PD-1 non-blocking clearing antibody and use thereof

Abstract

A PD-1 non-blocking antibody and a use thereof. The present invention relates to a clearing antibody, which specifically binds with high affinity to a PD-1, but does not block binding of the PD-1 and a ligand thereof, thereby achieving the effect of removing autoreactive and alloreactive T cells and B cells. Therefore, the clearing antibody is used for treating autoimmune diseases, rejection reaction of organ and cell transplantation, and inflammations.

Description

PD-1 non-blocking clearance antibodies and their uses Technical Field

[0001] The present invention belongs to the field of biomedicine and specifically relates to a PD-1 non-blocking clearance antibody and its uses. More specifically, the present invention relates to a clearance antibody that binds to PD-1 with high affinity but does not block the binding of PD-1 to its ligands. This antibody clears autoreactive and alloreactive T and B cells, and is used to treat autoimmune diseases, organ and cell transplant rejection, and inflammation. Background Art

[0002] Programmed cell death protein 1 (PD-1, also known as CD297) is a member of the CD28 receptor family. It is primarily expressed on activated CD4+ T cells, CD8+ T cells, and B cells, and its expression is primarily induced by T cell receptor (TCR) and B cell receptor (BCR) signals. The PD-1 molecule consists of an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain contains an immunoglobulin variable region (IgV) domain for ligand binding, while the intracellular domain contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM) to transmit inhibitory signals. PD-1 has two ligands: PD-L1 and PD-L2, both of which are B7 homologs and can bind to PD-1 but not to other CD28 family members. PD-L1 is widely expressed on activated T cells, B cells, macrophages, dendritic cells, and tumor cells, while PD-L2 expression is restricted, primarily on activated T cells and B cells and a small number of tumor cells. The immunosuppressive effect mediated by the binding of PD-1 to its ligand is an important mechanism of immune tolerance, tumor escape, and transplant tolerance. PD-1-deficient animals develop a variety of autoimmune diseases, including autoimmune cardiomyopathy and lupus-like syndrome. PD-1 signaling-mediated T cell dysfunction in tumors is the primary cause of tumor immune escape. Blocking PD-1 antibodies can block its binding to PD-L1 and PD-L2, reversing T cell dysfunction and significantly enhancing T cell activation and cytotoxicity, thereby achieving tumor immunotherapy. Currently, several PD-1 blocking antibody drugs have been approved for cancer treatment: nivolumab, pembrolizumab, cemiplimab, toripalimab, cindilimab, and camrelizumab. However, clinical use has shown that PD-1 blocking antibodies may induce and aggravate autoimmune diseases and organ transplant rejection.

[0003] Cellular and humoral immune responses mediated by reactive T cells and B cells are the main pathogenesis of autoimmune diseases and transplant rejection. Autoimmune diseases refer to diseases caused by the body's immune response to its own antigens, resulting in damage to its own tissues. Specifically, they refer to abnormal autoimmune tolerance of autoimmune cells, mainly T and B cells, leading to overactivation and organ-specific damage (such as chronic lymphocytic thyroiditis, hyperthyroidism, type 1 diabetes, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis, acute idiopathic polyneuritis) or systemic damage (multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, psoriasis, scleroderma, dermatomyositis, ulcerative colitis, etc.). Organ and cell transplant rejection is an immune response to foreign antigens. The primary antigen triggering this rejection is the major histocompatibility complex (MHC). Human MHC is known as HLA (human leukocyte antigen). Rejection primarily involves cellular immune responses mediated by activated T cells and humoral immune responses mediated by B cells, and is the primary cause of graft failure. In summary, autoimmune diseases and transplant rejection are primarily immune responses mediated by activated T and B cells. The specific mechanism is as follows: When the TCR of a T cell recognizes self-antigens or alloantigens, it becomes activated, highly expressing co-stimulatory and co-inhibitory molecules such as CD2, ICOS, and PD-1, and differentiating into effector T cells, including Th1, Th2, Th9, Th17, Th22, follicular T cells (Tfh), and cytotoxic T cells (Tc). These cells promote immune cell activation and aggravate the inflammatory response by secreting pro-inflammatory cytokines (IFN-γ, TNF-α, IL-2, IL-4, IL-17, etc.), assist B cells in activation and maturation to secrete specific antibodies, and induce cell death or apoptosis by secreting toxic proteins (such as perforin and granzymes) or expressing ligands (Fas-FasL). Immunosuppressants are currently the most important drugs for treating autoimmune diseases and suppressing rejection reactions.

[0004] The main strategy for the development of immunosuppressants is to inhibit and eliminate autoreactive T cells and B cells. In addition to small molecule compounds, the antibody drugs commonly used in clinical practice currently include: (1) Antithymocyte globulin (ATG) and antilymphocyte globulin (ALG): Both are polyclonal IgG components, prepared and collected from the serum of various animals (rabbits, horses, goats or pigs) that have been immunized against human thymocytes or lymphocytes. They are potent immunosuppressants. The mechanism of action is to inhibit the lymphocyte activation process after antigen recognition and, with the assistance of complement, produce a cytolytic effect on lymphocytes. (2) Anti-human CD3 antibody (Muromonab-CD3, trade name Orthoclone OKT3): It is an antibody targeting the CD3 complex of T lymphocytes, including chimeric antibodies and humanized antibodies. It can eliminate T cells and thus exert a strong immunosuppressive effect. (3) Anti-human CD25 (high affinity IL-2 receptor α chain) antibodies: including humanized CD25 monoclonal antibodies (daclizumab, xnapaxR) and human-mouse chimeric CD25 monoclonal antibodies (Basiliximab, simulect), which can specifically act on the activated T cell IL-2 receptor α chain, and inhibit T cell proliferation by competitively binding to the IL-2 receptor and antagonizing the binding of IL-2 to its receptor. However, ATG / ALG or OKT3 are rarely used in clinical practice because of their lack of specificity in target cells and large side effects. CD25 antibodies can only inhibit T cell proliferation, lack the effect of clearing specific T cells, and have the potential to affect the inhibitory T cells - regulatory T cells (Treg).

[0005] PD-1 is a molecule expressed by activated T and B cells. Currently available PD-1 antibodies are blocking antibodies and lack cytotoxicity. They competitively bind to PD-1, blocking its binding to PD-L1 and PD-L2, thereby blocking T cell negative regulatory signals and enhancing T cell immune function and anti-tumor effects. However, this can also induce and aggravate autoimmune diseases and transplant rejection. Clearing PD-1-positive cells may alleviate autoimmune diseases and transplant rejection. Therefore, there is an urgent need to develop PD-1-clearing antibodies in this field.

[0006] Summary of the Invention

[0007] To address the above-mentioned issues, the present invention provides a PD-1 non-blocking clearance antibody and its use. This novel PD-1 antibody, a high-affinity non-blocking clearance antibody against human PD-1, can bind to human PD-1 with high affinity and has no significant blocking effect on the binding of PD-1 to PD-L1 and PD-L2. The clearance antibody has good protective effects in both autoimmune disease models and heart transplant models of humanized PD-1 mice. The humanized Fc segment of the antibody is a human IgG1 with ADCC and CDC effects, which has a clearance effect on cells expressing PD-1.

[0008] Antibodies or antigen-binding fragments thereof that specifically bind to programmed death receptor-1 (PD-1)

[0009] In one aspect, the present invention provides a non-blocking clearance antibody or an antigen-binding fragment thereof that specifically binds to PD-1, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 1, 2 and 3, respectively, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 4, 5 and 6, respectively.

[0010] More specifically, the antibody or antigen-binding fragment thereof that specifically binds to PD-1 can be any one of a murine antibody, a chimeric antibody or a humanized antibody.

[0011] More specifically, the antibody or antigen-binding fragment thereof that specifically binds to PD-1 is a murine antibody comprising a light chain as set forth in SEQ ID NO: 7 and a heavy chain as set forth in SEQ ID NO: 9. This antibody is designated herein as murine antibody 3D6-mIgG2a. More specifically, the antibody or antigen-binding fragment thereof that specifically binds to PD-1 is a murine antibody comprising a light chain encoded by SEQ ID NO: 8 and a heavy chain encoded by SEQ ID NO: 10.

[0012] More specifically, the antibody or antigen-binding fragment thereof that specifically binds to PD-1 is a humanized antibody, and its Fc segment is the Fc segment of human IgG1 having ADCC and CDC effects.

[0013] More specifically, the antibody or antigen-binding fragment thereof that specifically binds to PD-1 is a humanized antibody comprising a light chain variable region as shown in SEQ ID NO: 11 and a heavy chain variable region as shown in SEQ ID NO: 13. More specifically, the antibody or antigen-binding fragment thereof that specifically binds to PD-1 is a humanized antibody comprising a light chain variable region encoded by SEQ ID NO: 12 and a heavy chain variable region encoded by SEQ ID NO: 14.

[0014] More specifically, the antibody or antigen-binding fragment thereof that specifically binds to PD-1 is a humanized antibody comprising a light chain as set forth in SEQ ID NO: 15 and a heavy chain as set forth in SEQ ID NO: 17. This antibody is designated herein as humanized antibody 3D6-hIgG1. More specifically, the antibody or antigen-binding fragment thereof that specifically binds to PD-1 is a humanized antibody comprising a light chain encoded by SEQ ID NO: 16 and a heavy chain encoded by SEQ ID NO: 18.

[0015] As used herein, the term "antibody" encompasses full-length antibodies (e.g., IgG1 or IgG4 antibodies), various functional fragments thereof (e.g., which may contain only the antigen-binding portion, such as Fab, F(ab')2, or scFv fragments), and modified antibodies (e.g., humanized, glycosylated, etc.).

[0016] As used herein, the term "monoclonal antibody" or "mAb" refers to an antibody produced from a single clonal cell line, which is not limited to eukaryotic, prokaryotic, or phage clones. Monoclonal antibodies or antigen-binding fragments can be recombinantly produced using hybridoma technology, recombinant technology, phage display technology, synthetic technology (e.g., CDR grafting), or other existing technologies.

[0017] As used herein, "antibody fragment" and "antigen-binding fragment" are used interchangeably and refer to antigen-binding fragments of antibodies and antibody analogs, which generally include at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of the parental antibody. Antibody fragments retain at least some of the binding specificity of the parental antibody. Typically, when activity is expressed on a molar basis, the antibody fragment retains at least 10% of the parental binding activity. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parental antibody for the target. Examples of antibody fragments include, but are not limited to: Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as ScFv, monobodies (technology from Genmab); nanobodies (technology from Domantis); domain antibodies (technology from Ablynx); and multispecific antibodies formed from antibody fragments. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0018] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. Antibodies contain three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system. For a given antibody, one skilled in the art will readily identify the CDRs defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art.

[0019] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its antigen. The strength or affinity of a specific binding interaction can be measured by the equilibrium dissociation constant (K) of the interaction. D ) indicates. In the present invention, the term "K D ” refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

[0020] As used herein, the term "murine antibody" in the present invention refers to a monoclonal antibody against human PD-1 prepared according to the knowledge and skill in the art. During preparation, a test subject is injected with the PD-1 antigen, and then a hybridoma expressing an antibody with the desired sequence or functional properties is isolated. In a preferred embodiment of the present invention, the murine PD-1 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a murine kappa or lambda chain, or variants thereof, or a heavy chain constant region of a murine IgG1, IgG2, IgG3, or IgG4, or variants thereof.

[0021] As used herein, the term "chimeric antibody" refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody. To create a chimeric antibody, one must first establish a hybridoma that secretes a murine-specific monoclonal antibody. Then, the variable region gene is cloned from the mouse hybridoma cell. The constant region gene of the human antibody is cloned as needed. The mouse variable region gene and the human constant region gene are connected to form a chimeric gene, which is then inserted into a vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic expression system or a prokaryotic expression system. In a preferred embodiment of the present invention, the antibody light chain variable region of the PD-1 chimeric antibody further comprises a light chain FR region of a murine lambda chain or a variant thereof. The antibody heavy chain variable region of the PD-1 chimeric antibody further comprises a heavy chain FR region of murine IgG1, IgG2, IgG3, or IgG4, or a variant thereof. The constant region of the human antibody can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or their variants, preferably the human IgG1 heavy chain constant region with ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity.

[0022] As used herein, the term "humanized antibody (antibody)" refers to a "humanized" form of a non-human (e.g., mouse) antibody that is a chimeric antibody containing minimal sequences derived from non-human immunoglobulin. The majority of humanized antibodies are human immunoglobulins, in which the residues in the hypervariable region of the antibody are replaced by residues from the hypervariable region of a non-human species having the desired specificity and affinity. In some cases, residues in the Fv framework region of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may contain residues that are not present in the recipient antibody or the donor antibody. These modifications are performed to further improve antibody performance.

[0023] It should be understood that the present invention encompasses variants of the PD-1 specific antibodies or antigen-binding fragments thereof as described herein, which have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the amino acid sequence of the PD-1 specific antibodies or antigen-binding fragments thereof as described herein, and substantially retain the biological function of the antibody from which it is derived (e.g., biological activity of specific binding to PD-1).

[0024] More specifically, the variant differs from the PD-1 specific antibody or antigen-binding fragment thereof as described herein only in conservative substitutions of one or more (e.g., conservative substitutions of up to 20, up to 15, up to 10, up to 5, or up to 1 amino acid) amino acid residues.

[0025] As used herein, the term "identity" refers to the match between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions match). Typically, two sequences are compared when they are aligned for maximum identity. Such an alignment can be achieved, for example, by using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol. Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0026] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions of residues physically or functionally similar to corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0027] polynucleotides

[0028] In another aspect, the present invention also relates to a polynucleotide encoding the antibody or antigen-binding fragment thereof as described herein.

[0029] The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand.

[0030] The term "polynucleotide encoding a polypeptide / protein / antibody" may include a polynucleotide encoding the polypeptide / protein / antibody, or may also include additional coding and / or non-coding sequences.

[0031] The present invention also relates to polynucleotides that hybridize to the polynucleotide sequences described herein and have at least 50%, preferably at least 70%, more preferably at least 80%, and most preferably at least 90% identity between the two sequences, and the polypeptides / proteins / antibodies they encode have substantially the same functions and activities. The present invention particularly relates to polynucleotides that can hybridize to the polynucleotides described herein under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, more preferably at least 95%.

[0032] The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can generally be obtained by PCR amplification, recombination, or synthetic methods. One feasible method is to synthesize the relevant sequence by synthetic methods, especially when the fragment length is relatively short. Generally, very long fragments can be obtained by synthesizing multiple small fragments and then ligating them.

[0033] carrier

[0034] In another aspect, the present invention also relates to a vector comprising the polynucleotide as described herein.

[0035] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector may also contain an origin of replication.

[0036] host cells

[0037] In another aspect, the present invention also relates to a host cell comprising the vector as described herein.

[0038] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or other human cells. A host cell may include a single cell or a cell population.

[0039] Vectors can be introduced into host cells using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCl2, using procedures well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0040] Conjugate

[0041] In another aspect, the present invention also relates to a conjugate comprising the antibody or antigen-binding fragment thereof as described herein and a coupling moiety.

[0042] The conjugated moiety is selected from a detectable label, a drug, a toxin, a cytokine, a radionuclide or an enzyme.

[0043] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention are conjugated to the coupling moiety, optionally via a linker.

[0044] In certain embodiments, the coupling moiety is selected from a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent material (e.g., a chemiluminescent material), or biotin. The detectable label of the present invention can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics, or chemical means. Such labels are well known in the art, and examples thereof include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa750)), luminescent substances (e.g., chemiluminescent substances such as acridinium ester compounds), magnetic beads, calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above-mentioned labels. In certain embodiments, such labels can be suitable for use in immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.). In certain embodiments, the detectable labels described above can be linked to the antibodies or antigen-binding fragments thereof of the present invention via linkers of varying lengths to reduce potential steric hindrance.

[0045] In certain embodiments, the conjugated moiety is selected from a drug, such as an anti-inflammatory drug or an immunosuppressant.

[0046] In certain embodiments, the conjugation moiety is selected from additional biologically active polypeptides.

[0047] Pharmaceutical composition

[0048] In another aspect, the present invention also relates to a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described herein and a pharmaceutically acceptable carrier, diluent or excipient.

[0049] In certain embodiments, the pharmaceutical composition may further comprise a second therapeutic agent. The second therapeutic agent is any agent that is advantageously combined with the PD-1 antibody. In some embodiments, the second therapeutic agent is another anti-inflammatory drug or an immunosuppressant.

[0050] In certain embodiments, in the pharmaceutical composition, the antibody or antigen-binding fragment thereof as described herein and the second therapeutic agent can be provided as separate components or as mixed components. Thus, the antibody or antigen-binding fragment thereof as described herein and the second therapeutic agent can be administered simultaneously, separately or sequentially.

[0051] In some embodiments, the pharmaceutically acceptable carrier and / or excipient may comprise a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffered solution (e.g., phosphate buffered solution), Ringer's solution, and any combination thereof.

[0052] The pharmaceutical compositions of the present invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody or antigen-binding fragment thereof as described herein and a second therapeutic agent. A "prophylactically effective amount" refers to an amount sufficient to prevent, arrest, or delay the onset of a disease. A "therapeutically effective amount" refers to an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount may vary depending on factors such as the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight, and sex, the mode of administration of the drug, and other concurrently administered therapies.

[0053] Therapeutic uses

[0054] In another aspect, the present invention also relates to the use of the antibody or antigen-binding fragment thereof, polynucleotide, vector, host cell, conjugate or pharmaceutical composition as described herein in the preparation of a medicament for depleting autoreactive and alloreactive T cells and B cells.

[0055] In another aspect, the present invention also relates to the use of the antibody or antigen-binding fragment thereof, polynucleotide, vector, host cell, conjugate or pharmaceutical composition as described herein in the preparation of a medicament for treating autoimmune diseases, organ or cell transplant rejection and / or inflammation.

[0056] Autoreactive T cells

[0057] As used herein, autoreactive T cells refer to T cells that attack one's own tissues, cells, and organs. Sometimes the body's immune system fails to recognize one or more of its own components as "self" and produces autoantibodies and autoreactive T cells, which attack its own cells, tissues, and / or organs (mistaking its own normal tissue cells for foreign antigens). This leads to inflammation and damage, causing autoimmune diseases.

[0058] Alloreactive T cells

[0059] As used herein, alloreactive T cells are key cells mediating transplant rejection. Differences in MHC molecules exist between allogeneic donors and recipients, and lymphocytes from both donors and recipients directly recognize the heterotypic MHC molecules on the surface of the other's lymphocytes, leading to their proliferation and activation. Distinct from responses to general antigens, rejection reactions resulting from direct recognition have two characteristics: 1) rapidity, as they require no antigen uptake, processing, and manipulation; and 2) high intensity, as in each individual, T cell clones reactive to alloantigens comprise 1%-10% of the total T cell pool, while T cell clones targeting general heterologous antigens comprise only 1 / 100,000-1 / 10,000 of the total.

[0060] Organ transplant rejection

[0061] The antibodies or antibody fragments of the present invention can be used to treat organ transplant rejection, which occurs after an allogeneic tissue or organ transplant in a recipient, when the recipient's immune system recognizes the foreign tissue or organ as a "foreign component," which then initiates an immunological response to attack, destroy, and eliminate the transplant. The antibodies or conjugates of the present invention can be used alone or in combination with other immunosuppressants to treat organ transplant rejection, including, but not limited to, heart transplants, kidney transplants, lung transplants, liver transplants, pancreas and islet transplants, parathyroid transplants, combined heart-lung transplants, bone marrow transplants, corneal transplants, small bowel transplants, hand transplants, and various tissue types including skin, fat, fascia, tendon, dura mater, blood vessels, lymphatic vessels, cartilage, and bone transplants.

[0062] Cell transplant rejection

[0063] Cell therapy involves the transplantation or infusion of normal or bioengineered human cells into a patient's body. The newly infused cells can replace damaged cells or enhance immune-killing capabilities, thereby treating the disease. Cell therapy can be categorized into stem cell therapy and immune cell therapy based on the cell type. The antibodies or conjugates of the present invention can be used alone or in combination with other immunosuppressants to treat cell transplant rejection.

[0064] Autoimmune diseases

[0065] The antibodies or antibody fragments of the present invention can be used to treat autoimmune diseases (AID), which are diseases in which immune tolerance is broken under certain conditions and the immune system produces a pathological immune response to self-antigens, causing damage to self-tissue cells or dysfunction. The antibodies or conjugates of the present invention can be used alone or in combination with other immunosuppressants to treat autoimmune diseases, such as, but not limited to, some examples of these, including: immune-mediated thrombocytopenia, such as acute idiopathic thrombocytopenic purpura and chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea, lupus nephritis, rheumatic fever, polyendocrine syndrome, Henoch-Schonlein purpura, post-streptococcal nephritis, erythema nodosum, Takayasu's arteritis, Addison's disease, and other autoimmune diseases. disease), erythema multiforme, polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome, thromboangiitis obliterans, primary biliary cirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, chronic active hepatitis, polymyositis / dermatomyositis, polychondritis, pemphigus vulgaris, Wegener's granulomatosis granulomatosis), membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, and fibrosing alveolitis; inflammatory skin diseases, including psoriasis and dermatitis (e.g., atopic dermatitis); systemic scleroderma and sclerosis; inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis); respiratory distress syndrome, including adult respiratory distress syndrome (ARDS); dermatitis; meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergic conditions, such as eczema and asthma, and other conditions involving T-cell infiltration and chronic inflammatory responses; atherosclerosis; leukocyte adhesion deficiency; rheumatoid arthritis; systemic lupus erythematosus (SLE); diabetes (e.g., type 1 diabetes or insulin-dependent diabetes mellitus); multiple sclerosis; Reynaud's syndrome syndrome); autoimmune thyroiditis; allergic encephalomyelitis; Sjorgen's syndrome; juvenile diabetes mellitus; and immune reactions associated with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes commonly found in tuberculous sarcoidosis, polymyositis, granulomatosis, and vasculitis; malignant amenorrhea (Addison's disease); diseases involving leukocytic infiltration;Central nervous system (CNS) inflammatory disorders; multiple organ damage syndrome; hemolytic anemia (including but not limited to cryoglobulinemia or Coombs-positive anemia); myasthenia gravis; antigen-antibody complex-mediated diseases: anti-glomerular basement membrane disease; antiphospholipid syndrome; allergic neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome; bullous pemphigoid; pemphigus; autoimmune polyendocrine disease; Reiter's disease; generalized myotonia syndrome: Behcedisease; giant cell arteritis; immune complex nephritis; IgA nephropathy; IgM polyneuropathy: immune thrombocytopenic purpura (ITP) or autoimmune thrombocytopenia, etc.

[0066] Abbreviations hPD-1 Human PD-1 protein CDR Complementarity determining regions in immunoglobulin variable regions defined using the Kabat numbering system EC50 Concentration resulting in 50% efficacy and binding ELISA Enzyme-linked immunosorbent assay IL-2 Interleukin-2 IC50 Concentration resulting in 50% inhibition IgG Immunoglobulin G Kabat Immunoglobulin alignment and numbering system pioneered by Elvin A Kabat FR Antibody framework region: immunoglobulin variable region excluding CDR regions HRP Horseradish peroxidase IFN-γ Interferon gamma mAb Monoclonal antibody PCR Polymerase chain reaction V region Segment of the IgG chain with variable sequence between different antibodies. It extends to Kabat residue 109 of the light chain and residue 113 of the heavy chain VH Immunoglobulin heavy chain variable region VK Immunoglobulin kappa light chain variable region Kd Equilibrium dissociation constant Ka Association rate constant Kd Dissociation rate constant BRIEF DESCRIPTION OF THE DRAWINGS

[0067] FIG1 shows a flow chart for preparing hPD-1 antibodies.

[0068] FIG2 shows the SDS-PAGE identification results of the murine antibody 3D6-mIgG2a.

[0069] FIG3 shows the ELISA binding test results of the murine antibody 3D6-mIgG2a.

[0070] FIG4 shows the FCM detection results of the murine antibody 3D6-mIgG2a.

[0071] FIG5 shows the results of competition ELISA detection of the murine antibody 3D6-mIgG2a.

[0072] FIG6 shows that the murine antibody 3D6-mIgG2a prolongs the survival of PD-1 humanized mouse heart allografts.

[0073] FIG. 7 shows the ELISPOT results of the murine antibody 3D6-mIgG2a.

[0074] Figure 8 shows a humanized antibody plasmid map.

[0075] FIG9 shows the SDS-PAGE detection results of the humanized antibody 3D6-hIgG1.

[0076] FIG10 shows the BLI affinity detection results of the humanized antibody 3D6-hIgG1.

[0077] FIG11 shows the effect of humanized antibody 3D6-hIgG1 on mixed lymphocyte reaction.

[0078] FIG12 shows the ADCC activity test results of the humanized antibody 3D6-hIgG1. DETAILED DESCRIPTION

[0079] The present invention is further described below with reference to specific examples, which, however, are not intended to limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art.

[0080] Example 1: Generation of mouse anti-hPD-1 antibodies

[0081] To generate mouse antibodies against human PD-1, 6-8 week old female Balb / c mice were immunized with human PD-1, and anti-hPD-1 antibodies were collected by the method shown in FIG1 .

[0082] Immunization of mice

[0083] The immunogen was recombinant human PD-1 protein (ABclonal, RP00067). For the first immunization, 100 μg of the diluted immunogen was emulsified with an equal amount of complete Freund's adjuvant (CFA) and injected subcutaneously. For the second and third immunizations, 50 μg of the diluted immunogen was emulsified with an equal amount of incomplete Freund's adjuvant (IFA) and injected subcutaneously. Three days after the intravenous shock immunization, 100 μg of the immunogen was injected, and the serum antibody titer of the mice was determined by ELISA. The spleen cells of the mice with the highest titer were selected for cell fusion.

[0084] Cell fusion

[0085] Balb / c-derived myeloma cells sp2 / 0 in the logarithmic growth phase were used to enrich B cells with the highest titer from the spleen of immunized mice using CD19 magnetic beads. B cells and myeloma cells were mixed at a ratio of 1:5-1:10, 1 ml of 37°C 50% PEG (pH 8.0) was added dropwise, and incomplete culture medium and the remaining stop solution were added. After centrifugation and discarding the supernatant, HAT culture medium was added to suspend and mix, and the cells were cultured in a 37°C, 5% CO2 constant temperature incubator.

[0086] Combined ELISA primary screening and subcloning

[0087] Hybridoma cell clones were selected within 7-10 days of fusion, and the culture supernatant was taken for ELISA binding detection: PD-1 protein 0.1ug / ml, 1ug / ml and 5ug / ml, 100ul / well, coated at 4℃ overnight; the liquid in the plate was shaken off and patted dry, 2% BSA 300ul per well, sealed and incubated at room temperature for 1h; 300ul per well of washing solution, washed twice, and patted dry for the last time; the culture supernatant was diluted and added to the corresponding well plate, 100ul per well, at room temperature for 2 hours; the plate was washed repeatedly; the secondary antibody was diluted to the working concentration, 100ul per well, at room temperature for 1 hour; the plate was washed repeatedly; the color development solution A and solution B were mixed in a 1:1 ratio, 200ul was added to each well, and incubated at room temperature in the dark for 20min; 50ul of stop solution was added to each well, and the OD value was immediately measured at a wavelength of 450nm. Perform limiting dilution on the cells in the positive wells, measure the ELISA value 5-6 days after each limiting dilution, and pick the monoclonal wells with higher OD450 positive values ​​detected by ELISA for limiting dilution until the results of the entire 96-well plate are positive by ELISA.

[0088] FCM binding screening

[0089] Use the MOLT-4 cell line in the logarithmic growth phase and divide it into 100,000 cells per tube. Add 10ul of the culture supernatant of the monoclonal cells with positive results in the above-mentioned ELISA assay. Incubate at room temperature for 30 minutes, wash once with PBS, centrifuge, add 0.5ul of anti-mouse IgG fluorescent secondary antibody to each tube, incubate at room temperature for 30 minutes, wash once with PBS, and detect on the machine. Select several clones with higher average fluorescence intensity for further screening.

[0090] Competitive ELISA screening

[0091] Coat PDCD1-Fc, 100uL / well, overnight at 4℃; shake off the liquid in the plate and pat dry; add 2% BSA, 300μL / well, seal and incubate at room temperature for 1h; wash the plate twice with 300μL / well washing solution and pat dry for the last time; dilute the competitor PD-L2-His to 0.2ug / mL and the competitor B7-H1-His to 3ug / mL, take 10% of the diluted sample (the clone with higher mean fluorescence intensity) and competitor 0ul was added to the corresponding well plate, mixed evenly, and reacted at room temperature for 2h; 300μL / well wash solution was added, and the plate was washed three times, and the final pat was dried; HRP-labeled anti-His detection secondary antibody was diluted to the working concentration, 100μL / well, mixed evenly, and incubated at room temperature for 1h; the plate was washed, and solution A and solution B were mixed in a 1:1 ratio, and 200μL was added to each well. Incubate at room temperature in the dark for 10min; 50μL of stop solution was added to each well, and the OD value was immediately measured at a wavelength of 450nm. Clone 3D6, which has a low efficiency in blocking PD-1 and PD-L1 / 2 binding, was screened for sequencing (it contains the light chain shown in SEQ ID NO: 7 and the heavy chain shown in SEQ ID NO: 9) and the production of 3D6-mIgG2a antibody.

[0092] Hybridoma cell gene extraction and sequencing

[0093] The hybridoma cells of clone 3D6 were lysed and RNA was extracted. Using the extracted RNA as a template, a reverse transcription kit was used to obtain cDNA in two steps. The reverse-transcribed cDNA was used as a template for PCR amplification using multiple primers. The amplified fragment was inserted into an expression vector and sequenced to obtain a plasmid containing the correct sequence.

[0094] Cell culture and antibody purification

[0095] Transfer 1 mL of hybridoma cells into a 100 mL culture flask, regularly add a certain amount of 1640 medium for cell expansion, and culture for 10-12 days. Rinse the Protein A affinity chromatography column with ultrapure water and then equilibrate with equilibration buffer. Load the supernatant of the cultured hybridoma cells onto the affinity chromatography column and rinse with equilibration buffer. Elute with elution buffer, collect the elution peak, and neutralize with Tris buffer. Desalt to PBS 7.4. Prepare the sample, perform gel electrophoresis, and determine the correct band size. Then, perform SDS-PAGE to determine the purity. The results show that the antibody is successfully expressed with a purity greater than 90% (Figure 2).

[0096] Example 2: Binding and blocking activity testing of the produced mouse antibody 3D6-mIgG2a

[0097] Combined with ELISA detection

[0098] Coat PD-1 protein at 0.1ug / ml, 1ug / ml and 5ug / ml, 100ul / well, and coat overnight at 4℃; shake off the liquid in the plate and pat dry, add 300ul of 2% BSA to each well, seal and incubate at room temperature for 1h; add 300ul of washing solution to each well, wash the plate twice, and pat dry for the last time; dilute the antibody to 1ug / ml and add it to the corresponding well plate, 100ul per well, and incubate at room temperature for 2 hours; repeat the plate washing process; dilute the secondary antibody to the working concentration, 100ul per well, and incubate at room temperature for 1 hour; repeat the plate washing process; mix the color development solution A and solution B in a 1:1 ratio, add 200ul to each well, and incubate at room temperature in the dark for 20min; add 50ul of stop solution to each well, and immediately measure the OD value at a wavelength of 450nm (Figure 3). The results showed that the antibody 3D6-mIgG2a (represented by 3D6 in Figure 3) bound well to the PD-1 protein.

[0099] FCM detection

[0100] Using the MOLT-4 cell line in the logarithmic growth phase, 100,000 cells were divided into each tube, and the antibody was added and diluted to 1ug / ml. The cells were incubated at room temperature for 30 minutes, washed once with PBS, and centrifuged. 0.5ul of anti-mouse IgG fluorescent secondary antibody was added to each tube, and the cells were incubated at room temperature for 30 minutes. After washing once with PBS, the cells were tested on the microscope (Figure 4). The results showed that the antibody 3D6-mIgG2a (represented by 3D6 in Figure 4) bound well to cells expressing human PD-1.

[0101] Competitive ELISA assay

[0102] Coating PDCD1-Fc, 100uL / well, coating at 4℃ overnight; shake off the liquid in the plate and pat dry, 2% BSA, 300μL / well, seal and incubate at room temperature for 1h; 300μL / well washing solution, wash the plate twice, and pat dry for the last time; competitor PD-L2-His is diluted to 0.2ug / mL and competitor B7-H1-His is diluted to 3ug / mL, 0.1ug / ml and 5ug / ml antibodies and 100ul of competitor are added to the corresponding wells of the plate, mixed evenly, and reacted at room temperature for 2h; 300μ L / well washing solution, wash the plate three times, and pat dry the last time; dilute the HRP-labeled anti-His detection secondary antibody to the working concentration, 100 μL / well, mix evenly, and incubate at room temperature for 1 hour; wash the plate, mix solution A and solution B at a ratio of 1:1, add 200 μL to each well, and incubate at room temperature in the dark for 10 minutes; add 50 μL of stop solution to each well, and immediately measure the OD value at a wavelength of 450 nm (Figure 5). The results show that the antibody 3D6-mIgG2a (represented by 3D6 in Figure 5) hardly blocks the binding of PD-1 to PD-L1 / 2.

[0103] Example 3: Detection of the in vivo effect of 3D6-mIgG2a antibody in a PD-1 humanized mouse heart transplant model

[0104] Mice were anesthetized, and hearts from BALB / c mice were transplanted into the abdomens of B6hPD-1 (Jicui Yaokang, T003095) recipient mice. The donor mouse's abdomen was opened, and 1 ml of heparinized saline was injected via the inferior vena cava. One minute later, the thorax was opened, and icy heparinized saline was immediately dripped onto the heart until it stopped beating. The superior and inferior vena cava were ligated with 6-0 silk sutures. The thymus was removed, and the aorta and pulmonary artery were exposed. These were then opened with Venus scissors. The heart was then washed with 2 ml of icy heparinized saline via aortic pulse until bloodless. The pulmonary veins were uniformly ligated, and the heart was removed en bloc and placed in icy heparinized saline until ready for use. The recipient mouse's abdominal cavity was opened, the abdominal artery and vein were isolated and exposed, and the branch vessels were ligated. The abdominal artery and vein were blocked proximally and distally. A small incision was made in each artery and vein using Venus scissors. The donor heart's aorta was sutured to the abdominal artery and the pulmonary artery was sutured to the abdominal vein using 10-0 silk suture. The circulation was opened. After the heart was re-beating, the abdomen was closed and fluids were replenished. The antibody (500 μg) was injected intraperitoneally into the recipient mouse on days 0, 3, and 6 after transplantation, followed by 250 μg every 5 days. Rejection was assessed by palpating the heart daily. Complete rejection was considered complete when the heart stopped beating, and this was confirmed by laparotomy. Cardiac arrest within 3 days was considered a technical error (less than 5%) and was excluded from subsequent analysis (Figure 6). The results showed that the antibody significantly prolonged heart graft survival.

[0105] Example 4: ELISPOT detection of alloreactive T cell numbers

[0106] A heart transplant model was established by transplanting BALB / c mouse hearts into B6hPD-1 recipient mice. The mice were treated with antibodies (500 μg intraperitoneally on days 0, 3, and 6). On day 6 after transplantation, the recipient spleens were harvested and ground into single-cell suspensions. T cells were isolated using a T cell negative selection kit (Miltenyi Biotec, 130-095-130) and mixed with stimulatory BALB / c APCs at a ratio of 1:1. 2x10 5 Cells were seeded per well on mouse IFN-γ pre-coated ELISpot plates and incubated at 37°C for 16 hours. After lysis with cold ddH2O at 4°C for 10 minutes, the cells were washed six times with wash buffer and then incubated with biotinylated anti-IFN-γ for 1 hour at 37°C. Following washes, the cells were incubated with streptavidin-HRP for 1 hour at 37°C. After repeated washes, the cells were incubated with AEC solution for 10 minutes at 37°C and terminated with cold ddH2O. Single-cell counting was performed using RAWspot technology, and the results were analyzed using a Mabtech IRIS FluoroSpot / ELISpot reader (Figure 7). The results showed that the antibody significantly reduced the number of alloreactive T cells in the recipient spleen.

[0107] Example 5: Humanization of murine antibodies and affinity testing of humanized antibodies

[0108] The humanized antibody 3D6-hIgG1 (comprising the light chain shown in SEQ ID NO: 15 and the heavy chain shown in SEQ ID NO: 17) was obtained by performing antibody sequence analysis, 3D structural modeling, CDR structure analysis, and humanized design. Gene synthesis, vector construction (Figure 8), and HEK293 transient expression and purification were then performed. Purity of the target protein was assessed by SDS-PAGE (Figure 9), showing bands of the correct size and position, with a purity of 98.7%. Specific methods for constructing humanized antibodies can be performed according to methods disclosed in numerous literatures in the art.

[0109] Determination of affinity between humanized antibodies and antigen proteins using BLI

[0110] The ForteBio Octet RED384 system biosensor was first immersed in a buffer solution for equilibrium, then immersed in a 5 μg / ml solution of the humanized antibody 3D6-hIgG1. The biosensor was then immersed in a gradient dilution of the hPD-1 protein solution (0.125 μg / ml, 0.25 μg / ml, 0.5 μg / ml, 1 μg / ml, and 2 μg / ml). The sensor, already bound to the test antibody, was then immersed in the buffer solution for dissociation. This caused the test antibody to detach from the biosensor surface, resulting in a decrease in the biofilm thickness. By monitoring the biofilm thickness of the biosensor in real time during the experiment, the kinetic constants of the test sample were determined. The results showed that the affinity of the humanized antibody 3D6-hIgG1 was 5.29E-10, maintaining a high affinity (Figure 10).

[0111] Example 6: Humanized antibody 3D6-hIgG1 does not enhance mixed lymphocyte reaction

[0112] Peripheral blood was collected from healthy individuals and peripheral blood mononuclear cells were isolated using lymphocyte separation medium (Tianjin Haoyang Biotechnology Co., Ltd.). Monocytes were further isolated using a monocyte isolation kit (Miltenyi Biotec), added with 25 ng / ml GM-CSF and 50 ng / ml IL-4, and inoculated into 96-well plates. Dendritic cells were cultured at 37°C for 7 days to induce dendritic cells. On day 7 of the experiment, peripheral blood was collected from another healthy individual and peripheral blood mononuclear cells were isolated using lymphocyte separation medium. CD4-positive T cells were further isolated using a CD4 cell isolation kit (Miltenyi Biotec). The cell concentration was adjusted and the cells were inoculated into the aforementioned dendritic cell-induced culture plate. 1 μg / ml of hIgG1 isotype negative control antibody (hIgG1isotype), nivolumab positive control antibody (Nivolumab), and 3D6-hIgG1 were added. After 5 days of routine culture, the supernatant was collected and the IFN-γ concentration in each supernatant was detected using a human IFN-gamma ELISA kit (ABclonal RK00015). The results showed that the PD-1 blocking antibody nivolumab significantly increased IFN-γ secretion in the mixed lymphocyte reaction compared to the hIgG1 isotype negative control antibody, while 3D6-hIgG1 did not increase IFN-γ secretion (Figure 11).

[0113] Example 7: In vitro ADCC activity detection of humanized antibody 3D6-hIgG1

[0114] Peripheral blood mononuclear cells (PBMCs) were isolated from healthy individuals using lymphocyte separation medium. NK cells were further isolated using a NK cell isolation kit (Miltenyi Biotec) and cultured in vitro with IL-2 stimulation. In vitro activated CD4-positive T cells were labeled using a PKH26 cell ligation kit (Sigma) and then plated with NK cells at a 1:5 ratio. 3D6-hIgG1 and hIgG1 isotype antibodies were added in a 10-fold dilution gradient (0.001 μg / ml, 0.01 μg / ml, 0.1 μg / ml, 1 μg / ml, and 10 μg / ml) and incubated at 37°C for 4 hours. The proportion of live CD4-positive T cells was analyzed by flow cytometry, and the lysis rate was calculated. The results showed that the lysis rate of CD4-positive T cells gradually increased with increasing 3D6-hIgG1 antibody concentration. At a 3D6-hIgG1 concentration of 0.074 μg / ml, approximately 50% of the CD4-positive T cells underwent lysis (Figure 12).

[0115] Throughout the specification, references to terms such as "one embodiment," "specific embodiment," and "example" indicate that a particular feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of these terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0116] The above contents are merely examples and explanations of the present invention. Those skilled in the art may make various modifications or additions to the described specific implementation plans or replace them in similar ways. As long as they do not deviate from the invention or exceed the scope defined by the claims, they should all fall within the scope of protection of the present invention.

[0117] It should be noted that the preferred embodiments of the present invention are given in the specification and drawings of the present invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. These embodiments are not intended to be additional limitations on the content of the present invention. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. In addition, the above-mentioned technical features can be combined with each other to form various embodiments not listed above, which are all considered to be within the scope of the description of the present invention. Furthermore, it is obvious to those skilled in the art that improvements or changes can be made based on the above description, and all such improvements and changes should fall within the scope of protection of the claims attached to the present invention.

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

Claims

1. A non-blocking clearance antibody or antigen-binding fragment thereof that specifically binds to PD-1, characterized in that: It comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 1, 2 and 3, respectively, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 4, 5 and 6, respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof is a mouse antibody, Preferably, it comprises a light chain as shown in SEQ ID NO:7 and a heavy chain as shown in SEQ ID NO:

9.

3. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof is a humanized antibody, Preferably, the Fc segment of the humanized antibody is the Fc segment of human IgG1 having ADCC and CDC effects. Preferably, the humanized antibody comprises a light chain variable region as shown in SEQ ID NO: 11 and a heavy chain variable region as shown in SEQ ID NO: 13, Preferably, the humanized antibody comprises a light chain as shown in SEQ ID NO:15 and a heavy chain as shown in SEQ ID NO:

17.

4. A polynucleotide, characterized in that The polynucleotide encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

5. A carrier, characterized in that Comprising the polynucleotide of claim 4.

6. A host cell, characterized in that Comprising the vector according to claim 5.

7. A conjugate, characterized in that Comprising the antibody or antigen-binding fragment thereof and a coupling portion as described in any one of claims 1 to 3, Preferably, the conjugated moiety is selected from a detectable label, a drug, a toxin, a cytokine, a radionuclide or an enzyme.

8. A pharmaceutical composition, characterized in that The invention comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier, diluent or excipient.

9. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the polynucleotide according to claim 4, the vector according to claim 5, the host cell according to claim 6, the conjugate according to claim 7 or the pharmaceutical composition according to claim 8 in the preparation of a medicament for eliminating autoreactive and alloreactive T cells and B cells.

10. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the polynucleotide according to claim 4, the vector according to claim 5, the host cell according to claim 6, the conjugate according to claim 7 or the pharmaceutical composition according to claim 8 in the preparation of a medicament for treating autoimmune diseases, rejection reactions and / or inflammation of organ or cell transplantation.

Images