required doses to benefit more patients .

Contents of the invention

In the present invention, anti-PD-1 antibodies with high affinity for both human and monkey PD-1 are screened by constructing a phage immune library, which can block the interaction between human PD-1 and PD-L1 or PD-L2, thereby providing more many choices.

In a first aspect, the present invention provides an anti-PD-1 antibody or an antigen-binding fragment thereof. In certain embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises: a heavy chain variable region (VH) and /or light chain variable region (VL).

In certain embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises the following complementarity determining regions (CDRs): (a) VH CDR1, VH CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1 or composed of Its composition, or its variant; VH CDR2, VH CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2 or consists of it, or its variant; VH CDR3, VH CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3 Or consist of it, or a variant thereof; and/or (b) VL CDR1, VL CDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 4, or a variant thereof; VL CDR2, VL CDR2 comprising SEQ ID The amino acid sequence shown in NO: 5 or consists of it, or a variant thereof; VL CDR3, VL CDR3 comprises the amino acid sequence shown in SEQ ID NO: 6 or consists of it, or a variant thereof.

In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3, the amino acid sequences of which are shown in SEQ ID NO: 1-6, respectively .

In some preferred embodiments, the antibody or antigen-binding fragment thereof is a chimeric or humanized antibody or antigen-binding fragment thereof.

In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 8, 15, 16, 17 or 18 or a variant thereof, or having at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the above sequence.

In some embodiments, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 9, 19, 20, 21, 22, 23, 24, 25 or 26 or a variant thereof, or has a At least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

In a preferred embodiment, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 8, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 9 Show.

In a preferred embodiment, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment is as shown in any one of SEQ ID NO: 15-18, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: ID NO: any one of 19-26.

In some embodiments, the antibody or antigen-binding fragment thereof may further comprise one or more of a heavy chain constant region, a light chain constant region, and an Fc region. In a further preferred embodiment, the light chain constant region is a lambda chain or kappa chain constant region. In some preferred embodiments, the heavy chain constant region is selected from IgG1, IgG2, IgG3 or IgG4 type.

In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region, the amino acid sequence of which is shown in SEQ ID NO: 27 (human IgG4 heavy chain constant region) or a variant thereof, or at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and/or,

The antibody or antigen-binding fragment thereof comprises a light chain constant region, the amino acid sequence of which is shown in SEQ ID NO: 28 (human IgG4 light chain constant region) or a variant thereof, or at least 90%, at least 95% identical to the above sequence , at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

In some embodiments, the antibody or antigen-binding fragment thereof includes, but is not limited to: Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, Fd fragment, Fd' fragment, single chain Fv (scFv) , a single chain Fab (scFab), and an epitope-binding fragment of any of the above.

In a preferred embodiment, the antibody or antigen-binding fragment thereof is a scFv, the amino acid sequence of which is shown in SEQ ID NO: 7 or a variant thereof, or at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

In a second aspect, the present invention provides a biological material selected from:

(1), a nucleic acid encoding the antibody or antigen-binding fragment thereof of the present invention;

(2), a vector, host cell or microorganism comprising (1);

(3) The expression product, suspension or supernatant of the above (2).

Those skilled in the art can easily select and prepare vectors, host cells or microorganisms containing the coding sequences of the antibodies according to the amino acid sequences of the antibodies, and know how to cultivate such host cells or microorganisms to obtain the corresponding expression products, suspension Solution, supernatant, etc., to obtain the corresponding antibody. These are all conventional technical means in this field.

In a preferred embodiment, the biological material is nucleic acid, which encodes the single-chain antibody of the present invention, and its nucleotide sequence is shown in SEQ ID NO:29.

In a preferred embodiment, the biological material is a nucleic acid encoding the heavy chain variable region or the light chain variable region of the antibody or antigen-binding fragment of the present invention; preferably, the nucleic acid sequence encoding the heavy chain variable region is such as Shown in SEQ ID NO:30, the nucleic acid sequence encoding the light chain variable region is shown in SEQ ID NO:31.

In a third aspect, the present invention provides a composition comprising the antibody or antigen-binding fragment thereof of the present invention; preferably, the composition is a pharmaceutical composition, further comprising a pharmaceutically acceptable carrier.

In a preferred embodiment, the dosage form of the pharmaceutical composition is oral, injection or transfusion, and the administration site of the pharmaceutical composition is parenteral, intravenous, mucosal, sublingual, intramuscular, intradermal , nasal cavity, intraperitoneal cavity or arterial or subcutaneous.

In the fourth aspect, there is provided a method for preparing the antibody or antigen-binding fragment thereof of the present invention, comprising: culturing the above-mentioned host cells to express the antibody or antigen-binding fragment, and isolating the antibody or antigen-binding fragment from the host cells.

In the fifth aspect, the application of the antibody or antigen-binding fragment thereof of the present invention or the biological material of the present invention or the composition of the present invention in the preparation of drugs for treating tumors, preventing or treating infections and infectious diseases is provided.

Preferably, the tumor is a tumor expressing PD-L1 or PD-L2. Further preferably, the tumor is prostate cancer, pancreatic adenocarcinoma, breast cancer, colorectal cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, thyroid cancer, glioblastoma, glioma, Leukemia, lymphoma and other malignancies.

Preferably, the infection or infectious disease is caused by viruses, bacterial or fungal parasites and other pathogens such as but not limited to human immunodeficiency virus, hepatitis A/B/C virus, Epstein-Barr virus, human Cytomegalovirus, human papillomavirus, herpesvirus.

In the sixth aspect, the application of the antibody or antigen-binding fragment thereof of the present invention or the biological material of the present invention or the composition of the present invention in the preparation of a preparation for binding to PD-1 is provided.

In the seventh aspect, there is provided the antibody or antigen-binding fragment thereof of the present invention or the biological material of the present invention or the composition of the present invention in the preparation of a preparation for blocking the binding of PD-1 to PD-L1 or PD-L2 application.

In an eighth aspect, the present invention provides a method of preventing and/or treating tumors, infections or autoimmune diseases in a subject, the method comprising administering to a subject in need thereof an effective amount of the present invention The antibody or its antigen-binding fragment, or the biological material of the present invention, or the composition of the present invention, etc.

In a ninth aspect, the present invention provides a conjugate comprising the antibody of the present invention or an antigen-binding fragment thereof and a coupling moiety, wherein the coupling moiety is another molecule; preferably, the coupling moiety is radioactive Isotopes, fluorescent substances, luminescent substances, colored substances or enzymes, etc.

In a tenth aspect, the present invention provides a kit comprising an antibody of the present invention or an antigen-binding fragment or conjugate thereof.

In an eleventh aspect, the present invention provides a method for detecting the presence or level of PD-1 in a sample, comprising the step of using the antibody of the present invention or an antigen-binding fragment or conjugate thereof.

In the twelfth aspect, the use of the antibody of the present invention or its antigen-binding fragment or conjugate in the preparation of a kit for detecting the presence or level of PD-1 in a sample is provided.

In the thirteenth aspect, there is provided an antibody or its antigen-binding fragment, biological material, composition or conjugate for use as a medicine or for prevention or treatment, which is the above-mentioned antibody or its antigen-binding fragment, biological material of the present invention , composition or conjugate.

The anti-PD-1 antibody of the present invention can specifically bind to PD-1, block the interaction between PD-1 and PD-L1 or PD-L2 to activate the immune response, and the antibody of the present invention can activate T cells in a mixed lymphatic reaction It is stronger than pembrolizumab, and its drug effect in animal models is slightly better than pembrolizumab, and it has a good prospect of becoming a drug.

Description of drawings

Figure 1: ELISA identification of the binding activity of phage library positive clones to PD-1; in the figure, the target represents the PD1-Fc antigen, irrelevant represents the SIRPA antigen, casein represents the blocking solution, HUFC represents the human IgG1Fc, and the vertical axis is the ELISA reading value, OD450nm Absorbance;

Figure 2: The binding curve between CQ1-hIgG4 and rhPD-1 detected by ELISA; the calculated EC ₅₀ of CQ1-hIgG4 antibody = 0.13 μg/ml;

Figure 3: The result of the chimeric antibody CQ1-hIgG4 inducing the secretion of IL-2 in mixed lymphocytes;

Figure 4: The results of chimeric antibody CQ1 and humanized antibody blocking PD-L1 binding to PD-1, A~E represent the results of different antibodies;

Figure 5: Chimeric antibody CQ1 and CQ1 humanized antibody block the binding of PD-L1 to PD-1 on 293T-PD-1 cells, A~E represent the results of antibody inhibition at different concentrations;

Figure 6: The results of chimeric antibody CQ1 and humanized antibody binding to PD-1 protein on the surface of 293T cells (average fluorescence intensity);

Figure 7: Humanized antibody CQ1-3+CQ1-11 blocks the binding of human PD-L2 to human PD-1;

Figure 8: The results of binding activity of humanized antibody CQ1-3+CQ1-11 to cynomolgus monkey PD-1;

Figure 9: Chimeric antibody CQ1 promotes SEB to stimulate PBMC T cells to secrete IL-2, A: PBMC donor Lot#A10Z707023, B: donor Lot#A10Z952082;

Figure 10: Chimeric antibody CQ1 and humanized antibody promote the secretion of IL-2 in MLR, A: DC donor Lot#LP200623, PBMC donor Lot#LP201019A; B: DC donor Lot#LP200623, PBMC donor Lot# LP200701;

Figure 11: Anti-PD-1 antibody inhibits the proliferation of MC38-huPDL1 in mice;

Figure 12: Effect of anti-PD-1 antibody treatment on mouse body weight in MC38-huPDL1 model.

Detailed ways

The content of the present invention will be described below in conjunction with specific embodiments. If not clearly stated, the reagents and instruments used in the following methods are all commonly used reagents and instruments in the art, and can be obtained commercially; the methods used are all conventional methods in the art, and those skilled in the art described according to the examples The content can carry out the described method without doubt and obtain the corresponding result.

definition:

In the context of the present invention, the term "antibody" refers to immunoglobulins and immunoglobulin fragments, whether native or partially or fully synthetically (e.g. recombinantly) produced, including those comprising at least part of the variable region of an immunoglobulin molecule that retains full length Any fragment of the binding specificity of an immunoglobulin. Thus, an antibody includes any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen binding domain (antibody combining site). Antibodies encompass a variety of antibody constructs including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, synthetic antibodies, recombinantly produced antibodies, human antibodies, non-human antibodies ( Such as murine antibody), humanized antibody, chimeric antibody, intrabody and antibody fragments, such as but not limited to Fab fragment, Fab' fragment, F(ab') ² fragment, Fv fragment, Fd fragment, Fd' fragment , single chain Fv (scFv), single chain Fab (scFab), or an antigen-binding fragment of any of the above antibodies. Antibodies provided herein include any immunoglobulin class (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgGI, IgG2, IgG3, IgG4, IgAl, and IgA2) or subclass (e.g., IgG2a and IgG2b) members. In preferred embodiments, the antibodies of the invention are murine antibodies, chimeric antibodies or humanized antibodies.

As used herein, an "antibody fragment" or "antigen-binding fragment" of an antibody refers to any portion of a full-length antibody that is less than full-length but includes at least a portion of the variable region (e.g., one or more CDRs and/or one or more antibody combining sites), and thus retain binding specificity and at least part of the specific binding ability of the full-length antibody. Thus, an antigen-binding fragment refers to an antibody fragment that comprises an antigen-binding portion that binds the same antigen as the antibody from which the antibody fragment is derived. Antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as derivatives produced synthetically, eg, recombinantly. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments and other fragments, including modified fragments (see, For example Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). The fragments may comprise multiple chains linked together, for example by disulfide bonds and/or by peptide linkers. Antibody fragments generally comprise at least or about 50 amino acids, and typically at least or about 200 amino acids. Antigen-binding fragments include any fragment of an antibody which, when inserted into an antibody framework (eg, by replacing the corresponding region), results in an antibody that immunospecifically binds an antigen.

As used herein, an "antibody" is generally an antibody comprising two heavy chains (which may be designated H and H') and two light chains (which may be designated L and L') and two antigen binding sites, wherein each The first heavy chain can be a full-length immunoglobulin heavy chain or any functional region thereof that retains antigen-binding ability (e.g., heavy chains include, but are not limited to, VH chains, VH-CH1 chains, and VH-CH1-CH2-CH3 chains), and each The light chain can be a full-length light chain or any functional region (eg, light chains include, but are not limited to, VL chains and VL-CL chains). Each heavy chain (H and H') is paired with one light chain (L and L', respectively).

As used herein, a full-length antibody is an antibody having two full-length heavy chains (e.g., VH-CH1-CH2-CH3) and two full-length light chains (VL-CL) and a hinge region, e.g., naturally produced by antibody-secreting B cells. produced antibodies as well as synthetically produced antibodies with the same domains.

As used herein, dsFv refers to a Fv with an engineered intermolecular disulfide bond that stabilizes the VH-VL pair.

As used herein, a Fab fragment is an antibody fragment obtained by papain digestion of a full-length immunoglobulin, or a fragment having the same structure produced synthetically, for example, by recombinant methods. The Fab fragment comprises a light chain (comprising VL and CL) and another chain comprising the variable domain (VH) of the heavy chain and one constant region domain (CH1) of the heavy chain.

As used herein, a F(ab')2 fragment is an antibody fragment obtained by pepsin digestion of an immunoglobulin at pH 4.0-4.5, or a fragment having the same structure produced synthetically, for example, by recombinant methods. An F(ab')2 fragment essentially consists of two Fab fragments, where each heavy chain portion contains an additional few amino acids, including a cysteine that forms a disulfide bond linking the two fragments.

As used herein, a Fab' fragment is a fragment comprising half (one heavy chain and one light chain) of an F(ab') ² fragment.

As used herein, a ScFv fragment refers to an antibody fragment comprising a light chain variable region (VL) and a heavy chain variable region (VH) covalently linked by a polypeptide linker in any order.

As used herein, a variable domain or variable region is a domain of an antibody heavy or light chain that recognizes and specifically binds an antigen, comprising amino acid sequences that vary from antibody to antibody. Each light chain and each heavy chain has one variable region domain, VL and VH, respectively. The variable domains provide antigen specificity and are thus responsible for antigen recognition. Each variable region comprises CDRs and framework regions (FRs), the CDRs being part of the antigen binding site domain.

As used herein, "antigen-binding domain" and "antigen-binding site" are used synonymously to refer to the domain within an antibody that recognizes and physically interacts with an alloantigen. A natural conventional full-length antibody molecule has two conventional antigen binding sites, each comprising a heavy chain variable region portion and a light chain variable region portion. The conventional antigen binding site comprises loops linking antiparallel beta strands within the variable region domains. The antigen binding site may comprise other portions of the variable region domain. Each conventional antigen binding site contains 3 hypervariable regions from the heavy chain and 3 hypervariable regions from the light chain. Hypervariable regions are also known as complementarity determining regions (CDRs).

As used herein, "hypervariable region", "complementarity determining region" and "CDR" are used interchangeably and refer to one of the portions within each variable region that together form the antigen binding site of an antibody. Each variable region domain contains 3 CDRs, named CDR1, CDR2 and CDR3. For example, the light chain variable region domain contains 3 CDRs, named VL CDR1, VL CDR2 and VL CDR3; the heavy chain variable region domain contains 3 CDRs, named H CDRI (or VH CDR1), H CDR2 ( or VH CDR2) and H CDR3 (or VH CDR3). The three CDRs in the variable region are not contiguous along the linear amino acid sequence, but are close together in the folded polypeptide. The CDRs are located within loops connecting the parallel strands of the beta sheets of the variable domains. As described herein, those skilled in the art know and can identify CDRs based on Kabat or Chotnia numbering (see, e.g., Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, AIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917).

As used herein, the framework regions (FRs) are the domains within the antibody variable region domains located within the beta sheet; the FR regions are relatively more conserved than the hypervariable regions in terms of amino acid sequence.

As used herein, a "constant region" domain is a domain in an antibody heavy or light chain that comprises a relatively more conserved amino acid sequence than that of a variable region domain. In a conventional full-length antibody molecule, each light chain has a single light chain constant region (CL) domain, and each heavy chain contains one or more heavy chain constant region (CH) domains, including but not limited to CH1, CH2 , CH3 and CH4. Antibody constant regions may serve effector functions such as, but not limited to, clearance of antigens, pathogens and toxins to which the antibody specifically binds, such as by interacting with various cells, biomolecules and tissues.

As used herein, a functional region of a VH domain is at least a portion of a complete VH domain that retains at least a portion of the binding specificity of the complete VH domain (e.g., by retaining one or more CDRs of the complete VH domain), such that the VH A functional region of a domain binds antigen alone or in combination with another antibody domain (eg a VL domain) or region thereof. Exemplary functional regions of the VH domain are regions comprising CDR1, CDR2 and/or CDR3 of the VH domain.

As used herein, a functional region of a VL domain is at least a portion of a complete VL domain that retains at least a portion of the binding specificity of the complete VL domain (e.g., by retaining one or more CDRs of the complete VL domain), such that the VL domain A functional region of a domain binds antigen alone or in combination with another antibody domain (eg a VH domain) or region thereof. Exemplary functional regions of the VL domain are regions comprising CDRI, CDR2 and/or CDR3 of the VL domain.

As used herein, the term "variant" refers to a substitution, deletion or addition of one or more amino acids, or any combination thereof, compared to its original sequence.

As used herein, the term "chimeric antibody" refers to an antibody whose light chain and/or part of the heavy chain is derived from an antibody (which may be derived from a specific species or belong to a specific class or subclass of antibodies), and another part of the light chain and/or heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains the target antigen binding activity.

As used herein, the term "humanized antibody" refers to a non-human antibody that has been modified by techniques such as genetic engineering, and its amino acid sequence has been modified to increase the sequence homology with the human antibody. Generally speaking, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (for example, variable region FR and/or constant region) is derived from a human Immunoglobulin (receptor antibody). Humanized antibodies generally retain the expected properties of the donor antibody, including but not limited to antigen specificity, affinity, ability to enhance immune response, etc. The donor antibody can be a mouse, rat, rabbit, or non-human primate (eg, cynomolgus monkey) antibody of the desired property.

As used herein, the terms "polynucleotide" and "nucleic acid molecule" refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including nucleotides linked together usually by phosphodiester bonds. Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

As used herein, an isolated nucleic acid molecule is a nucleic acid molecule that is separated from other nucleic acid molecules that exist in the natural source of the nucleic acid molecule. An "isolated" nucleic acid molecule, such as a cDNA molecule, may be substantially free of other cellular material or culture medium when prepared by recombinant techniques, or substantially free of chemical precursors or other chemical components when chemically synthesized. Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding provided antibodies or antigen-binding fragments.

Sequence "identity" has an art-recognized meaning, and the percent sequence identity between two nucleic acid or peptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the entire length of a polynucleotide or polypeptide, or along a region of the molecule. (See, for example: Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana PressS, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov , M. and Devereux, J., eds., M Stockton Press, New York, 1991). Although there are many methods of measuring the identity between two polynucleotides or polypeptides, the term "identity" is well known to those of skill (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988) ).

As used herein, "expression" refers to the process by which a polypeptide is produced by the transcription and translation of a polynucleotide. Expression levels of a polypeptide can be assessed by any method known in the art, including, for example, methods for determining the amount of polypeptide produced by a host cell. Such methods may include, but are not limited to, quantification of polypeptides in cell lysates by ELISA, Coomassie blue staining after gel electrophoresis, Lowry protein assay, and Bradford protein assay.

As used herein, a "host cell" is a cell for receiving, maintaining, replicating and amplifying a vector. Host cells can also be used to express polypeptides encoded by vectors. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. Host cells can be eukaryotic or prokaryotic. Suitable host cells include, but are not limited to, CHO cells, HeLa cells, HEK cells such as HEK 293 cells.

As used herein, a "vector" is a replicable nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into an appropriate host cell. Reference to vectors includes those into which a nucleic acid encoding a polypeptide or fragment thereof can be introduced, typically by restriction digestion and ligation. Reference to vectors also includes those vectors comprising a nucleic acid encoding a polypeptide. Vectors are used to introduce nucleic acids encoding polypeptides into host cells for amplifying nucleic acids or expressing/displaying polypeptides encoded by nucleic acids. Vectors usually remain episomal, but can be designed to allow integration of a gene, or part thereof, into the chromosome of the genome. Also contemplated are vectors for artificial chromosomes, such as yeast artificial vectors and mammalian artificial chromosomes. The selection and use of such vehicles are well known to those skilled in the art.

As used herein, "expression vector" includes a vector capable of expressing DNA operably linked to regulatory sequences, such as a promoter region, capable of affecting the expression of such DNA fragments. Such additional fragments may include promoter and terminator sequences, and optionally may include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus or other vector, which, when introduced into an appropriate host cell, results in the expression of cloned DNA. Appropriate expression vectors are well known to those skilled in the art and include expression vectors replicable in eukaryotic cells and/or prokaryotic cells as well as expression vectors that remain episomal or integrate into the host cell genome.

As used herein, "treating" an individual suffering from a disease or condition means that the individual's symptoms are partially or fully alleviated, or remain unchanged following treatment. Thus, treatment includes prophylaxis, treatment and/or cure. Prevention refers to preventing an underlying disease and/or preventing worsening of symptoms or development of a disease. Treatment also includes any pharmaceutical use of any provided antibodies or antigen-binding fragments thereof and compositions provided herein.

As used herein, "therapeutic effect" means the effect resulting from a treatment in an individual that alters, usually ameliorates or ameliorate the symptoms of, or cures a disease or condition.

As used herein, "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of a substance, compound, material, or composition comprising a compound that is at least sufficient to produce a therapeutic effect when administered to a subject. Thus, it is the amount necessary to prevent, cure, ameliorate, arrest or partially arrest the symptoms of a disease or disorder. Also, as used herein, a "prophylactically effective amount" or "prophylactically effective dose" refers to the amount of a substance, compound, material, or composition comprising a compound that, when administered to a subject, will have the desired prophylactic effect, e.g., prevent or delay disease occurrence or recurrence of disease or symptoms, and reduce the likelihood of occurrence or recurrence of disease or symptoms. A full prophylactically effective dose does not have to occur by administering one dose, and can only occur after administering a series of doses. Thus, a prophylactically effective amount can be administered in one or more administrations.

As used herein, the term "individual" refers to a mammal, such as a human.

As used herein, the term "acceptable carrier" includes: buffers, such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethyl chloride Benzylammonium; Hexamethylammonium Chloride; Benzalkonium Chloride, Benzethonium Chloride; Phenol, Butanol, or Benzyl Alcohol; Alkylparabens such as Methyl or Propylparaben esters; catechol; resorcinol; cyclohexanol; 3-propanol; and m-cresol); low molecular weight (fewer than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immune globulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, These include glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes such as Zn-protein complexes substances); and/or nonionic surfactants such as Tween, PluronicsTM or polyethylene glycol (PEG).

Example 1: Screening of murine antibodies

Using immunized mice and phage library to screen anti-PD-1 antibodies, the procedure is as follows: 3 mice of Bab/c strain (provided by Zhejiang University of Traditional Chinese Medicine) were immunized with human PD1-Fc (Novoprotein, catalog number: C754) as antigen. ELISA was used to detect the titer of immunized mice, and anti-PD-1 antibodies could be detected in all three mice. The mouse spleen RNA with positive titer was extracted and reverse-transcribed, and the antibody gene was amplified using cDNA as a template and electrotransformed to construct a phage antibody library (scFv antibody library). After three rounds of solid-phase screening, the positive identification of phage ELISA was carried out , select the sequences with the highest chromogenicity of 3 phages by ELISA for small-scale expression, and name them respectively No. 4, 7, and No. 10. Bacterial supernatants were identified by ELISA. At the same time, SIRPA antigen (Novoprotein, product number: C385), blocking solution casein: (Thermo Fisher Scientific, product number: 37528), HUFC (human IgG1Fc, from Novoprotein, product number: CX82) were used as controls. The results are shown in Figure 1, No. 10 The clone with the best binding activity excludes false positives and other non-specific binding.

Sequence analysis was performed on clone No. 10, its amino acid sequence is shown in SEQ ID NO: 7, the amino acid sequences of the three CDRs of the heavy chain and the three CDRs of the light chain are respectively shown in SEQ ID NO: 1-6; its nucleotide The acid sequence is shown in SEQ ID NO:29.

Embodiment 2: Preparation of chimeric antibody CQ1-hIgG4

According to the amino acid sequence of No. 10 clone, it was split into a heavy chain variable region and a light chain variable region, respectively fused with human IgG4, and the heavy chain constant region was a human immunoglobulin IgG4 heavy chain constant region (UniProtKB-P01861, IGHG4_HUMAN), the light chain constant region adopts the human immunoglobulin K constant region (UniProtKB-P01834, IGKC_HUMAN, SEQ ID NO: 28) to construct the chimeric antibody CQ1-hIgG4, wherein, the heavy chain variable region and the light chain variable region of CQ1-hIgG4 The amino acid sequences of the chain variable region are shown in SEQ ID NO: 8 and 9 respectively, and the amino acid sequences of the three CDRs of the heavy chain and the three CDRs of the light chain are shown in SEQ ID NO: 1-6 respectively; the variable heavy chain The nucleotide sequence of the region is shown in SEQ ID NO:30, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:31.

Example 3: Detection of affinity between CQ1-hIgG4 and human PD-1 by ELISA

The reagents used in this example are routine reagents for ELISA experiments, and the process is as follows:

(1) Coating plate: Dilute antibody CQ1-hIgG4 to 2 μg/mL with coating solution, add 100 μL to each well, coat the plate at 37°C for 90 minutes, and make a negative control (isotype control antibody NC, recombinant human IgG4 Fc, Novoprotein, FC03 );

(2) Plate washing: discard the liquid in the wells and pat dry the remaining liquid, add 260 μL of cleaning solution to each well, place on a decolorization shaker and shake and wash for 3 minutes, then discard the cleaning solution and pat dry; repeat this step three times;

(3) Blocking: Add 100 μL of blocking solution to each well, block overnight at 4°C, and wash the plate;

(4) Protein incubation: use diluent to dilute PD-1 protein (Nearshore Protein Technology Co., Ltd., product number: CX91) (20μg/mL starts with 5-fold gradient dilution and 11 gradients), incubate at 37°C for 1 hour, and make a negative control at the same time (PBS), wash plate;

(5) Secondary antibody incubation: HRP-Anti-His (Biolegend) was diluted at a packaging concentration of 1:20000, and the diluted secondary antibody was added to the corresponding well, 100 μL per well, and the plate was washed;

(6) Color development: add 100 μL of color development solution to each well, and incubate at 37°C for 10 minutes to develop color;

(7) Termination: Add 100 μL of stop solution to each well to stop the reaction.

The results are shown in Figure 2, CQ1-hIgG4 can bind human PD-1 in a dose-dependent manner.

Example 4: Mixed Lymphatic Reaction

Take the peripheral blood of 8 volunteers, separate PBMC with lymphocyte separation medium (GE), mix, count, spread 96-well plates according to 1×10 ⁵ cells/well; add 50 μg/ml and 10 μg/ml of CQ1- hIgG4 antibody, no antibody sample was used as blank control, after 48 hours of culture, the supernatant was taken; IL-2 content in the supernatant was detected with IL-2ELISA Kit (R&D). The results are shown in Figure 3, both 50 μg/ml and 10 μg/ml chimeric antibody CQ1-hIgG4 can increase the IL-2 secretion of mixed lymphocytes.

Example 5: Design and preparation of humanized antibodies

The variable region sequence of chimeric antibody CQ1-hIgG4 was analyzed, compared with the human germ line (germ line) amino acid sequence of IMGT, and IGKV3-11*01 (provided light chain FR1-FR3 framework region sequence) (SEQ ID NO: 12) or the framework region sequence of IGKV1-39*01 (providing light chain FR1-FR3 framework region sequence) (SEQ ID NO:13) and IGKJ4*02 (providing light chain FR4 framework region sequence) (SEQ ID NO:14) For the humanized framework sequence of the light chain, IGHV3-23*04 (providing the sequence of the heavy chain FR1-FR3 framework region) (SEQ ID NO: 10) and IGHJ1*01 (providing the sequence of the heavy chain FR4 framework region) (SEQ ID The framework region sequence of NO:11) is the humanized framework sequence of the heavy chain. Through CDR transplantation (CDR is determined by the KABAT coding method), the CDRs of the heavy chain and the light chain are respectively juxtaposed to the selected humanized framework sequence, and at the same time, reverse mutation design is carried out on the key sites on the framework region to obtain several humanized The variable region of the derivatized antibody, the specific sequence of the variable region is shown in SEQ ID NO: 15-26. The heavy and light chain variable regions of the humanized antibodies are shown in Table 1. The variable regions of the heavy and light chains are respectively linked with the human IgG4 heavy and light chain constant regions shown in SEQ ID NO: 27 and 28, and expressed and purified in the ExpiCHO expression system (Gibco, catalog number: A29129) to obtain humanized antibodies. At the same time, the variable regions of the chimeric antibody CQ1-hIgG4 were linked to the human IgG4 heavy and light chain constant regions shown in SEQ ID NO: 27 and 28, and expressed and purified in the ExpiCHO expression system (Gibco, catalog number: A29129) , to obtain the chimeric antibody CQ1. The chimeric antibody CQ1 and the humanized antibody were sequenced and detected, and were consistent with the designed sequence.

Table 1: Humanized antibody numbers and their corresponding heavy and light chain variable region sequences

Example 6: Detection of anti-PD-1 chimeric antibody CQ1 and humanized antibody blocking PD-L1 binding to PD1 by competitive ELISA

Coat high adsorption 96-well ELISA plate with 1 μg/mL human PD-1Fc protein (Acro, PD1-H5257), overnight at 4°C, block with 1% BSA-PBS at 37°C for 1 hour the next day, and then block with 1% BSA-PBS for 1 hour at 37°C. Prepare antibody concentration gradient dilution (300nM-0.05nM, 3-fold dilution) and 2μg/mL biotin-labeled PD-L1 protein (Acro, PD1-H82F3) in PBST diluent, and mix the antibody dilution with biotin-labeled PD- L1 protein was mixed at 1:1 (volume ratio), and the antibody protein mixture was added to the sealed 96-well ELISA plate at 100 μL per well, incubated at 37 °C for 1 hour, washed 4 times with PBST, and then injected at 100 μL per well. Add Strep-HRP (BD, 554066, 1:5000) to the microplate plate, incubate at 37°C for 1 hour, wash 4 times with PBST, develop color with TMB, detect the absorbance at 450nm and 630nm with a microplate reader, and calculate OD450-OD630 , using a four-parameter model for fitting, and calculating the IC50 value of each antibody (Table 2). Pembrolizumab (Merk & Co.) and hIgG4 (anti-HEL-human IgG4, Taizhou Baiying Biotechnology Co., Ltd., product number: B107804) were used as positive and negative independent photos.

As shown in Figure 4, both the chimeric antibody CQ1 and the humanized antibody can effectively block the binding of PD-L1 to human PD-1 Fc, and the blocking efficiency is dose-dependent. The IC ₅₀ values of chimeric antibody CQ1 and humanized antibody against PD-L1 (Table 2) are comparable to those of pembrolizumab.

Table 2: Anti-PD-1 chimeric antibody CQ1 and CQ1 humanized antibody block PDL1 binding to PD1 (IC ₅₀ , nM)

Antibody	IC 50 nM	Antibody	IC 50 nM	Antibody	IC 50 nM
pembrolizumab	2.941	pembrolizumab	3.594	pembrolizumab	2.956
CQ1	4.066	CQ1	3.305	CQ1	3.879
CQ1-2+CQ1-7	3.697	CQ1-3+CQ1-7	3.782	CQ1-4+CQ1-7	3.384
CQ1-2+CQ1-8	3.715	CQ1-3+CQ1-8	3.184	CQ1-4+CQ1-8	3.116
CQ1-2+CQ1-9	3.348	CQ1-3+CQ1-9	3.243	CQ1-4+CQ1-9	3.471
CQ1-2+CQ1-10	4.122	CQ1-3+CQ1-10	3.303	CQ1-4+CQ1-10	3.343
CQ1-2+CQ1-12	3.47	CQ1-3+CQ1-12	3.292	CQ1-4+CQ1-12	3.461
CQ1-2+CQ1-13	3.256	CQ1-3+CQ1-13	3.249	CQ1-4+CQ1-13	3.355
CQ1-2+CQ1-14	4.507	CQ1-3+CQ1-14	2.904	CQ1-4+CQ1-14	2.824
hIgG4	N/A	-	-	-	-
pembrolizumab	3.046	pembrolizumab	3.569	the	the
CQ1	2.925	CQ1	2.587	the	the
CQ1-5+CQ1-7	2.844	CQ1-2+CQ1-11	7.084	the	the
CQ1-5+CQ1-8	3.509	CQ1-3+CQ1-11	3.19	the	the
CQ1-5+CQ1-9	3.277	CQ1-4+CQ1-11	3.888	the	the

CQ1-5+CQ1-10	3.075	CQ1-5+CQ1-11	3.055	the	the
CQ1-5+CQ1-12	4.47	hIgG4	N/A	the	the
CQ1-5+CQ1-13	3.318	the	the	the	the
CQ1-5+CQ1-14	2.969	-	the	the	the

N/A: not applicable; - blank

Example 7: Detection of anti-PD-1 chimeric antibody CQ1 and humanized antibody blocking PD-L1 binding to PD-1 by flow cytometry

293T cells overexpressing human PD-1 (293T-PD-1, Kangyuan Biotech, KC-0204) were used to detect PD-1 chimeric antibody CQ1 and humanized antibody blocking cell surface antigens PD-1 and PD- Binding activity of L1. The obtained 293T-PD-1 cells were digested with trypsin (Gibco, 10091-148), washed twice with FACS buffer (PBS containing 1% FBS), and resuspended in FACS buffer at a cell concentration of 2×10 ⁶ cells/ ml. Add 50 μL of cells into a 96-well U-bottom plate. After centrifugation at 300g for 5 minutes, the supernatant was discarded. Add 50 μL of biotin-labeled human PD-L1 protein (1.2 μg/ml, Acro, Cat. No.: PD1-H82F3) diluted in FACS buffer, incubate at 4°C for 10 minutes, then add 50 μL of FACS buffer (1% FBS-PBS ) antibody in gradient dilution (400nM-0.009nM, 3-fold dilution). Mix well, incubate at 4°C for 1 hour, wash 3 times with FACS buffer, add PE streptomycin (1:700, BDbioscience, catalog number: 554061) at 50 μL/well, incubate at 4°C for 1 hour, and then wash with FACS buffer After three washes, 100 μL/well of FACS buffer was added to resuspend the cells, and the fluorescent signal was detected on a flow cytometer (BECKMAN COULTER cytoFLEX). According to the formula: inhibition rate = (fluorescence intensity when the antibody concentration is 0 - fluorescence intensity at each antibody concentration) / fluorescence intensity when the antibody concentration is 0 x 100%, calculate the anti-PD-1 antibody against PD-1 and PD- Inhibition rate of L1 binding. The inhibition rate was fitted using a four-parameter model, and the EC ₅₀ value of each antibody was calculated.

As shown in Figure 5, both the chimeric antibody CQ1 and the humanized antibody can effectively block the binding of PD-L1 to PD-1 on the surface of 293T-PD-1 cells, and the blocking efficiency is dose-dependent. The EC ₅₀ values (Table 3) of chimeric antibody CQ1 and humanized antibody blocking the binding of PD-1 and PD-L1 are comparable to those of pembrolizumab (Merk & Co.).

Table 3: Blocking rate of chimeric antibody CQ1 and humanized antibody on the binding of PD-L1 and PD-1 (EC ₅₀ )

N/A: not applicable

Example 8: Detection of binding activity of chimeric antibody CQ1 and humanized antibody to cell surface PD-1 antigen by flow cytometry

Using 293T cells overexpressing human PD-1 (293T-PD-1, Kangyuan Biotech, KC-0204) to detect PD-1 chimeric antibody CQ1 and humanized antibody with better blocking activity and cell surface antigen PD -1 binding activity. 293T-PD-1 cells were obtained by digestion with trypsin (Gibco, 10091-148), washed twice with FACS buffer (PBS containing 1% FBS), and resuspended in FACS buffer at a cell concentration of ¹¹⁰ cells/ml . Add 100 μL of cells into a 96-well U-bottom plate (1x10 ⁵ cells/well). After centrifugation at 300g for 5 minutes, the supernatant was discarded. Add 100 μL of antibodies diluted in series (400 nM-0.009 nM, 3-fold dilution) with FACS buffer (1% FBS-PBS). Mix well, incubate at 4°C for 60 minutes, wash 3 times with FACS buffer, add AF647 goat anti-hIgG (H+L) (1:1000, Jackson, catalog number: 109-605-003) at 100 μL/well, and incubate at 4°C After 60 minutes, wash 3 times with FACS buffer, add 100 μL/well of FACS buffer to resuspend the cells, and detect fluorescence signal on a flow cytometer (BECKMAN COULTER cytoFLEX). The inhibition rate was fitted using a four-parameter model, and the EC ₅₀ value of each antibody was calculated. In this experiment, pembrolizumab (Merk & Co.) was used as a positive control, and hIgG4 (anti-HEL-human IgG4, Taizhou Baiying Biotechnology Co., Ltd., catalog number: B107804) was used as a negative control.

As shown in Figure 6, both the anti-PD-1 chimeric antibody CQ1 and the humanized antibody can effectively bind to the PD-1 protein on the cell surface, and the binding efficiency is dose _- dependent. Monoclonal antibody (Merk & Co.) is comparable (Table 4).

Table 4: The binding activity of anti-PD-1 chimeric antibody CQ1 and humanized antibody to PD-1 on the surface of 293T-PD-1 cells (EC ₅₀ )

Antibody	EC 50 ,nM
pembrolizumab	5.05
CQ1	5.64
CQ1-2+CQ1-11	9.51
CQ1-3+CQ1-11	4.34
CQ1-4+CQ1-11	4.66
CQ1-5+CQ1-11	3.90
hIgG4	N/A

N/A: not applicable

Example 9: ELISA method to detect the blocking activity of humanized antibody CQ1-3+CQ1-11 on the binding of hPD-1 and hPD-L2

Coat a high-adsorption 96-well microtiter plate with 1 μg/mL human PD1-his (Acro, Cat. No. PD1-H5221) and leave overnight at 4°C. The next day, after blocking with 1% BSA-PBST at 37°C for 2 hours and washing the plate, use this solution to prepare a series of antibody concentration dilution gradients (400nM-0.007nM, 3-fold dilution) and 1.6μg/mL biotin labeling human PD-L2 protein (Acro, Cat. No. PD2-H82F6). Add 50 μL of antibody per well and 50 μL of biotinylated human PD-L2 protein per well to the microtiter plate, and incubate at 37°C for 1 hour. After machine-washing the enzyme-labeled plate 3 times with PBST solution, add Strep-HRP (BD pharmingen, Cat. No. 554066, diluted at 1:5000) to the plate at 100 μL per well, incubate at 37°C for 45 minutes, and machine-wash the enzyme-labeled plate 3 times with PBST After the plate, the color was developed with TMB, the absorbance value at 450nm was detected with a microplate reader, and the IC ₅₀ value of the antibody was calculated by fitting with a four-parameter model.

As shown in Figure 7 and Table 5, the humanized antibody CQ1-3+CQ1-11 can effectively block the binding of human PD-L2 to human PD-1, and the blocking efficiency is dose-dependent, with an IC ₅₀ of 0.41 nM.

Table 5: Blocking rate of humanized antibody CQ1-3+CQ1-11 on the binding of PD-L2 and PD-1 (IC ₅₀ )

Antibody	IC 50 ,nM
CQ1-3+CQ1-11	0.41
hIgG4	N/A

N/A: not applicable

Example 10: Detection of binding activity of humanized antibody CQ1-3+CQ1-11 to Cynomolgus Monkey PD-1 by ELISA

Cynomolgus monkey PD-1 (ACRO, cat: PD1-C5223) was added to the ELISA plate, coated at 37°C for 2 hours, washed 4 times with PBS, blocked with 1% BSA at 37°C for 1 hour, washed 4 times with PBS, respectively Add antibody pembrolizumab and CQ1-3+CQ1-11 (11 points of 4-fold dilution from 50nM, add 0 concentration point), incubate at 37°C for 1h, wash with PBS 4 times, add HRP-labeled goat anti-human IgG Fc secondary antibody (abcam, ab97225), incubated at 37°C for 1 h, washed 4 times with PBS, then used TMB (BD, 555214) for color reaction, and detected the absorbance at 450 nm and 630 nm wavelength in a microplate reader.

As shown in Figure 8 and Table 6, the humanized antibody CQ1-3+CQ1-11 can bind to cynomolgus monkey PD-1, and its binding efficiency is dose-dependent, and its EC ₅₀ value is comparable to that of pembrolizumab quite.

Table 6: Binding activity of humanized antibody CQ1-3+CQ1-11 to cynomolgus monkey PD-1.

Antibody	EC 50 ,pM
pembrolizumab	36.1
CQ1-3+CQ1-11	48.8

Example 11: Fortebio detects the affinity of anti-PD1 antibodies to human, mouse and non-human primate PD-1

AHC sensor (Sartorius, catalog number: 18-5060) was activated with 0.02% Tween-containing PBS (Gbico, REF: 10010-023) buffer for 10 minutes. Dilute anti-PD-1 antibody CQ1-3+CQ1-11 with buffer to a concentration of 5 μg/mL. Human PD-1 (ACRO, catalog number: PD1-H5221), cynomolgus monkey PD-1 (ACRO, catalog number: PD1-C5223) and mouse PD-1 (ACRO, catalog number: PD1-M5228) were used as analytes, with buffer The concentration of human PD-1 is 100, 50, 25, 12.5 and 6.25nM; the concentration of monkey and mouse PD-1 is 100, 50, 25 and 12.5nM. Antibody CQ1-3+CQ1-11 and different species of PD-1 proteins were added to corresponding plate wells at 200 μL/well. Fortebio program parameter settings: Loading: 180s; Association: 180s; Dissociation: 600s; Regeneration: 30s. Use Data Analysis11.1r software to analyze the experimental results. Humanized antibody CQ1-3+CQ1-11 does not bind to mouse PD-1, and there is no fitting curve.

As shown in Table 7, the affinities of CQ1-3+CQ1-11 to human and cynomolgus monkey PD-1 were 1.39 and 16.7nM, respectively, and CQ1-3+CQ1-11 did not bind to mouse PD-1.

Table 7: Affinity determination of anti-PD-1 antibodies to human, mouse and non-human primate PD-1

antigen	Antibody	KD(M)	kon(1/Ms)	kdis(1/s)	Full R^2
Monkey PD-1	CQ1-3+CQ1-11	1.67E-08	1.09E+05	1.81E-03	0.9967
hPD-1	CQ1-3+CQ1-11	1.39E-09	1.99E+05	2.76E-04	0.9986
mouse PD-1	CQ1-3+CQ1-11	-	-	-	-

-: No fitting curve, not applicable

Example 12: Chimeric antibody CQ1 enhances the activity of T cells secreting hIL-2 under SEB stimulation of PBMC

Superantigen staphylococcal enterotoxin B (SEB) can directly bind T cell receptors and MHC molecules without being processed into antigenic peptides to activate T cells. Anti-PD-1 antibodies can promote SEB-stimulated T cells to express and secrete IL-2 (Changyu Wang et al. Cancer Immunol Res. 2014). In this experiment, the effect of chimeric CQ1 on SEB-stimulated T cells to secrete IL-2 was detected. Quickly resuscitate PBMC (Miaoshun (Shanghai) Biotechnology Co., Ltd.) in a water bath at 37°C, using RPMI 1640+10% FBS (fetal bovine serum)+1% P/S (Pen-Strep, Gibco, catalog number: 15140122) Cells were resuspended in medium and centrifuged at 300 g for 10 minutes. After removing the supernatant, resuspended in medium and centrifuged again. According to the number of viable cells (counted by NucleoCounter NC-200, viability >90%), adjust the cell concentration of PBMC to 1×10 ⁶ cells/mL, and add SEB (Toxin technology, product number: 92815B) to the cell suspension so that the concentration is 400ng/mL. A volume of 100 μL was taken from the cell suspension mixed with SEB into a round-bottom 96-well plate (Corning, catalog number: 3799). Chimeric antibody CQ1 and control antibody (133.3-0.013nM, 10-fold dilution) were diluted into a series of different concentrations with RPMI 1640+10%FBS+1*P/S solution, and 100 μL was added to the corresponding wells. Place them in a CO ₂ incubator at 37°C for 3 days. After 3 days, the supernatant was taken, and the IL-2 secretion was detected (CLARIOstar Plus reading value) using a human IL-2 detection kit (purchased from CisBio, product number: 62HIL02PEH).

The results are shown in Figure 9. Compared with the control negative antibody hIgG4 (anti-HEL-human IgG4, Taizhou Baiying Biotechnology Co., Ltd., Cat. No.: B107804), the anti-PD-1 chimeric antibody CQ1 can increase the SEB-stimulated PBMC The expression of IL-2 is secreted, and its activity is comparable to that of pembrolizumab.

Example 13: Anti-PD1 antibody enhances the activity of T cells secreting hIL-2 in mixed lymphocyte reaction (MLR)

When dendritic cells (DCs) from different donors are incubated with PBMCs, DCs will activate PBMCs (mainly T cells) through their binding mode donor antigen peptide-donor MHC-recipient TCR, and promote IL- 2 expression and secretion. PD-1 on T cells will bind to PD-L1, which is highly expressed on mature DC, and reduce the expression of hIL-2 cytokines. Anti-PD-1 antibodies can promote the secretion of cytokines in the MLR response (Changyu Wang et al.Cancer Immunol Res. 2014). In this experiment, the effects of anti-PD-1 chimeric antibody CQ1 and humanized antibody on IL-2 secretion in MLR were detected. Quickly revive DC (Allcells) and PBMC (Allcells) in a water bath at 37°C, resuspend the cells in X-VIVO15 (purchased from Lonza) medium and centrifuge at 200g for 15 minutes, remove the supernatant, and reconstitute with the medium Suspend and centrifuge again. Adjust the cell concentration of DC and PBMC to 1x10 ⁵ /mL and 2x10 ⁶ /mL respectively according to the number of viable cells (counted by NucleoCounter NC-200, the viability rate is greater than 85%), and mix DC and PBMC at a volume ratio of 1:1 After mixing, 200 μL was added to a round bottom 96-well plate (Corning, Cat. No. 3799). Chimeric antibody CQ1, humanized antibody and control antibody (1000-0.32nM, 5-fold dilution) were diluted with X-VIVO15 solution to a series of different concentrations, and 50 μL was added to the corresponding wells, and the total volume of the wells was 250 μL. Place them in a CO ₂ incubator at 37°C for 4 days. After 4 days, the supernatant was taken, and the IL-2 secretion was detected (CLARIOstar Plus reading value) using a human IL-2 detection kit (purchased from CisBio).

The results are shown in FIG. 10 , the chimeric antibody CQ1 and the humanized antibody, compared with the control negative antibody, can increase the expression of DC-stimulated PBMC to secrete IL-2. The activity of chimeric antibody CQ1 and humanized antibody was superior to or comparable to that of positive antibody Pembrolizumab.

Example 14: Anti-PD-1 antibody inhibits the activity of MC38 proliferation in mice

The human PD-1 gene knock-in mouse C57BL/6-hPD1 was constructed by Shanghai Southern Model Biotechnology Co., Ltd., and the mice were subcutaneously inoculated with MC38-huPDL1 colorectal cancer cells (provided by Shanghai Southern Model Biotechnology Co., Ltd.) to establish MC38 - huPDL1 colorectal cancer model. On the 6th day after inoculation, the average volume of the tumor was about 82mm ³ , and the tumor-bearing mice were divided into 4 groups by random block method: including PBS group, CQ1-3+CQ1-7, CQ1-3+CQ1-11 and Paboli Zhuzumab group, 8 rats in each group. Each group was administered intraperitoneally, twice a week (10 mg/kg), and the tumor volume and body weight of the mice were measured twice a week. PBS group was given equal volume of PBS.

As shown in FIG. 11 , on the 14th day after the start of administration, the average tumor volume of the control PBS group was 1499.7±190.3 mm ³ . The tumor volumes of the CQ1-3+CQ1-7, CQ1-3+CQ1-11 and pembrolizumab groups were 399.7±200.8mm ³ , 421.7±165.0mm ³ and 443.1±130.3mm ³ respectively, and the tumor volume inhibition rate (TGI ) were 77.6%, 76.1% and 74.6% respectively. Compared with the tumor volume of the control group, there were significant statistical differences in the CQ1-3+CQ1-7, CQ1-3+CQ1-11 and pembrolizumab groups (P<0.01). As shown in Figure 12, the effects of CQ1-3+CQ1-7 and CQ1-3+CQ1-11 groups on the body weight changes of mice during the treatment were no different from the effects of pembrolizumab on the body weight changes of mice Significant differences.

Sequence information:

Claims (18)

1. An anti-PD-1 antibody or an antigen-binding fragment thereof, comprising the following complementary determining regions: (a) VH CDR1, VH CDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1, or a variant thereof; VH CDR2, VH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 2, or a variant thereof; VH CDR3, VH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 3, or a variant thereof; and /or (b) VL CDR1, VL CDR1 comprises the amino acid sequence shown in SEQ ID NO: 4 or consists of it, or a variant thereof; VL CDR2, VL CDR2 comprises the amino acid sequence shown in SEQ ID NO: 5 or consists of it Composition, or its variant; VL CDR3, VL CDR3 comprises the amino acid sequence shown in SEQ ID NO: 6 or consists of it, or its variant;

   Preferably, the antibody or antigen-binding fragment thereof includes VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3, the amino acid sequences of which are shown in SEQ ID NO: 1-6, respectively.
2. The antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region and/or a light chain variable region, wherein,

   The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 8, 15, 16, 17 or 18 or its variants, or at least 90%, at least 95%, at least 96%, at least 97% of the above sequence %, at least 98%, or at least 99% sequence identity; and/or

   The amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 9, 19, 20, 21, 22, 23, 24, 25 or 26 or variants thereof, or at least 90%, at least 95% of the above sequence %, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
3. The antibody or antigen-binding fragment thereof according to claim 2, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:8, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:9; or

   The amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NO: 15-18, and the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NO: 19-26.
4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, further comprising one or more of a heavy chain constant region, a light chain constant region, and an Fc region; preferably, the light chain constant region is the lambda chain or kappa chain constant region; the heavy chain constant region is selected from IgG1, IgG2, IgG3 or IgG4 type.
5. The antibody or antigen-binding fragment thereof according to claim 4, wherein,

   The amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO: 27 or a variant thereof, or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the above sequence consistency; and/or,

   The amino acid sequence of the light chain constant region is as shown in SEQ ID NO: 28 or its variants, or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence with the above sequence consistency.
6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, which is a Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, Fd fragment, Fd' fragment, single-chain Fv ( scFv), single-chain Fab (scFab) and any of the above-mentioned epitope-binding fragments; preferably, the antibody or its antigen-binding fragment is scFv, and its amino acid sequence is as shown in SEQ ID NO: 7 or a variant thereof, Or having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the above sequence.
7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, which is a chimeric antibody or antigen-binding fragment thereof, or a humanized antibody or antigen-binding fragment thereof.
8. A biological material selected from:

   (1) A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1-7;

   (2), a vector, host cell or microorganism comprising (1);

   (3), the expression product, suspension or supernatant of the above-mentioned (2);

   Preferably, the biological material is a nucleic acid encoding the heavy chain variable region or light chain variable region of the antibody or antigen-binding fragment according to any one of claims 1 to 7; more preferably, encoding the heavy chain variable region The nucleic acid sequence is shown in SEQ ID NO:30, and the nucleic acid sequence encoding the light chain variable region is shown in SEQ ID NO:31; further preferably, the nucleic acid encodes a single-chain antibody, and its nucleotide sequence is shown in SEQ ID NO:29.
9. A composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-7; preferably, the composition is a pharmaceutical composition, further comprising a pharmaceutically acceptable carrier; further preferably, the The dosage form of the pharmaceutical composition is oral, injection or transfusion, and the administration site of the pharmaceutical composition is parenteral, intravenous, mucosal, sublingual, intramuscular, intradermal, nasal cavity, abdominal cavity or intraarterial or subcutaneous.
10. The method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, comprising: culturing the host cell of claim 8 to express the antibody or antigen-binding fragment, and isolating the antibody or antigen from the host cell Combine fragments.
11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 or the biological material according to claim 8 or the composition according to claim 9 is used in the preparation of treating tumors, preventing or treating infections and infectious diseases application in medicine;

    Preferably, the tumor is a tumor expressing PD-L1 or PD-L2; further preferably, the tumor is prostate cancer, pancreatic adenocarcinoma, breast cancer, colorectal cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer , ovarian cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma and other malignancies;

    Preferably, the infection or infectious disease is caused by viruses, bacterial or fungal parasites and other pathogens, such as human immunodeficiency virus, hepatitis A/B/C virus, Epstein-Barr virus, human cytomegalovirus Viruses, human papillomavirus, herpesvirus.
12. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 or the biological material according to claim 8 or the composition according to claim 9 in the preparation of a preparation for binding to PD-1.
13. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 or the biological material according to claim 8 or the composition according to claim 9 is used in the preparation of blocking PD-1 and PD-L1 or PD - Use in L2-bound formulations.
14. A method for preventing and/or treating tumors, infections or autoimmune diseases in a subject, the method comprising administering an effective amount of the antibody according to any one of claims 1 to 7 to a subject in need or an antigen-binding fragment thereof or the biological material of claim 8 or the composition of claim 9.
15. A conjugate comprising the antibody or antigen-binding fragment thereof and a coupling moiety according to any one of claims 1 to 7, wherein the coupling moiety is another molecule; preferably, the coupling moiety is radioactive Isotopes, fluorescent substances, luminescent substances, colored substances or enzymes.
16. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-7 or the conjugate according to claim 15.
17. The method for detecting the presence or level of PD-1 in a sample comprises the step of using the antibody or antigen-binding fragment thereof according to any one of claims 1-7 or the conjugate according to claim 15.
18. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 or the conjugate of claim 15 in the preparation of a kit for detecting the presence of PD-1 in a sample or its level.

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