Anti-canine PD-1 antibody and use thereof

Abstract

Provided are an anti-canine PD-1 antibody and the use thereof in the prevention and treatment of tumors in mammals, especially the use in the prevention and treatment of canine tumors. The canine PD-1 antibody having high affinity and high activity can block the binding of canine PD-1 and a ligand thereof, thus achieving the purpose of treating canine tumor diseases.

Description

An anti-canine PD-1 antibody and its use Technical Field

[0001] The present invention relates to the field of antibody technology, and in particular to an anti-canine PD-1 antibody and uses thereof. Background Art

[0002] Tumors are a common and frequently occurring disease in humans and animals, among which canine malignant tumors have become the main cause of death in dogs. Relevant research data show that one in every four dogs is diagnosed with a tumor, and the proportion of dogs over 10 years old suffering from tumors is as high as over 50%. Currently, the clinical treatments for canine tumors mainly include surgery, chemotherapy and radiotherapy. Common chemotherapy drugs include cyclophosphamide, doxorubicin, vincristine and prednisolone, all of which have disadvantages such as poor tumor specificity and large toxic side effects. Immunotherapy is an emerging treatment method. It may be effective and have few side effects for patients who do not respond well to chemotherapy. The research and application of immunotherapy have made major breakthroughs in human tumors, and it is of great significance to promote immunotherapy to animal tumors.

[0003] Programmed cell death receptor 1 (PD-1) is a type I transmembrane glycoprotein of approximately 55 kDa, belonging to the CD28 superfamily of receptors. It is primarily expressed on the surfaces of T cells, B lymphocytes, and activated macrophages. The PD-1 protein has two ligands: PD-L1 and PD-L2. Under normal physiological conditions, the binding of PD-1 to PD-L1 / PD-L2 inhibits T cell activation, thereby protecting the body from autoimmune attack. However, PD-L1 is also expressed abundantly in various solid tumors and some hematologic malignancies, including melanoma, breast cancer, various digestive system tumors, lymphomas, and leukemias. PD-L1 on the tumor cell membrane binds to PD-1 on T cells, inhibiting T cell activation and thereby evading immune recognition and attack, achieving immune escape. Studies have also found that PD-L1 expression on tumor cells is associated with poor prognosis in multiple tumor types. Therefore, blocking the binding of PD-1 and PD-L1 is a promising approach for tumor immunotherapy. Summary of the Invention

[0004] The present invention addresses the deficiencies of the prior art and aims to provide a high-affinity, high-activity canine PD-1 antibody that blocks the binding of canine PD-1 to its ligand, thereby achieving the purpose of treating canine tumor diseases.

[0005] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0006] The first aspect of the present invention provides a CDR sequence of an antibody or an antigen-binding fragment thereof that can bind to canine PD-1.

[0007] The antibody or antigen-binding fragment thereof capable of binding to canine PD-1 comprises CDR1, 2, 3 of the heavy chain variable region and / or CDR1, 2, 3 of the light chain variable region.

[0008] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof capable of binding to canine PD-1 comprises a CDR sequence selected from at least one of the following or an amino acid sequence having at least 85% identity thereto: the CDR sequence of the heavy chain variable region is selected from SEQ ID NOs: 2-4, 8-11, and 14, and the CDR sequence of the light chain variable region is selected from SEQ ID NOs: 5-7, 12-13, and 15.

[0009] It is worth noting that the scope of protection in the above description is all possible CDR sequences within the above sequence range defined by the variable region: CDR1-3 of the above-mentioned heavy and light chain variable regions can be obtained by different encoding systems in the prior art, including but not limited to Kabat, Chothia, IMGT, North, AbM, Contact and other encoding systems. CDRs determined using other known or unknown encoding systems are also within the scope of protection of this application. In addition, in different databases (such as different commercial, non-commercial, scientific research databases for predicting CDR positions, including but not limited to abYsis database, IMGT database, TABS database, wisdom bud BIO database, etc.), using encoding systems including but not limited to Kabat, Chothia, IMGT, North, AbM, Contact and other encoding systems, the CDR sequences predicted are also within the scope of protection of this application. If there are certain differences in the CDRs predicted by different databases using the same encoding system for the same antibody variable region sequence, they are also within the scope of protection of the present invention.

[0010] Furthermore, the antibody or antigen-binding fragment thereof capable of binding to canine PD-1 comprises a heavy chain variable region and / or a light chain variable region, the sequence of which is: the heavy chain variable region shown in SEQ ID NO: 16, the light chain variable region shown in SEQ ID NO: 17;

[0011] Or, the heavy chain variable region shown in SEQ ID NO: 18, the light chain variable region shown in SEQ ID NO: 21;

[0012] Or, the heavy chain variable region shown in SEQ ID NO: 18, the light chain variable region shown in SEQ ID NO: 22;

[0013] Or, the heavy chain variable region shown in SEQ ID NO: 18, the light chain variable region shown in SEQ ID NO: 23;

[0014] Or, the heavy chain variable region shown in SEQ ID NO: 19, and the light chain variable region shown in SEQ ID NO: 21;

[0015] Or, the heavy chain variable region shown in SEQ ID NO: 19, and the light chain variable region shown in SEQ ID NO: 22;

[0016] Or, the heavy chain variable region shown in SEQ ID NO: 19, and the light chain variable region shown in SEQ ID NO: 23;

[0017] Or, the heavy chain variable region shown in SEQ ID NO: 20, and the light chain variable region shown in SEQ ID NO: 21;

[0018] Or, the heavy chain variable region shown in SEQ ID NO: 20, and the light chain variable region shown in SEQ ID NO: 22;

[0019] Or, the heavy chain variable region shown in SEQ ID NO: 20, and the light chain variable region shown in SEQ ID NO: 23;

[0020] or an amino acid sequence at least 85% identical thereto.

[0021] Furthermore, the present invention provides an antibody or antigen-binding fragment thereof capable of binding to canine PD-1. According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof capable of binding to canine PD-1 comprises: a CDR sequence selected from at least one of the following, or an amino acid sequence having at least 85% identity thereto: a heavy chain variable region CDR sequence selected from SEQ ID NOs: 2-4, 8-11, and 14; a light chain variable region CDR sequence selected from SEQ ID NOs: 5-7, 12-13, and 15, or an amino acid sequence having at least 85% identity thereto.

[0022] Furthermore, the antibody or antigen-binding fragment thereof capable of binding to canine PD-1 comprises a CDR sequence selected from at least one of the following or an amino acid sequence having at least 85% identity therewith:

[0023] Selected from the heavy chain variable region CDR1 as shown in any one of SEQ ID NOs: 2, 8, and 14, selected from the heavy chain variable region CDR2 as shown in any one of SEQ ID NOs: 3, 9, and 10, selected from the heavy chain variable region CDR3 as shown in any one of SEQ ID NOs: 4 and 11; selected from the light chain variable region CDR1 as shown in any one of SEQ ID NOs: 5 and 12, selected from the light chain variable region CDR2 as shown in any one of SEQ ID NOs: 6, 13, and 15, selected from the light chain variable region CDR3 as shown in SEQ ID NO: 7, or an amino acid sequence having at least 85% identity thereto.

[0024] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof capable of binding to canine PD-1 comprises a heavy chain variable region CDR and an amino acid sequence with an identity of more than 85%: the heavy chain variable region CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NOs: 2, 3, and 4, respectively (the above sequences are obtained according to the Kabat encoding system); the heavy chain variable region CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NOs: 8, 9, and 4, respectively (the above sequences are obtained according to the Chothia encoding system); the heavy chain variable region CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NOs: 14, 10, and 11, respectively (the above sequences are obtained according to the IMGT encoding system).

[0025] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof capable of binding to canine PD-1 comprises a light chain variable region CDR and an amino acid sequence with an identity of more than 85%: the light chain variable region CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NOs: 5, 6, and 7, respectively (the above sequences are obtained according to the Kabat or Chothia coding system); the light chain variable region CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NOs: 12, 13, and 7, respectively; and the light chain variable region CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NOs: 12, 15, and 7, respectively (the above sequences are obtained according to the IMGT coding system, but are obtained by analysis using different databases).

[0026] Specifically, the antibody or antigen-binding fragment thereof capable of binding to canine PD-1 includes:

[0027] The heavy chain variable region CDR1, CDR2, and CDR3 sequences are as shown in the amino acid sequences of SEQ ID NOs: 2, 3, and 4, respectively, and the light chain variable region CDR1, CDR2, and CDR3 sequences are as shown in the amino acid sequences of SEQ ID NOs: 5, 6, and 7, respectively (these sequences are obtained according to the Kabat numbering system); or, the heavy chain variable region CDR1, CDR2, and CDR3 sequences are as shown in the amino acid sequences of SEQ ID NOs: 8, 9, and 4, respectively, and the light chain variable region CDR1, CDR2, and CDR3 sequences are as shown in the amino acid sequences of SEQ ID NOs: 5, 6, and 7, respectively (these sequences are obtained according to the Chothia numbering system); or, the heavy chain variable region CDR1, CDR2, and CDR3 sequences are as shown in the amino acid sequences of SEQ ID NOs: 14, 10, and 11, respectively, and the light chain variable region CDR1, CDR2, and CDR3 sequences are as shown in the amino acid sequences of SEQ ID NOs: 12, 13, and 7, respectively; or The heavy chain variable region CDR1, CDR2, and CDR3 sequences are shown in the amino acid sequences of SEQ ID NOs: 14, 10, and 11, and the light chain variable region CDR1, CDR2, and CDR3 sequences are shown in the amino acid sequences of SEQ ID NOs: 12, 15, and 7, respectively.

[0028] The above sequences all contain amino acid sequences that can reach 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity.

[0029] In a second aspect, the present invention provides a variable region sequence of an antibody or antigen-binding fragment thereof that can bind to canine PD-1.

[0030] The antibody or antigen-binding fragment thereof capable of binding to canine PD-1 comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region is selected from SEQ ID NOs: 16, 18-20, and the light chain variable region is selected from SEQ ID NOs: 17, 21-23;

[0031] Furthermore, the antibody or antigen-binding fragment thereof capable of binding to canine PD-1 may be:

[0032] the heavy chain variable region set forth in SEQ ID NO: 16, and the light chain variable region set forth in SEQ ID NO: 17;

[0033] or, the heavy chain variable region set forth in SEQ ID NO: 18, and the light chain variable region set forth in SEQ ID NO: 21;

[0034] or, the heavy chain variable region set forth in SEQ ID NO: 18, and the light chain variable region set forth in SEQ ID NO: 22;

[0035] or, the heavy chain variable region set forth in SEQ ID NO: 18, and the light chain variable region set forth in SEQ ID NO: 23;

[0036] or, the heavy chain variable region set forth in SEQ ID NO: 19, and the light chain variable region set forth in SEQ ID NO: 21;

[0037] or, the heavy chain variable region set forth in SEQ ID NO: 19, and the light chain variable region set forth in SEQ ID NO: 22;

[0038] or, the heavy chain variable region set forth in SEQ ID NO: 19, and the light chain variable region set forth in SEQ ID NO: 23;

[0039] or, the heavy chain variable region set forth in SEQ ID NO: 20, and the light chain variable region set forth in SEQ ID NO: 21;

[0040] or, the heavy chain variable region set forth in SEQ ID NO: 20, and the light chain variable region set forth in SEQ ID NO: 22;

[0041] or, the heavy chain variable region set forth in SEQ ID NO: 20, and the light chain variable region set forth in SEQ ID NO: 23;

[0042] or an amino acid sequence that is at least 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identical thereto.

[0043] The third aspect of the present invention is to provide a constant region of an antibody or antigen-binding fragment thereof that can bind to canine PD-1.

[0044] The heavy chain constant region used in the antibody or antigen-binding fragment thereof capable of binding to canine PD-1 may be canine IgG A, IgG B, IgG C, IgG D, or a mutant sequence that ensures the same function. Furthermore, the IgG B mutant sequence is preferred. The specific heavy chain constant region domain amino acid sequence may be shown in SEQ ID NO: 24. The light chain constant region used in the antibody may be a canine kappa chain or lambda chain domain constant region sequence. Furthermore, the canine kappa chain is preferred, and the amino acid sequence is shown in SEQ ID NO: 25.

[0045] However, the inventive concept of the present invention is not limited to the constant regions shown in the above example sequences. As long as the constant region design can achieve the prevention, treatment, detection and other effects based on the PD-1 target of the present invention, it is within the scope of protection of the present invention.

[0046] The fourth aspect of the present invention provides a full-length sequence of an antibody or antigen-binding fragment thereof that is capable of binding to canine PD-1.

[0047] The antibody or antigen-binding fragment thereof capable of binding to canine PD-1 comprises a full-length heavy chain and / or a full-length light chain, wherein the full-length heavy chain sequence used is selected from any one of SEQ ID NOs: 26, 28-30, and the full-length light chain used in the antibody or antigen-binding fragment capable of binding to canine PD-1 is selected from any one of SEQ ID NOs: 27, 31-33;

[0048] The antibody or antigen-binding fragment thereof capable of binding to canine PD-1 has an affinity constant KD range of 8.76E-10 to 3.83E-09M.

[0049] Furthermore, the full length of the heavy chain of the antibody or antigen-binding fragment capable of binding to canine PD-1 is shown in SEQ ID NO: 26, and the full length of the light chain is shown in SEQ ID NO: 27;

[0050] Or, the full length heavy chain is as shown in SEQ ID NO:28, and the full length light chain is as shown in SEQ ID NO:31;

[0051] Or, the full length heavy chain is as shown in SEQ ID NO:28, and the full length light chain is as shown in SEQ ID NO:32;

[0052] Or, the full length heavy chain is as shown in SEQ ID NO:28, and the full length light chain is as shown in SEQ ID NO:33;

[0053] Or, the full length heavy chain is as shown in SEQ ID NO:29, and the full length light chain is as shown in SEQ ID NO:31;

[0054] Or, the full length heavy chain is as shown in SEQ ID NO:29, and the full length light chain is as shown in SEQ ID NO:32;

[0055] Or, the full length heavy chain is as shown in SEQ ID NO:29, and the full length light chain is as shown in SEQ ID NO:33;

[0056] Or, the full length of the heavy chain is as shown in SEQ ID NO: 30, and the full length of the light chain is as shown in SEQ ID NO: 31;

[0057] Or, the full length of the heavy chain is as shown in SEQ ID NO: 30, and the full length of the light chain is as shown in SEQ ID NO: 32;

[0058] Or, the full length of the heavy chain is as shown in SEQ ID NO: 30, and the full length of the light chain is as shown in SEQ ID NO: 33,

[0059] or an amino acid sequence that is at least 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0060] The fifth aspect of the present invention provides a type of antibody or antigen-binding fragment thereof capable of binding to canine PD-1.

[0061] The antibody or antigen-binding fragment thereof capable of binding to canine PD-1 comprises at least one selected from a monoclonal antibody, a polyclonal antibody, a multimeric antibody, and a CDR-grafted antibody;

[0062] Optionally, the antibody or antigen-binding fragment capable of binding to canine PD-1 comprises at least one selected from a single-chain antibody, a Fab antibody, a Fv antibody, a single-chain antibody, a single-domain antibody, and a minimum recognition unit; when used as an antigen-binding fragment, the CDR sequence is not limited to 6, and can be less than or more than 6, as long as it can affinity bind to the target antigen.

[0063] Optionally, the antibody or antigen-binding fragment capable of binding to canine PD-1 comprises at least one of a Fab fragment, a (Fab)2 fragment, a scFv-Fc fusion protein, a scFv-Fv fusion protein, an Fv fragment, and a minimal recognition unit.

[0064] The sixth aspect of the present invention provides a nucleic acid element.

[0065] The element may be a nucleic acid molecule encoding one of the aforementioned antibodies or antigen-binding fragments thereof that can bind to canine PD-1. The nucleic acid molecule includes, but is not limited to, DNA and RNA; preferably, the nucleic acid molecule is DNA. However, the scope of protection of the present invention includes all nucleic acids and nucleotide sequences that can express the aforementioned amino acid sequence, including nucleic acid sequences derived from various codon preferences. Those skilled in the art can infer other nucleotide sequences that can express the aforementioned amino acid sequence using conventional techniques in the art. The following sequences are merely examples:

[0066] Specifically, the heavy chain variable region sequence of the antibody or antigen-binding fragment thereof that can bind to canine PD-1 can be the DNA coding sequence shown in SEQ ID NOs: 49, 51-53, and the light chain variable region sequence of the antibody or antigen-binding fragment thereof that can bind to canine PD-1 can be the DNA coding sequence shown in SEQ ID NOs: 50, 54-56.

[0067] Furthermore, the antibody or antigen-binding fragment thereof capable of binding to canine PD-1 may have a heavy chain variable region having a DNA coding sequence as shown in SEQ ID NO:49, and a light chain variable region nucleotide sequence having a DNA coding sequence as shown in SEQ ID NO:50;

[0068] The antibody or antigen-binding fragment thereof capable of binding to canine PD-1 may have a heavy chain variable region having a DNA coding sequence as shown in SEQ ID NO: 51, and a light chain variable region nucleotide sequence having a DNA coding sequence as shown in SEQ ID NO: 54;

[0069] The antibody or antigen-binding fragment thereof capable of binding to canine PD-1 may have a heavy chain variable region having a DNA coding sequence as shown in SEQ ID NO: 51, and a light chain variable region nucleotide sequence having a DNA coding sequence as shown in SEQ ID NO: 55;

[0070] The antibody or antigen-binding fragment thereof capable of binding to canine PD-1 may have a heavy chain variable region having a DNA coding sequence as shown in SEQ ID NO: 51, and a light chain variable region nucleotide sequence having a DNA coding sequence as shown in SEQ ID NO: 56;

[0071] The antibody or antigen-binding fragment thereof capable of binding to canine PD-1 may have a heavy chain variable region having a DNA coding sequence as shown in SEQ ID NO: 52, and a light chain variable region nucleotide sequence having a DNA coding sequence as shown in SEQ ID NO: 54;

[0072] The antibody or antigen-binding fragment thereof capable of binding to canine PD-1 may have a heavy chain variable region having a DNA coding sequence as shown in SEQ ID NO: 52, and a light chain variable region nucleotide sequence having a DNA coding sequence as shown in SEQ ID NO: 55;

[0073] The antibody or antigen-binding fragment thereof capable of binding to canine PD-1 may have a heavy chain variable region having a DNA coding sequence as shown in SEQ ID NO: 52, and a light chain variable region nucleotide sequence having a DNA coding sequence as shown in SEQ ID NO: 56;

[0074] The antibody or antigen-binding fragment thereof capable of binding to canine PD-1 may have a heavy chain variable region having a DNA coding sequence as shown in SEQ ID NO: 53, and a light chain variable region having a DNA coding sequence as shown in SEQ ID NO: 54;

[0075] The antibody or antigen-binding fragment thereof capable of binding to canine PD-1 may have a heavy chain variable region having a DNA coding sequence as shown in SEQ ID NO: 53, and a light chain variable region having a DNA coding sequence as shown in SEQ ID NO: 55;

[0076] The antibody or antigen-binding fragment thereof capable of binding to canine PD-1 may have a heavy chain variable region having a DNA coding sequence as shown in SEQ ID NO: 53, and a light chain variable region having a DNA coding sequence as shown in SEQ ID NO: 56.

[0077] Specifically, the heavy chain constant region canine IgG B mutant of the antibody or antigen-binding fragment thereof capable of binding to canine PD-1 can be the DNA coding sequence shown in SEQ ID NO: 57, and the light chain constant region canine kappa chain can be the DNA coding sequence shown in SEQ ID NO: 58.

[0078] Specifically, the full-length DNA sequence of the heavy chain of the antibody or antigen-binding fragment thereof that can bind to canine PD-1 can be shown in any one of SEQ ID NOs: 59, 61-63, and the full-length DNA sequence of the antibody light chain can be shown in any one of SEQ ID NOs: 60, 64-66.

[0079] Specifically, the antibody or antigen-binding fragment thereof capable of binding to canine PD-1 may have a heavy chain full-length DNA sequence as shown in SEQ ID NO: 59, and a light chain full-length DNA sequence as shown in SEQ ID NO: 60;

[0080] or, the full-length DNA sequence of the heavy chain is shown in SEQ ID NO:61, and the full-length DNA sequence of the light chain is shown in SEQ ID NO:64;

[0081] or, the full-length DNA sequence of the heavy chain is shown in SEQ ID NO:61, and the full-length DNA sequence of the light chain is shown in SEQ ID NO:65;

[0082] or, the full-length DNA sequence of the heavy chain is shown in SEQ ID NO:61, and the full-length DNA sequence of the light chain is shown in SEQ ID NO:66;

[0083] or, the full-length DNA sequence of the heavy chain is shown in SEQ ID NO: 62, and the full-length DNA sequence of the light chain is shown in SEQ ID NO: 64;

[0084] or, the full-length DNA sequence of the heavy chain is shown in SEQ ID NO: 62, and the full-length DNA sequence of the light chain is shown in SEQ ID NO: 65;

[0085] or, the full-length DNA sequence of the heavy chain is shown in SEQ ID NO: 62, and the full-length DNA sequence of the light chain is shown in SEQ ID NO: 66;

[0086] or, the full-length DNA sequence of the heavy chain is shown in SEQ ID NO: 63, and the full-length DNA sequence of the light chain is shown in SEQ ID NO: 64;

[0087] or, the full-length DNA sequence of the heavy chain is shown in SEQ ID NO: 63, and the full-length DNA sequence of the light chain is shown in SEQ ID NO: 65;

[0088] Alternatively, the full-length DNA sequence of the heavy chain is shown in SEQ ID NO: 63, and the full-length DNA sequence of the light chain is shown in SEQ ID NO: 66.

[0089] Specifically, the CDR amino acid sequences shown in SEQ ID NOs: 2-15 may correspond to DNA coding sequences shown in SEQ ID NOs: 35-48.

[0090] The element may be a recombinant vector, including but not limited to a cloning vector, an expression vector, a shuttle vector, or a viral vector (e.g., a lentiviral vector, an adeno-associated viral vector, a poxvirus-associated vector, etc.), carrying the aforementioned nucleic acid molecule. Preferably, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector. The expression vector is a plasmid expression vector, such as pcDNA3.1 or pcDNA3.4.

[0091] The element can be a recombinant cell carrying the aforementioned nucleic acid molecule, or a recombinant vector capable of expressing one of the aforementioned antibodies or antigen-binding fragments thereof capable of binding to canine PD-1; the recombinant cell is obtained by introducing the aforementioned expression vector into a host cell; the recombinant cell is preferably a eukaryotic cell, preferably a mammalian cell, including but not limited to Chinese hamster ovary cells (CHOK1) and HEK293 cells.

[0092] The seventh aspect of the present invention relates to a pharmaceutical composition and use.

[0093] The pharmaceutical composition comprises the aforementioned antibody or antigen-binding fragment thereof capable of binding to canine PD-1, or the aforementioned nucleic acid molecule, or the aforementioned expression vector, or the aforementioned recombinant cell.

[0094] Optionally, the pharmaceutical composition further comprises pharmaceutically acceptable adjuvants and excipients.

[0095] The pharmaceutical composition can be prepared as a combined drug or a drug kit, which comprises the aforementioned antibody or antigen-binding fragment thereof or pharmaceutical composition capable of binding to canine PD-1 as a first active ingredient; and optionally, a second active ingredient for combined use. The first active ingredient and the second active ingredient may be the same or different, and the second active ingredient may include but is not limited to paladin, cyclophosphamide, doxorubicin, vincristine and prednisolone.

[0096] The use of the anti-canine PD-1 antibody or its antigen-binding fragment, nucleic acid molecule, expression vector, recombinant cell, pharmaceutical composition, combined drug or drug kit in preventing and / or treating tumors, or in preparing a drug, or in preparing a detection kit for detecting PD-1 protein in a biological matrix; the tumor is a tumor of a mammal, including a human, monkey, mouse (rats, mice), horse, cattle, sheep, pig, dog, cat, etc., and more preferably a canine tumor.

[0097] The types of tumors include, but are not limited to, "tumors," but rather refer to a class of diseases characterized by the development of abnormal cells that proliferate uncontrollably and have the ability to infiltrate and destroy normal body tissues. Exemplary tumors include, but are not limited to, squamous cell carcinoma (SCC); papilloma; fibropapilloma; intraosseous carcinoma; invasive nasal carcinoma; malignant melanoma (MM); fibrosarcoma (FSA); fibroma; rhabdomyosarcoma; angiosarcoma (HAS); granular cell tumor; mixed mesenchymal sarcoma; neurofibrosarcoma; undifferentiated sarcoma; myxosarcoma; chondrosarcoma (CSA); chondrosarcoma phyllodes (MLO); osteosarcoma (OSA); transmissible venereum tumor (TVT); mast cell tumor (MCT); lymphoma (LSA); ameloblastoma; ameloblastic adenoma; odontogenic epithelial calcification tumor; odontoma; fibromyxoma; cementoma; odontogenic fibroma; acanthoderma; ossification Encephaloderma; fibroma of the eminence; basal cell tumor; cerumenoma; viral papilloma, salivary gland tumor, nasal lymphoma; cerumen gland carcinoma (CGC); benign oncocytic adenoma (rhabdomyomas); chondroma; osteoma; osteochondroma; tracheal cancer; leiomyoma; benign laryngeal tumors (e.g., rhabdomyomas or oncocytic adenomas); mediastinal tumors (e.g., thymoma and mediastinal lymphoma); malignant chest wall tumors (sarcomas); benign chest wall tumors (osteomas and chondromas); primary lung tumors; secondary (metastatic) lung tumors; cardiac tumors (e.g., angiosarcomas; mesotheliomas; myxomas; ectopic thyroid cancer); esophageal cancer; gastric tumors (e.g., gastric adenocarcinoma and lymphoma); gastric leiomyosarcoma; gastric smooth muscle tumors; extramedullary plasmacytoma of the stomach; mast cell tumor of the gastrointestinal tract; nonlymphoid intestinal tumors; intestinal lymphoma; intestinal adenocarcinoma; extramedullary plasmacytoma; carcinoid tumor; adenocarcinomatous polyps; colorectal adenocarcinoma; colorectal leiomyomas and leiomyosarcomas; perianal adenomas; perianal adenocarcinomas; anal sac adenocarcinomas; liver tumors (e.g., liver sarcoma, hepatocarcinoid, or carcinoid); hepatocellular carcinoma (HCC); hepatocellular adenoma; hepatoblastoma; biliary tract cancer; bile duct adenocarcinoma; gallbladder tumors; pancreatic adenocarcinoma; breast tumors; malignant breast tumors (breast cancer); malignant mixed tumors; breast sarcoma; extraskeletal breast osteosarcoma; uterine tumors; vaginal and vulvar tumors; ovarian tumors (e.g., adenocystoma, granulosa cell tumor); germ cord stromal tumors (e.g., theca uterine carcinoma (e.g., ovarian cystoma, ovarian cyst, or ovarian cyst); cervical cyst (e.g., ovarian cyst, ovarian cyst, or ovarian cyst); testicular tumors (e.g., Sertoli cell tumor, seminoma, and Leydig cell tumor); histiocytic reticuloendothelioma, transitional cell carcinoma (TCC); prostate tumors; kidney tumors (e.g., renal carcinoma, fibrosarcoma, and angiosarcoma); ureteral tumors; bladder tumors; urethral tumors; cutaneous hemangioma; subungual squamous cell carcinoma; basal cell tumors (including basal cell carcinoma, basal cell epithelial cave, and basal cell-like tumor); cerumen tumors; intradermal keratoepithelioma (keratoacanthoma); hair follicle tumors (trichoblastoma, trichoepithelioma, and nail bed cell tumor); cutaneous plasma cell neoplasms; cutaneous lymphoma; sebaceous adenoma;Sweat gland tumors; epitheliotropic lymphoma (ELSA); cutaneous histiocytoma; cutaneous neuroendocrine (Merkel cell) tumor; cutaneous smooth muscle tumor; soft tissue or spindle cell sarcoma (STS); lymphangiosarcoma; synovial cell sarcoma; histiocytic sarcoma (HS); extremity osteosarcoma; axial osteosarcoma; extraskeletal osteosarcoma; superficial osteosarcoma; multilobular chondrosarcoma of bone (MLO); multiple cartilaginous exostoses (MCE); lymphocytic leukemia (CLL); non-lymphocytic leukemia (granulocytic leukemia); chronic myeloid leukemia (CML); acute myeloid leukemia (AML); splenic angiosarcoma (HSA); splenic sarcoma (non-lymphoid, non-vascular); glioma; meningioma; ganglioneuroma (pheochromocytoma and non-pheochromocytic paraganglioma); neuroendocrine tumors (NSA); Ganglioneuroglioma; spinal cord tumors (OSA, CSA, FSA, HSA, hemangioendothelioma, myeloma); peripheral nerve sheath tumors (PNSTs); neuroepithelial tumors (ependymomas, medullary epithelioma, nephroblastoma, and spinal blastoma); intramedullary spinal tumors; epidural sarcomas; epidural sarcomas; nerve sheath tumors; peripheral nervous system tumors (e.g., neuroblastoma, medulloblastoma, retinoblastoma); eye cancer tumors; meibomian adenoma; myoblastoma; third eye cancer tumors; conjunctival tumors; extraoral (corneal and scleral) tumors; orbital (the space surrounding the eyeball) and retroorbital tumors; thyroid tumors; parathyroid tumors; APUD cell tumors (glial precursor uptake decarboxylation cell tumors); insulinomas; glucagonomas; gastrinomas; adrenal cortical tumors; pheochromocytoma; pituitary tumors. Melanoma, mast cell tumor, and colon cancer are preferred.

[0098] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0099] 1. The anti-PD-1 chimeric antibodies and caninized antibodies described in the present invention have good affinity. Among them, the affinity constant KD of the chimeric antibody is 8.76E-10M, and the affinity constant KD of various caninized antibodies is lower than 3.83E-09M.

[0100] 2. The anti-PD-1 chimeric antibody and caninized antibody described in the present invention can effectively block the canine T cell activation inhibitory pathway (cPD-1 / cPD-L1) in vitro, thereby enhancing T cell activation and having biological activity.

[0101] 3. The anti-PD-1 chimeric antibody and caninized antibody described in the present invention can inhibit the growth of mouse MC38 tumors at the in vivo level and also have good anti-tumor efficacy against canine tumors, which is of great significance for the development of efficient cancer treatment drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0102] The drawings described herein are used to provide a further understanding of the embodiments of the present invention, constitute a part of this application, and do not constitute a limitation of the embodiments of the present invention. In the drawings:

[0103] Figure 1 is a flow cytometry analysis of the competitive binding of 3334 chimeric antibody to canine PD-1 / PD-L1 (A is a flow cytometry result graph; B is a quantitative graph of the APC-positive cell rate).

[0104] Figure 2 shows the results of in vitro activity detection of 3334 chimeric antibody and caninized antibody by luciferase reporter gene method (A is the group of 3334 chimeric antibody and 3334-H1K1 / H1K2 / H1K3-canIgG B caninized antibody; B is the group of 3334 chimeric antibody and 3334-H2K1 / H2K2 / H2K3-canIgG B caninized antibody; C is the group of 3334 chimeric antibody and 3334-H3K1 / H3K2 / H3K3-canIgG B caninized antibody).

[0105] FIG3 is a graph showing the results of the in vitro activity detection-T cell activation experiment of 3334 chimeric antibody and caninized antibody.

[0106] FIG4 shows the effects of 3334 chimeric antibody and 3334-H1K2-canIgG B caninized antibody on mouse body weight.

[0107] FIG5 is a graph showing the effects of 3334 chimeric antibody and 3334-H1K2-canIgG B caninized antibody on the body weight change rate of mice.

[0108] FIG6 shows the effects of 3334 chimeric antibody and 3334-H1K2-canIgG B caninized antibody on mouse tumor volume. DETAILED DESCRIPTION

[0109] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0110] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0111] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0112] The present invention will be described in detail below through embodiments in conjunction with the accompanying drawings, but this does not limit the present invention and is only used as an example.

[0113] To better understand this disclosure, some terms are first defined. The terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0114] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0115] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0116] In this article, the term "antibody" generally refers to an antibody that can recognize one or more antigenic epitopes, including but not limited to monoclonal antibodies, polyclonal antibodies, multimeric antibodies and CDR-grafted antibodies. It is an immunoglobulin molecule that can bind to a specific antigen. It includes two light chains with a relatively low molecular weight and two heavy chains with a relatively high molecular weight. The heavy chain (H chain) and the light chain (L chain) are connected by disulfide bonds to form a tetrapeptide chain molecule. Among them, the amino acid sequence at the amino terminus (N terminus) of the peptide chain varies greatly and is called the variable region (V region), while the carboxyl terminus (C terminus) is relatively stable and varies very little, and is called the constant region (C region). The V regions of the L chain and H chain are called VL and VH, respectively. In the variable region, certain areas have a higher degree of variation in amino acid composition and arrangement order, which are called hypervariable regions (HVRs). The hypervariable region is the location where the antigen and antibody bind, and is therefore also called the complementarity-determining region (CDR). There are three CDRs in both the heavy chain variable region and the light chain variable region.

[0117] As used herein, the term "Fab antibody" generally refers to an antibody containing only Fab molecules, which are composed of VH and CH1 of the heavy chain and a complete light chain, with the light chain and heavy chain connected by a disulfide bond.

[0118] In this article, the term "Fv antibody" generally refers to an antibody composed only of a light chain variable region (VL) and a heavy chain variable region (VH) connected by non-covalent bonds. It is the smallest functional fragment of an antibody that retains a complete antigen-binding site.

[0119] In this article, the terms "single domain antibody", "nanoantibody" and "VHH antibody" are used interchangeably, which were originally described as antigen-binding immunoglobulin (variable) domains of "heavy chain antibodies" (i.e., "antibodies lacking light chains") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)), which only contain a heavy chain variable region (VH) and conventional CH2 and CH3 regions, and specifically bind to an antigen through the heavy chain variable region.

[0120] Herein, the term "single-chain antibody" generally refers to an antibody composed of a heavy chain variable region (VH) and a light chain variable region (VL) connected by a linker peptide.

[0121] In this article, the terms "minimum recognition unit" and "MRU" both refer to an antibody consisting of only one CDR, which has a very small molecular weight accounting for only about 1% of the complete antibody.

[0122] As used herein, the terms "identity," "homology," or "similarity" are used to describe an amino acid sequence or nucleic acid sequence relative to a reference sequence, and the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 10:106); the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 10:116; the similarity search method of Pearson et al. (1990) Proc. Natl. Acad. Sci. 10:117; the similarity search method of Pearson et al. (1990 ... .70:173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs that utilize these algorithms are also available and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0123] As used herein, the term "at least 85% identity" refers to at least 85%, and may be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity to the respective reference sequence.

[0124] As used herein, “binding to PD-1”, “antibody that specifically binds to PD-1” or “anti-PD-1 antibody” refers to an antibody that binds to PD-1 with a KD value of 1.0×10 -8 Binding affinity was determined using a standard binding assay, such as biomembrane interferometry (Gator), using the PD-1 extracellular domain.

[0125] As used herein, the term "expression vector" generally refers to a self-replicating nucleic acid molecule capable of insertion into a suitable host and transferring the inserted nucleic acid molecule into and / or between host cells. Such expression vectors may include vectors primarily used to insert DNA or RNA into cells, vectors primarily used to replicate DNA or RNA, and vectors primarily used for expression through transcription and / or translation of the DNA or RNA. Such expression vectors also include vectors that have multiple of these functions. Such expression vectors may be polynucleotides capable of being transcribed and translated into polypeptides when introduced into suitable host cells. Generally, by culturing suitable host cells containing such expression vectors, such expression vectors can produce the desired expression product. Such expression vectors carry the aforementioned nucleic acid molecules. When such nucleic acid molecules are linked to a vector, they may be directly or indirectly linked to control elements on the vector, as long as these control elements are capable of controlling the translation and expression of the nucleic acid molecule. These control elements may be derived directly from the vector itself or exogenously, i.e., not from the vector itself. Of course, it suffices for the nucleic acid molecule to be operably linked to the control elements. As used herein, "operably linked" refers to linking an exogenous gene to a vector so that control elements within the vector, such as transcriptional and translational control sequences, can function as intended to regulate the transcription and translation of the exogenous gene. Commonly used vectors include plasmids and bacteriophages. Expression vectors according to certain embodiments of the present invention, upon introduction into appropriate recipient cells, can effectively express the aforementioned antibodies or antigen-binding fragments under the mediation of a regulatory system, thereby enabling the in vitro production of large quantities of the antibodies or antigen-binding fragments.

[0126] As used herein, the term "recombinant cell" generally refers to a cell that has been modified or recombined using genetic engineering techniques or cell fusion techniques to modify or reorganize the genetic material of a host cell to obtain a cell with unique traits that are stably inherited. The term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector can be introduced. The terms "transformed" or "transfected" as used herein refer to the introduction of a nucleic acid (e.g., a vector) into a cell by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequences of the present invention and can be used for the expression and / or secretion of a target protein.

[0127] As used herein, the term "pharmaceutical composition" generally refers to unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. Generally, the compositions are prepared by uniformly and thoroughly combining the active compound with liquid carriers, finely divided solid carriers, or both.

[0128] As used herein, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for the specific target dosage form. Except to the extent that any conventional excipient is incompatible with the compound of the present invention, such as any adverse biological effect produced or interaction with any other component of the pharmaceutically acceptable composition in a harmful manner, their use is also contemplated by the present invention.

[0129] As used herein, the term "administration" refers to the introduction of a predetermined amount of a substance into a subject for treatment by a suitable means. The antibody, antigen-binding fragment, or pharmaceutical composition of the present invention can be administered by any common route as long as it reaches the desired tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intravenous, intravenous, intramuscular, and subcutaneous injections, but the present invention is not limited to these exemplified modes of administration. The subject for treatment can be a mammal, preferably a dog.

[0130] As used herein, the term "treatment" refers to any agent used to obtain a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly dogs, and includes: (a) preventing the occurrence of a disease or condition in individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or compound to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a drug containing a compound described herein to an individual in need.

[0131] Example 1 Preparation and identification of mouse anti-canine PD-1 antibodies

[0132] 1. Antigen preparation

[0133] The canine PD-1 protein (UniProtKB: A0A024FCJ9) was searched in the UniProt and GenBank libraries. The amino acid sequence was shown in SEQ ID NO: 1. The corresponding nucleotide sequence with a His tag was artificially synthesized and constructed in the pcDNA3.4 vector. It was transiently transfected into HEK293 cells. Five days after transfection, the cell supernatant was collected and subjected to affinity chromatography on a Ni column to elute the purified canine PD-1 recombinant protein.

[0134] 2. Mouse immunization

[0135] Balb / c mice or C57 mice were immunized with 7-8 mg of canine PD-1 recombinant protein as an antigen. Serum was collected after four immunizations and analyzed for antibody titers using an enzyme-linked immunosorbent assay (ELISA).

[0136] 3. Spleen RNA Extraction and Reverse Transcription

[0137] RNA was extracted from mouse spleen tissue using the Trizol method, and the integrity of the RNA was identified by denaturing agarose gel electrophoresis. The concentration and OD of the RNA were determined using an ultramicro spectrophotometer. 260 / OD 280 RNA of qualified quality was selected and reverse transcribed according to the instructions of Superscript II reverse transcriptase (Thermo Scientific) to obtain cDNA.

[0138] 4. Antibody library construction and phage display

[0139] Antibody heavy and light chains were amplified from cDNA to construct scFv antibody library and Fab antibody library, respectively, with a library capacity of 1×10 8 The antibody library was electroporated into TG1 E. coli, and helper phage M13KO7 was added to infect TG1 E. coli. After overnight incubation, phages were precipitated using PEG / NaCl to prepare the phage-displayed antibody library.

[0140] 5. Phage display antibody library screening

[0141] Phage-displayed antibody libraries were screened using immunotubes and magnetic bead screeners for solid-phase screening and solid-phase / liquid-phase cross-screening, respectively. For solid-phase screening, immunotubes were coated with canine PD-1 recombinant protein, while for liquid-phase screening, Dynabeads magnetic beads were coated with biotinylated canine PD-1 recombinant protein. After three rounds of screening, the enriched phage pool and the isolated single clones were identified using Phage ELISA.

[0142] 6. Antibody construction, expression and purification

[0143] Phage ELISA-positive monoclonal phagemids were sequenced, and the correct reading frame mouse antibody variable regions were selected from the sequencing results for gene synthesis to obtain active mouse antibodies. The amino acid sequences of the complementarity determining regions (CDRs) 1, 2, and 3 of the heavy and light chain variable regions of the mouse antibodies were encoded using Kabat, Chothia, and IMGT (encoded in the abYsis and IMGT databases, respectively).

[0144] The murine antibody heavy chain variable region (SEQ ID NO: 16) was fused to the heavy chain constant region (including CH1, CH2, and CH3) from a canine IgG B mutant, and the light chain variable region (SEQ ID NO: 17) was fused to the canine kappa chain constant region (CL). The resulting fragments were inserted into the pcDNA3.4 vector to construct a full-length chimeric antibody expression plasmid. The chimeric antibody was transiently transfected and expressed in HEK293 cells. The culture supernatant was subjected to recombinant protein A affinity chromatography to obtain a purified chimeric antibody, designated 3334. The full-length heavy chain of the chimeric antibody is shown in SEQ ID NO: 26, and the full-length light chain is shown in SEQ ID NO: 27.

[0145] Example 2 Screening of antibodies that block the binding of canine PD-1 to its ligand

[0146] The full-length canine PD-L1 (UniProtKB: E2RKZ5) was constructed and transfected into CHO-K1 cells. A monoclonal cell line with high expression (hereafter referred to as canine PD-L1 / CHO-K1 cells) was obtained through screening. The ability of canine PD-1 antibodies to block the binding of canine PD-1 to canine PD-L1 / CHO-K1 cells was detected by flow cytometry.

[0147] Biotinylated canine PD-1 recombinant protein was incubated with various concentrations of antibody for 5 minutes and then added to PD-L1 / CHO-K1 cells. The cells were incubated at 4°C for 60 minutes, rinsed twice with cold PBS containing 1% FBS, and then incubated with streptavidin-APC fluorescent secondary antibody at 4°C for 30 minutes. The cells were then rinsed twice with cold PBS containing 1% FBS and resuspended in 200 mL of PBS containing 1% FBS. The proportion of cells labeled with APC was analyzed using flow cytometry (Beckman Coulter, CytoFLEX). The APC-positive cell rate reflects the binding ability of biotinylated canine PD-1 recombinant protein to PD-L1 / CHO-K1 cells. A lower APC-positive cell rate indicates a stronger ability of the antibody to block the binding of biotinylated canine PD-1 recombinant protein to its ligand.

[0148] As shown in Figure 1, as the concentration of 3334 chimeric antibody increased, the APC-positive cell rate gradually decreased, indicating that 3334 chimeric antibody can block the binding of canine PD-1 and PD-L1.

[0149] Example 3 Construction of caninized antibodies

[0150] The 3334 chimeric antibody was caninized based on CDR grafting and backmutation based on framework region homology. First, caninized heavy chain variable region variants (SEQ ID NOs: 18-20) and caninized light chain variable region variants (SEQ ID NOs: 21-23) were constructed. The variable regions of the caninized antibodies were freely combined with caninized heavy and light chain variable regions from the same antibody. For example, the heavy chain 3334-H1 (SEQ ID NO: 18) could be combined with the light chains 3334-K1 (SEQ ID NO: 21), 3334-K2 (SEQ ID NO: 22), or 3334-K3 (SEQ ID NO: 23), labeled 3334-H1K1, 3334-H1K2, and 3334-H1K3, respectively. The constant region of the caninized antibody is composed of the constant domain of the canine IgG B mutant and the canine kappa constant region domain. For example, 3334-H1K1-canIgG B has a caninized variable region heavy chain H1, a caninized variable region light chain K1, a canine IgG B mutant constant region, and a canine kappa constant region. The specific sequence combination names are detailed in Table 1. The constant regions of the antibody shown are, respectively, the constant region domain of the canine IgG B mutant heavy chain, the amino acid sequence of which is shown in SEQ ID NO: 24, and the constant region domain of the canine kappa chain, the amino acid sequence of which is shown in SEQ ID NO: 25.

[0151] Table 1 Sequence numbering table of 3334 chimeric and caninized antibodies

[0152] Example 4 Affinity determination of chimeric antibodies and caninized antibodies

[0153] The affinity of 3334 chimeric antibodies and 9 3334 caninized antibodies was determined using a Gator instrument based on bio-layer interferometry (BLI) technology. First, the antibody was biotinylated (ThermoFisher, 21343), and the biotinylated detection antibody was diluted to 100nM and added to a black 96-well plate as the stationary phase; then the analyte (canine PD-1-His) was serially diluted (200, 100, 50, 25, 12.5, 6.25, 3.13nM) and added to the black 96-well plate; finally, a streptavidin probe (Gator) was used to detect the binding of PD-1-His to the analyte. TM Streptavidin Probes) were used to determine the affinity of the antibody for canine PD-1 according to the mobile procedure of equilibrium, stationary phase, equilibrium, analyte, equilibrium.

[0154] The results are shown in Table 2. The affinity of the 3334 chimeric antibody is 8.76E-10. The affinity of the 3334 caninized antibody is slightly weaker than that of the 3334 chimeric antibody, but the change is not obvious.

[0155] Table 2 Affinity data of 3334 chimeric antibody and 3334 caninized antibody Note: E is scientific notation, which is ×10. The value after E is the power of 10.

[0156] Example 5 In vitro activity determination of chimeric antibodies and caninized antibodies

[0157] 1. Detection of in vitro biological activity of new antibodies using canine PD-1 / PD-L1 luciferase reporter gene cell lines

[0158] Canine PD-1 / PD-L1 luciferase reporter gene monoclonal cell lines, Canine_PD-L1 aAPC CHO-K1 and Canine_PD-1Reporter Jurkat, based on the canine PD-1 / PD-L1 signaling pathway, were constructed to evaluate the ability of antibodies to affect the PD-1 / PD-L1 signaling pathway. The tested antibodies were 3334 chimeric antibodies and 9 caninized antibodies.

[0159] First, the target cells Canine_PD-L1 aAPC CHO-K1 Cell Line were plated in a 96-well cell plate at a density of 1E4 cells / well 16-24 hours in advance. Then, 50 μL 2E6 cells / mL effector cells Canine_PD-1Reporter Jurkat Cell Line were incubated with 50 μL of a series of concentrations of 3334 chimeric antibodies and caninized antibodies for 1 hour. Afterwards, the culture medium of the target cells in the 96-well cell plate was removed, and the mixture of the above-mentioned effector cells and antibodies was added, and the incubation continued for 16 hours. Finally, the suspended cells in the supernatant were taken, 50 μL one-glo enzyme reaction substrate (Promega, E6120) was added, and after incubation at room temperature in the dark for 15 minutes, the fluorescence signal was detected on the machine. EC was calculated using Prism software. 50 .

[0160] The results are shown in Table 3 and Figure 2. 3334 chimeric antibodies and 9 caninized antibodies can effectively block the PD-1 / PD-L1 signaling pathway. 50 The values ​​are between 0.36 and 1.23 μg / mL.

[0161] Table 3 In vitro cell biological activity data of 3334 chimeric antibody and caninized antibody

[0162] 2. Further testing the in vitro biological activity of 3334 chimeric antibodies and caninized antibodies using T cell activation assays

[0163] Healthy adult beagle dogs were selected for whole blood collection, and canine PBMCs were isolated using Ficoll (GE Healthcare, 17-1440-02) density gradient centrifugation. Canine PBMCs were plated into 96-well plates at a density of 4E5 cells / well and stimulated with 10 ng / mL of Staphylococcus aureus enterotoxin B (SEB, Toxin Technology, BT202) as a superantigen. A negative control (canIgG B), 3334 chimeric antibody, and caninized antibody were also added. The cells were cultured in a 37°C CO2 incubator for 72 hours, and the cell supernatants were collected by centrifugation. The supernatants were assayed for cIFN-γ (R&D, DY781B) and cIL-2 (R&D, DY1815) using ELISA kits.

[0164] The results, as shown in Figure 3, show that in vitro, the chimeric 3334 antibody and nine caninized antibodies all increased T cell cIFN-γ and cIL-2 secretion, with significant differences compared to the negative control. This suggests that the 3334 antibody effectively blocks the canine T cell activation inhibitory pathway (cPD-1 / cPD-L1) in vitro, thereby enhancing T cell activation and demonstrating biological activity.

[0165] Example 6 In vivo efficacy evaluation of 3334 chimeric antibody and 3334-H1K2-canIgG B antibody

[0166] Previous studies have shown that canine PD-1 can cross-bind with human PD-L1 (hPD-L1). Therefore, the anti-tumor effects of the 3334 chimeric antibody and 3334-H1K2-canIgG B were further evaluated using a female canine PD-1 C57BL / 6 transgenic mouse model subcutaneously transplanted with the colon cancer MC38 / hPD-L1 recombinant cell line.

[0167] MC38 / hPD-L1 cells were collected during the exponential growth phase and resuspended in PBS buffer to 1E7 / mL. 0.1 mL of MC38 / hPD-L1 cells were subcutaneously inoculated on the right side of the back of canine PD-1 C57BL / 6 transgenic mice. All animals were weighed and tumor volumes were measured with a vernier caliper. The tumors were grown to an average volume of 102.10 mm. 3 The mice were randomly divided into groups according to the size of the tumor and given drugs. The experimental groups were negative control group (vehicle control), 3334 chimeric antibody group and 3334-H1K2-canIgG B antibody group, with 6 mice in each group. Drug administration began on the day of grouping (day 0). The corresponding antibody was injected into the tail vein of the mice in the antibody group at 3 mg / kg, and the same volume of vehicle was injected into the tail vein of the mice in the negative control group. The frequency of administration was 3 times a week for a total of 3 weeks. After administration, the activity, food and water intake, weight gain and loss, eyes, fur and other abnormalities of the experimental animals were observed every day, and the weight and tumor size of the mice were measured three times a week. Tumor volume calculation formula: Tumor volume (mm 3 )=1 / 2×(a×b 2 ) (where a represents the major diameter and b represents the minor diameter). The experiment was terminated after 9 administrations.

[0168] The anti-tumor effect of the 3334 antibody was evaluated using absolute tumor proliferation rate (T / C%) and absolute tumor inhibition rate (TGI%). T / C% = Ti / Ci × 100%, TGI% = (Ci-Ti) / Ci × 100%, where Ti represents the average tumor volume of the dosing group on day i, and Ci represents the average tumor volume of the negative control group on day i.

[0169] The results are shown in Figures 4-6. Figures 4 and 5 indicate that the weight of mice in each group increased during the drug administration period, and there was no significant difference in weight change among the groups. No accidental death of mice occurred during the treatment process, indicating that each drug administration group was well tolerated and had no significant toxic reactions. Figure 6 shows the tumor growth of each group. Compared with the negative control group, the tumor growth rate of mice in the 3334 antibody group and the 3334-H1K2-canIgG B antibody group was significantly slowed.

[0170] The statistical results of T / C% and TGI% after the last administration are shown in Table 4. The mice in the 3334 chimeric antibody and 3334-H1K2-canIgG B antibody groups survived on day 19, with an average tumor volume of 1333.08 mm 3 and 1044.91mm 3 , TGI were 55.54% and 65.15% respectively, which were statistically significant and extremely significant differences compared with the negative control group.

[0171] Table 4 Data on inhibition of mouse MC38 tumor growth by 3334 chimeric antibody and 3334-H1K2-canIgG B antibody

[0172] Example 7 In vivo efficacy evaluation of 3334 chimeric antibody and 3334-H1K2-canIgG B antibody in target animals

[0173] This example is used to illustrate the therapeutic effects of 3334 and 3334-H1K2-canIgG B on canine malignant tumors.

[0174] A single-arm study was conducted in 40 canine tumor-bearing dogs (20 with melanoma and 20 with mast cell tumors) from a Beijing veterinary hospital. The efficacy of the 3334 chimeric antibody and the 3334-H1K2-canIgG B antibody in treating these malignant tumors was studied. The dogs included Schnauzers, Labradors, Chihuahuas, and Golden Retrievers, with an average age of 12 years (range 10-15 years). All dogs had advanced malignant tumors.

[0175] All dogs were divided into two groups: the 3334 chimeric antibody group and the 3334-H1K2-canIgG B group. Each group included 10 dogs with melanoma and 10 dogs with mast cell tumors. All dogs received an intravenous infusion of the corresponding 3334 chimeric antibody and 3334-H1K2-canIgG B antibody (diluted in normal saline for injection) at a dose of 5 mg / kg body weight every two weeks for eight consecutive doses, with the study terminated one week after the last dose. Tumor burden was assessed by gross examination and computed tomography scans, and tumor size was measured every two weeks to assess efficacy. The objective response rate (ORR), complete response rate (CR), and partial response rate (PR) were compared. ORR = dogs with complete response and partial response / total dogs × 100%; CR = dogs with disappearance of all detectable tumors / total dogs × 100%; PR = dogs with at least a 30% reduction in the sum of the largest diameters of target lesions / total dogs × 100%.

[0176] The preliminary clinical treatment effects are shown in Table 5 below: both 3334 chimeric antibody and 3334-H1K2-canIgG B have good anti-tumor efficacy; and the therapeutic effect of 3334-H1K2-canIgG B is better than that of 3334 chimeric antibody.

[0177] Table 5 Clinical anti-tumor efficacy data of 3334 and 3334-H1K2-canIgG B

[0178] The above results show that the antibodies obtained by screening using the present invention have high affinity and high activity, and achieve the purpose of treating canine tumor diseases by blocking the binding of canine PD-1 and its ligand.

[0179] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

[0180] Sequence Listing

Claims

1. An anti-PD-1 antibody or antigen-binding fragment, characterized in that: Capable of binding to canine PD-1, the antibody or antigen-binding fragment comprises CDR1, 2, 3 of the heavy chain variable region and / or CDR1, 2, 3 of the light chain variable region; wherein the antibody or antigen-binding fragment comprises an amino acid sequence having at least 85% identity with at least one of the following CDR sequences: the CDR sequence of the heavy chain variable region is selected from SEQ ID NOs: 2-4, 8-11, 14, and the CDR sequence of the light chain variable region is selected from SEQ ID NOs: 5-7, 12-13, 15.

2. The antibody or antigen-binding fragment according to claim 1, characterized in that The antibody or antigen-binding fragment comprises at least one of the following CDR sequences: the CDR sequence of the heavy chain variable region is selected from SEQ ID NOs: 2-4, 8-11, and 14, and the CDR sequence of the light chain variable region is selected from SEQ ID NOs: 5-7, 12-13, and 15.

3. An anti-PD-1 antibody or antigen-binding fragment, characterized in that: Capable of binding to canine PD-1, the antibody or antigen-binding fragment comprises CDR1, 2, 3 of the heavy chain variable region and / or CDR1, 2, 3 of the light chain variable region; the heavy chain variable region is selected from SEQ ID NOs: 16, 18-20, and the light chain variable region is selected from SEQ ID NOs: 17, 21-23.

4. The antibody or antigen-binding fragment according to claim 1, characterized in that The antibody or antigen-binding fragment comprises a heavy chain variable region and / or a light chain variable region, wherein The heavy chain variable region comprises CDR1 shown in SEQ ID NO:2, CDR2 shown in SEQ ID NO:3, and CDR3 shown in SEQ ID NO:4, and the light chain variable region comprises CDR1 shown in SEQ ID NO:5, CDR2 shown in SEQ ID NO:6, and CDR3 shown in SEQ ID NO:7; or The heavy chain variable region comprises CDR1 shown in SEQ ID NO:8, CDR2 shown in SEQ ID NO:9, and CDR3 shown in SEQ ID NO:4, and the light chain variable region comprises CDR1 shown in SEQ ID NO:5, CDR2 shown in SEQ ID NO:6, and CDR3 shown in SEQ ID NO:7; or The heavy chain variable region comprises CDR1 shown in SEQ ID NO:14, CDR2 shown in SEQ ID NO:10 and CDR3 shown in SEQ ID NO:11, and the light chain variable region comprises CDR1 shown in SEQ ID NO:12, CDR2 shown in SEQ ID NO:13 or 15 and CDR3 shown in SEQ ID NO:

7.

5. The antibody or antigen-binding fragment according to claim 1, characterized in that The antibody or antigen-binding fragment comprises a heavy chain variable region and / or a light chain variable region: a heavy chain variable region as shown in SEQ ID NO: 16, a light chain variable region as shown in SEQ ID NO: 17; Or, the heavy chain variable region shown in SEQ ID NO: 18, the light chain variable region shown in SEQ ID NO: 21; Or, the heavy chain variable region shown in SEQ ID NO: 18, the light chain variable region shown in SEQ ID NO: 22; Or, the heavy chain variable region shown in SEQ ID NO: 18, the light chain variable region shown in SEQ ID NO: 23; Or, the heavy chain variable region shown in SEQ ID NO: 19, the light chain variable region shown in SEQ ID NO: 21; Or, the heavy chain variable region shown in SEQ ID NO: 19, the light chain variable region shown in SEQ ID NO: 22; Or, the heavy chain variable region shown in SEQ ID NO: 19, the light chain variable region shown in SEQ ID NO: 23; Or, the heavy chain variable region shown in SEQ ID NO:20, and the light chain variable region shown in SEQ ID NO:21; Or, the heavy chain variable region shown in SEQ ID NO:20, the light chain variable region shown in SEQ ID NO:22; Or, the heavy chain variable region shown in SEQ ID NO:20, and the light chain variable region shown in SEQ ID NO:

23.

6. The antibody or antigen-binding fragment according to claim 1, characterized in that The heavy chain constant region of the antibody or antigen-binding fragment is a canine IgG B mutant, and the amino acid sequence is shown in SEQ ID NO:24; the light chain constant region of the antibody or antigen-binding fragment is a canine kappa chain domain constant region, and the amino acid sequence is shown in SEQ ID NO:

25.

7. The antibody or antigen-binding fragment according to claim 1, characterized in that The antibody or antigen-binding fragment comprises a full-length heavy chain and / or a full-length light chain. The full-length heavy chain sequence used by the antibody or antigen-binding fragment is selected from any one of SEQ ID NOs: 26, 28-30, and the full-length light chain used by the antibody or antigen-binding fragment is selected from any one of SEQ ID NOs: 27, 31-33.

8. The antibody or antigen-binding fragment according to claim 1, characterized in that The full length of the heavy chain of the antibody or antigen-binding fragment is shown in SEQ ID NO: 26, and the full length of the light chain is shown in SEQ ID NO: 27; Or, the full length of the heavy chain is as shown in SEQ ID NO:28, and the full length of the light chain is as shown in SEQ ID NO:31; Or, the full length of the heavy chain is as shown in SEQ ID NO:28, and the full length of the light chain is as shown in SEQ ID NO:32; Or, the full length of the heavy chain is as shown in SEQ ID NO:28, and the full length of the light chain is as shown in SEQ ID NO:33; Or, the full length of the heavy chain is as shown in SEQ ID NO:29, and the full length of the light chain is as shown in SEQ ID NO:31; Or, the full length of the heavy chain is as shown in SEQ ID NO:29, and the full length of the light chain is as shown in SEQ ID NO:32; Or, the full length of the heavy chain is as shown in SEQ ID NO:29, and the full length of the light chain is as shown in SEQ ID NO:33; Or, the full length of the heavy chain is as shown in SEQ ID NO:30, and the full length of the light chain is as shown in SEQ ID NO:31; Or, the full length of the heavy chain is as shown in SEQ ID NO:30, and the full length of the light chain is as shown in SEQ ID NO:32; Alternatively, the full length of the heavy chain is as shown in SEQ ID NO:30, and the full length of the light chain is as shown in SEQ ID NO:

33.

9. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, and the nucleic acid molecule is DNA or RNA.

10. An expression vector, characterized in that: The nucleotide sequence of the expression vector comprises the nucleic acid molecule of claim 9.

11. A recombinant cell, characterized in that The recombinant cell is obtained by introducing the expression vector according to claim 10 into a host cell.

12. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the antibody or antigen-binding fragment according to any one of claims 1 to 8, or the nucleic acid molecule according to claim 9, or the expression vector according to claim 10, or the recombinant cell according to claim 11.

13. Use of the antibody or antigen-binding fragment of any one of claims 1 to 8, or the nucleic acid molecule of claim 9, or the expression vector of claim 10, or the recombinant cell of claim 11, or the pharmaceutical composition of claim 12 in preventing and / or treating mammalian tumors; or in preparing a drug for preventing and / or treating mammalian tumors; or in detecting PD-1 protein.

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