Anti-canine PD-1 antibody and application thereof

Abstract

The invention relates to the field of antibodies, and discloses an anti-canine PD-1 antibody and application thereof in prevention and treatment of mammal tumors, in particular to application in prevention and treatment of canine tumors. The high-affinity and high-activity canine PD-1 antibody can achieve the purpose of treating canine tumor diseases by blocking the binding of the canine PD-1 and the ligand of the canine PD-1.

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies, and particularly to an anti-canine PD-1 antibody and its uses. Background Art

[0002] Tumors are common and frequently-occurring diseases in humans and animals. Among them, 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 tumors, and the proportion of dogs over 10 years old suffering from tumors is as high as over 50%. Currently, the clinical treatment methods for canine tumors mainly include surgery, chemotherapy, and radiotherapy. Common chemotherapy drugs include cyclophosphamide, doxorubicin, vincristine, and prednisolone, etc. These all have disadvantages such as poor tumor specificity and large toxic and side effects. Immunotherapy is a new treatment method. Immunotherapy may be effective and have small 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 extend immunotherapy to animal tumors.

[0003] Programmed cell death receptor 1 (PD-1) is a type I transmembrane glycoprotein with a size of approximately 55 kDa and belongs to the CD28 superfamily of receptors. It is mainly expressed on the surface 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 the activation of T cells, thereby protecting the body from attack by the autoimmune system; however, a variety of solid tumors and some hematological tumors, including melanoma, breast cancer, various digestive system tumors, lymphoma, leukemia, etc., also highly express PD-L1 on cancer cells. The PD-L1 on the tumor cell membrane binds to PD-1 on T cells, inhibiting the activation of T cells, thereby successfully escaping the recognition and attack of the body's immune system and achieving immune escape of tumor cells. At the same time, studies have found that the expression of PD-L1 on tumor cells is related to poor prognosis in multiple tumor types. Therefore, blocking the binding of PD-1 and PD-L1 is an idea for tumor immunotherapy. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a canine PD-1 antibody with high affinity and high activity, and by blocking the binding of canine PD-1 to its ligand, the purpose of treating canine tumor diseases is achieved.

[0005] To achieve the above-mentioned invention purpose, the technical solution adopted by the present invention is as follows:

[0006] In the first aspect of the present invention, there is provided a CDR sequence of an antibody or its antigen-binding fragment capable of binding to canine PD-1.

[0007] The antibody or antigen-binding fragment thereof that can bind 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 that can bind 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 sequences of the heavy chain variable region are selected from SEQ ID NO: 2-4, 8-11, 14, and the CDR sequences of the light chain variable region are selected from SEQ ID NO: 5-7, 12-13, 15.

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

[0010] Furthermore, the antibody or antigen-binding fragment thereof that can bind to canine PD-1 comprises a heavy chain variable region and / or a light chain variable region, and the sequences are: the heavy chain variable region shown in SEQ ID NO: 16 and the light chain variable region shown in SEQ ID NO: 17;

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

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

[0013] or, the heavy chain variable region shown in SEQ ID NO: 18 and 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 having at least 85% identity with the above.

[0021] Furthermore, the present invention provides an antibody or antigen-binding fragment thereof that can bind to canine PD-1. According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof that can bind 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: the CDR sequences of the heavy chain variable region are selected from SEQ ID NO:2-4, 8-11, 14, and the CDR sequences of the light chain variable region are selected from SEQ ID NO:5-7, 12-13, 15, or an amino acid sequence having at least 85% identity therewith.

[0022] Furthermore, the antibody or antigen-binding fragment thereof that can bind 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] A heavy chain variable region CDR1 selected from any one of SEQ ID NO:2, 8, 14, a heavy chain variable region CDR2 selected from any one of SEQ ID NO:3, 9, 10, and a heavy chain variable region CDR3 selected from any one of SEQ ID NO:4, 11; a light chain variable region CDR1 selected from any one of SEQ ID NO:5, 12, a light chain variable region CDR2 selected from any one of SEQ ID NO:6, 13, 15, and a light chain variable region CDR3 selected from 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 includes heavy chain variable region CDRs and an amino acid sequence having an identity of 85% or more: the heavy chain variable region CDR1, CDR2, and CDR3 sequences shown by the amino acid sequences of SEQ ID NO:2, 3, and 4 respectively (the above sequences are obtained according to the Kabat coding system); the heavy chain variable region CDR1, CDR2, and CDR3 sequences shown by the amino acid sequences of SEQ ID NO:8, 9, and 4 respectively (the above sequences are obtained according to the Chothia coding system); the heavy chain variable region CDR1, CDR2, and CDR3 sequences shown by the amino acid sequences of SEQ ID NO:14, 10, and 11 respectively (the above sequences are obtained according to the IMGT coding system).

[0025] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof capable of binding to canine PD-1 includes light chain variable region CDRs and an amino acid sequence having an identity of 85% or more: the light chain variable region CDR1, CDR2, and CDR3 sequences shown by the amino acid sequences of SEQ ID NO: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 shown by the amino acid sequences of SEQ ID NO:12, 13, and 7 respectively, and the light chain variable region CDR1, CDR2, and CDR3 sequences shown by the amino acid sequences of SEQ ID NO:12, 15, and 7 respectively (the above sequences are obtained according to the IMGT coding system but obtained by analyzing different databases).

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

[0027] The CDR1, CDR2, and CDR3 sequences of the heavy chain variable region as shown by the amino acid sequences of SEQ ID NO: 2, 3, and 4 respectively, and the CDR1, CDR2, and CDR3 sequences of the light chain variable region as shown by the amino acid sequences of SEQ ID NO: 5, 6, and 7 respectively (the above sequences are obtained according to the Kabat coding system); or, the CDR1, CDR2, and CDR3 sequences of the heavy chain variable region as shown by the amino acid sequences of SEQ ID NO: 8, 9, and 4 respectively, and the CDR1, CDR2, and CDR3 sequences of the light chain variable region as shown by the amino acid sequences of SEQ ID NO: 5, 6, and 7 respectively (the above sequences are obtained according to the Chothia coding system); or, the CDR1, CDR2, and CDR3 sequences of the heavy chain variable region as shown by the amino acid sequences of SEQ ID NO: 14, 10, and 11 respectively, and the CDR1, CDR2, and CDR3 sequences of the light chain variable region as shown by the amino acid sequences of SEQ ID NO: 12, 13, and 7 respectively; or, the CDR1, CDR2, and CDR3 sequences of the heavy chain variable region as shown by the amino acid sequences of SEQ ID NO: 14, 10, and 11 respectively, and the CDR1, CDR2, and CDR3 sequences of the light chain variable region as shown by the amino acid sequences of SEQ ID NO: 12, 15, and 7 respectively.

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

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

[0030] The antibody or its antigen-binding fragment that can bind to canine PD-1 includes a heavy chain variable region and / or a light chain variable region. The heavy chain variable region is selected from those shown in SEQ ID NO: 16, 18 - 20, and the light chain variable region is selected from those shown in SEQ ID NO: 17, 21 - 23;

[0031] Furthermore, the antibody or its antigen-binding fragment that can bind to canine PD-1 can be:

[0032] The heavy chain variable region shown in SEQ ID NO: 16 and the light chain variable region shown in SEQ ID NO: 17;

[0033] Or, the heavy chain variable region shown in SEQ ID NO: 18 and the light chain variable region shown in SEQ ID NO: 21;

[0034] or, a heavy chain variable region as shown in SEQ ID NO:18 and a light chain variable region as shown in SEQ ID NO:22;

[0035] or, a heavy chain variable region as shown in SEQ ID NO:18 and a light chain variable region as shown in SEQ ID NO:23;

[0036] or, a heavy chain variable region as shown in SEQ ID NO:19 and a light chain variable region as shown in SEQ ID NO:21;

[0037] or, a heavy chain variable region as shown in SEQ ID NO:19 and a light chain variable region as shown in SEQ ID NO:22;

[0038] or, a heavy chain variable region as shown in SEQ ID NO:19 and a light chain variable region as shown in SEQ ID NO:23;

[0039] or, a heavy chain variable region as shown in SEQ ID NO:20 and a light chain variable region as shown in SEQ ID NO:21;

[0040] or, a heavy chain variable region as shown in SEQ ID NO:20 and a light chain variable region as shown in SEQ ID NO:22;

[0041] or, a heavy chain variable region as shown in SEQ ID NO:20 and a light chain variable region as shown in SEQ ID NO:23;

[0042] or an amino acid sequence having at least 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity with the above.

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

[0044] The heavy chain constant region used by the antibody or its antigen-binding fragment that can bind to canine PD-1 can be canine IgG A, IgG B, IgG C, IgG D or a mutant sequence that ensures the same function. Further, preferably, it is a mutant sequence of IgG B. The specific amino acid sequence of the heavy chain constant region domain can be as shown in SEQ ID NO:24; the light chain constant region used by the antibody can be a canine kappa chain or lambda chain domain constant region sequence. Further, preferably, it is a canine kappa chain, and the amino acid sequence is as 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. Any constant region design that can achieve the prevention, treatment, detection, etc. effects based on the PD-1 target of the present invention is within the protection scope of the present invention.

[0046] The fourth aspect of the present invention provides the full-length sequence of an antibody or its antigen-binding fragment that can bind to canine PD-1.

[0047] The antibody or its antigen-binding fragment that can bind to canine PD-1 includes the full-length heavy chain and / or the full-length light chain. The full-length heavy chain sequence used is selected from any one of SEQ ID NO:26, 28-30, and the full-length light chain used for the antibody or antigen-binding fragment that can bind to canine PD-1 is selected from any one of SEQ ID NO:27, 31-33;

[0048] The affinity constant KD of the antibody or its antigen-binding fragment that can bind to canine PD-1 with canine PD-1 ranges from 8.76E-10 to 3.83E-09 M;

[0049] Furthermore, the full-length heavy chain of the antibody or antigen-binding fragment that can bind to canine PD-1 is as shown in SEQ ID NO:26, and the full-length light chain is as 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 heavy chain is as shown in SEQ ID NO:30, and the full-length light chain is as shown in SEQ ID NO:31;

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

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

[0059] Or an amino acid sequence having at least 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the above.

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

[0061] The antibody or antigen-binding fragment thereof that can bind to canine PD-1 includes at least one selected from monoclonal antibodies, polyclonal antibodies, multimeric antibodies, and CDR-grafted antibodies;

[0062] Optionally, the antibody or antigen-binding fragment thereof that can bind to canine PD-1 includes at least one selected from single-chain antibodies, Fab antibodies, Fv antibodies, single-chain antibodies, single-domain antibodies, and minimal recognition units; 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 bind to the target antigen with affinity.

[0063] Optionally, the antibody or antigen-binding fragment thereof that can bind to canine PD-1 includes at least one of Fab fragments, (Fab) 2 fragments, scFv-Fc fusion proteins, scFv-Fv fusion proteins, Fv fragments, and minimal recognition units.

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

[0065] The element can be a nucleic acid molecule that encodes the aforementioned antibody or antigen-binding fragment 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 protection scope of the present invention includes all nucleic acids and nucleotide sequences that can express the aforementioned amino acid sequences, including nucleic acid sequences deduced from various different codon preferences. Those skilled in the art can infer other nucleotide sequences that can express the aforementioned amino acid sequences using conventional techniques in the art. The following sequences are only examples:

[0066] Specifically, the heavy chain variable region sequence of the antibody or its antigen-binding fragment capable of binding to canine PD-1 may be the DNA coding sequences shown in SEQ ID NO: 49, 51-53, and the light chain variable region sequence of the antibody or its antigen-binding fragment capable of binding to canine PD-1 may be the DNA coding sequences shown in SEQ ID NO: 50, 54-56.

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

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

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

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

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

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

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

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

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

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

[0077] Specifically, the canine IgG B mutant of the heavy chain constant region of the antibody or antigen-binding fragment thereof that can bind to canine PD-1 may have a DNA coding sequence as shown in SEQ ID NO: 57, and the canine kappa chain of the light chain constant region may have a DNA coding sequence as 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 may be any one of SEQ ID NO: 59, 61-63, and the full-length DNA sequence of the light chain of the antibody may be any one of SEQ ID NO: 60, 64-66.

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

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

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

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

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

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

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

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

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

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

[0089] Specifically, the CDR amino acid sequences shown in SEQ ID NOs: 2-15, the corresponding DNA coding sequences in order can be those shown in SEQ ID NOs: 35-48.

[0090] The element can be a recombinant vector, including but not limited to a cloning vector, an expression vector, a shuttle vector, a viral vector (such as a lentiviral vector, an adeno-associated viral vector, a poxvirus-related vector, etc.), carrying the above 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, pcDNA3.4.

[0091] The element can be a recombinant cell, the recombinant cell carrying the above nucleic acid molecule, or a recombinant vector capable of expressing one of the aforementioned antibodies or antigen-binding fragments thereof that can bind 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, and the recombinant cell is preferably a mammalian cell. Including but not limited to Chinese hamster ovary cells (CHO K1), HEK293 cells, etc.

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

[0093] The pharmaceutical composition comprises the aforementioned antibody or its antigen-binding fragment 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 excipients and carriers.

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

[0096] 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 kit in the prevention and / or treatment of tumors, or use in the preparation of drugs, or use in the preparation of a detection kit for detecting PD-1 protein in a biological matrix; the tumor is a tumor of a mammal, including human, monkey, mouse (rat, mouse), horse, cow, sheep, pig, dog, cat, etc., and further preferably a tumor of a dog.

[0097] The types of tumors include, but are not limited to, "tumors", and refer to a group of diseases characterized by the development of abnormal cells that proliferate uncontrollably and have the ability to invade 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; hemangiosarcoma (HAS); granular cell tumor; mixed mesenchymal sarcoma; neurofibrosarcoma; undifferentiated sarcoma; myxosarcoma; chondrosarcoma (CSA); metaplastic chondrosarcoma (MLO); osteosarcoma (OSA); transmissible venereal tumor (TVT); mast cell tumor (MCT); lymphoma (LSA); ameloblastoma; ameloblastic adenoma; odontogenic epithelial calcifying tumor; odontoma; fibromyxoma; cementoma; odontogenic fibroma; acanthomatous epulis; ossifying epulis; fibromatous epulis; basal cell carcinoma; ceruminous adenoma; viral papilloma, salivary gland tumors, nasal lymphoma; ceruminous adenocarcinoma (CGC); benign oncocytic adenoma (rhabdomyoma); chondroma; osteoma; osteochondroma; tracheal carcinoma; leiomyoma; benign laryngeal tumors (e.g., rhabdomyoma or oncocytic adenoma); mediastinal tumors (e.g., thymoma and mediastinal lymphoma); malignant chest wall tumors (sarcoma); benign chest wall tumors (osteoma and chondroma); primary lung tumors; secondary (metastatic lung tumors); cardiac tumors (e.g., hemangiosarcoma; mesothelioma; myxoma; ectopic thyroid carcinoma); esophageal carcinoma; gastric tumors (e.g., gastric adenocarcinoma and lymphoma); gastric leiomyosarcoma; gastric leiomyoma; extramedullary plasmacytoma of the stomach; gastrointestinal mast cell tumor; non-lymphoid intestinal tumors; intestinal lymphoma; intestinal adenocarcinoma; extramedullary plasmacytoma; carcinoid; adenomatous polyp; colorectal adenocarcinoma; colorectal leiomyoma and leiomyosarcoma; perianal adenoma; perianal adenocarcinoma; anal sac adenocarcinoma; liver tumors (e.g., liver sarcoma, hepatocellular carcinoma or carcinoid); hepatocellular carcinoma (HCC); hepatocellular adenoma; hepatoblastoma; biliary tract carcinoma; cholangiocarcinoma; gallbladder tumors; pancreatic adenocarcinoma; breast tumors; malignant breast tumors (breast cancer); malignant mixed tumors; breast sarcoma; extraskeletal osteosarcoma of the breast; uterine tumors; vaginal and vulvar tumors; ovarian tumors (e.g., adenocystoma, granulosa cell tumor); gonadal stromal tumors (e.g., theca cell tumor and luteoma); germ cell tumors (dysgerminoma, teratoma or teratocarcinoma); 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 cell carcinoma, fibrosarcoma and hemangiosarcoma); ureteral tumors; bladder tumors; urethral tumors; cutaneous hemangioma; subungual squamous cell carcinoma; basal cell carcinoma (including basal cell carcinoma, basal cell epithelioma and basal cell-like tumors); ceruminous gland tumors; intraepidermal keratinizing epithelioma (keratoacanthoma); follicular tumors (trichoblastoma, trichoepithelioma and nail bed tumor); cutaneous plasma cell tumors; 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 sarcoma; histiocytic sarcoma (HS); osteosarcoma of the extremities; osteosarcoma of the axial skeleton; extraskeletal osteosarcoma; surface osteosarcoma; multilobular osteochondrosarcoma (MLO); multiple cartilaginous exostoses (MCE); lymphocytic leukemia (CLL); non-lymphocytic leukemia (granulocytic leukemia); chronic myelogenous leukemia (CML); acute myelogenous leukemia (AML); hemangiosarcoma of the spleen (HSA); sarcoma of the spleen (non-lymphoid, non-vascular); glioma; meningioma; gangliocytoma (pheochromocytoma and non-chromaffin paraganglioma); ganglioglioma; spinal cord tumors (OSA, CSA, FSA, HSA, hemangioendothelioma, myeloma); peripheral nerve sheath tumors (PNSTs); neuroepithelial tumors (ependymoma, medulloepithelioma, nephroblastoma, and spinal cord blastoma); intramedullary spinal tumors; epidural sarcoma; epidural sarcoma; schwannoma; tumors of the peripheral nervous system (e.g., neuroblastoma, medulloblastoma, retinoblastoma); eyelid tumors; meibomian adenoma; rhabdomyoblastoma; third eyelid tumor; conjunctival tumors; extraocular (cornea and sclera) tumors; orbital (the cavity surrounding the eyeball) and retrobulbar tumors; thyroid tumors; parathyroid tumors; APUD cell tumors (amine precursor uptake and decarboxylation cell tumors); insulinoma; glucagonoma; gastrinoma; adrenal cortical tumors; pheochromocytoma; pituitary tumors. Preferably melanoma, mast cell tumor, colon cancer.;

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

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

[0100] 2. The anti-PD-1 chimeric antibody and the canineized antibody of 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 the canineized antibody of 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 highly effective cancer treatment drugs. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0105] Figure 3 It is a graph showing the results of the in vitro activity detection of the 3334 chimeric antibody and the canineized antibody - T cell activation experiment.

[0106] Figure 4 It is a graph showing the results of the effects of the 3334 chimeric antibody and 3334-H1K2-canIgG B canineized antibody on the body weight of mice.

[0107] Figure 5 It is a graph showing the results of the effects of the 3334 chimeric antibody and 3334-H1K2-canIgG B canineized antibody on the body weight change rate of mice.

[0108] Figure 6 It is a graph showing the results of the effects of the 3334 chimeric antibody and 3334-H1K2-canIgG B canineized antibody on the tumor volume of mice. Detailed implementation manners

[0109] The following embodiments are used to illustrate the present invention, but do not limit the scope of the present invention.

[0110] The experimental methods used in the following embodiments are all conventional methods unless otherwise specified.

[0111] The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0112] The present invention will be described in detail below through embodiments in conjunction with the accompanying drawings, but it is not a limitation of the present invention and is only for illustrative purposes.

[0113] To better understand the present disclosure, some terms are first defined. The terms "first" and "second" are for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present invention, unless otherwise specified, the meaning of "a plurality" is two or more.

[0114] In this document, the term "comprising" or "including" is an open-ended expression, that is, it includes the content specified by the present invention, but does not exclude other aspects.

[0115] In this document, the terms "optionally", "optional" or "option" generally mean that the subsequent event or condition may or may not occur, and this description includes the case where the event or condition occurs, and the case where the event or condition does not occur.

[0116] In this document, 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, and is an immunoglobulin molecule capable of binding to a specific antigen. It includes two light chains with relatively low molecular weights and two heavy chains with relatively high molecular weights. The heavy chain (H chain) and the light chain (L chain) are connected by disulfide bonds to form a four-peptide chain molecule. Among them, the amino-terminal (N-terminal) amino acid sequence of the peptide chain varies greatly and is called the variable region (V region), and the carboxyl-terminal (C-terminal) is relatively stable and varies little, which is called the constant region (C region). The V regions of the L chain and the H chain are called VL and VH respectively. In the variable region, some regions have a higher degree of amino acid composition and arrangement variation, which is called the hypervariable region (HVR). The hypervariable region is the position where the antigen and the antibody bind, so it is also called the complementarity-determining region (CDR). There are three CDRs on both the heavy chain variable region and the light chain variable region.

[0117] In this document, the term "Fab antibody" generally refers to an antibody containing only the Fab molecule, which is composed of VH and CH1 of the heavy chain and the complete light chain, and the light chain and the heavy chain are connected by a disulfide bond.

[0118] In this document, the term "Fv antibody" generally refers to an antibody formed by non-covalent bonding of only the light chain variable region (VL) and the heavy chain variable region (VH), and is the smallest functional fragment in which the antibody molecule retains the complete antigen-binding site.

[0119] In this text, the terms "single-domain antibody", "nanobody", and "VHH antibody" are used interchangeably and were originally described as the antigen-binding immunoglobulin (variable) domain 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 contain only the variable heavy-chain region (VH) and the conventional CH2 and CH3 regions and specifically bind to antigens through the variable heavy-chain region.

[0120] In this text, the term "single-chain antibody" generally refers to an antibody formed by linking the variable heavy-chain region (VH) and the variable light-chain region (VL) with a linker peptide.

[0121] In this text, the terms "minimal 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 a complete antibody.

[0122] In this text, when the terms "identity", "homology", or "similarity" are used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, 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, D.C.)). 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. 70: 173-187 (1997)); and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215: 403-410). Computer programs utilizing 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, 8th edition, 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% identity with each reference sequence, and can be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity.

[0124] As used herein, an antibody that "binds to PD-1", "specifically binds to PD-1", or "anti-PD-1 antibody" refers to having a KD value of 1.0×10 -8 mol / L or lower. Binding affinity is measured using a standard binding assay, such as Biolayer Interferometry (Gator) using the extracellular domain of PD-1.

[0125] As used herein, the term "expression vector" generally refers to a nucleic acid molecule capable of self-replicating after insertion into a suitable host, which transfers the inserted nucleic acid molecule into host cells and / or between host cells. The expression vector may include vectors mainly for inserting DNA or RNA into cells, vectors mainly for replicating DNA or RNA, and vectors mainly for the transcription and / or translation of DNA or RNA for expression. The expression vector also includes vectors having multiple of the above functions. The expression vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, by culturing a suitable host cell containing the expression vector, the expression vector can produce the desired expression product. The expression vector carries the aforementioned nucleic acid molecule. When the above nucleic acid molecule is ligated to the vector, the nucleic acid molecule can be directly or indirectly linked to the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule, etc. Of course, these control elements can be directly from the vector itself or exogenous, that is, not from the vector itself. Of course, the nucleic acid molecule and the control elements are operably linked. "Operably linked" as used herein means that an exogenous gene is linked to a vector such that the control elements within the vector, such as transcriptional control sequences and translational control sequences, etc., can perform their intended functions of regulating the transcription and translation of the exogenous gene. Commonly used vectors can be, for example, plasmids, phages, etc. After the expression vector according to some specific embodiments of the present invention is introduced into a suitable recipient cell, under the mediation of the regulatory system, the expression of the aforementioned antibody or antigen-binding fragment can be effectively achieved, and thus a large amount of the antibody or antigen-binding fragment can be obtained in vitro.

[0126] As used herein, the term "recombinant cell" generally refers to a cell obtained by modifying or recombining the genetic material of a host cell using genetic engineering techniques or cell fusion techniques, which has unique traits with stable inheritance. Among them, the term "host cell" refers to a prokaryotic cell or a eukaryotic cell that can be introduced with a recombinant expression vector. The term "transformed" or "transfected" as used herein refers to the introduction of nucleic acids (such as vectors) into cells 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 target proteins.

[0127] As used herein, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical field. All methods include the step of combining the active ingredient with a carrier that constitutes one or more accessory ingredients. Generally, the composition is prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a finely divided solid carrier, or both.

[0128] As used herein, the term "pharmaceutically acceptable excipients" can include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for a particular target dosage form. Their use is also contemplated within the scope of the present invention, except to the extent that any conventional excipient is incompatible with the compounds of the present invention, such as any adverse biological effects produced or any interaction with any other component of the pharmaceutically acceptable composition in a harmful manner.

[0129] As used herein, the term "administering" refers to introducing a predetermined amount of a substance into a subject to be treated by a suitable means. The antibodies or antigen-binding fragments or pharmaceutical compositions of the present invention can be administered by any common route as long as it can reach the intended tissue. Various modes of administration are contemplated, including intraperitoneal, intravenous injection, intravenous infusion, intramuscular injection, subcutaneous injection, etc., but the present invention is not limited to these exemplified modes of administration. The subject to be treated can be a mammal, preferably a canine.

[0130] As used herein, the term "treatment" refers to obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing the disease and / or the adverse effects caused by the disease. "Treatment" as used herein encompasses diseases in mammals, particularly canines, including: (a) preventing the occurrence of a disease or disorder in an individual who is susceptible but not yet diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as reducing the symptoms associated with the disease. "Treatment" as used herein encompasses any administration of a drug or compound to an individual to treat, cure, alleviate, improve, reduce or inhibit the disease of the individual, including but not limited to administering a drug containing the compounds described herein to an individual in need.

[0131] Example 1 Preparation and Identification of Mouse Anti - Canine PD - 1 Antibody

[0132] 1. Antigen Preparation

[0133] Search for canine PD - 1 protein (UniProtKB: A0A024FCJ9) in the UniProt library and GenBank library. The amino acid sequence is shown as SEQ ID NO:1. Manually synthesize the corresponding nucleotide sequence with a His - tag and construct it into the pcDNA3.4 vector. Transiently transfect it into HEK293 cells. After 5 days of transfection, collect the cell supernatant and perform affinity chromatography on the supernatant through a Ni column to elute and obtain the purified canine PD - 1 recombinant protein.

[0134] 2. Mouse Immunization

[0135] Use 7 - 8 mg of canine PD - 1 recombinant protein as the antigen to immunize Balb / c mice or C57 mice. After four immunizations, collect the serum and analyze the antibody titer using enzyme - linked immunosorbent assay (ELISA).

[0136] 3. RNA Extraction and Reverse Transcription of Spleen

[0137] Extract the RNA of mouse spleen tissue using the Trizol method, identify the integrity of the RNA by denaturing agarose gel electrophoresis, and measure the concentration and OD 260 / OD 280 of the RNA using a ultra - micro spectrophotometer. Select the RNA with qualified quality and perform RNA reverse transcription according to the instructions of reverse transcriptase Superscript II reverse transcriptase (Thermo Scientific) to obtain cDNA.

[0138] 4. Antibody Library Construction and Phage Display

[0139] Amplify the heavy and light chains of the antibody from the cDNA, and construct scFv antibody library and Fab antibody library respectively. The library capacity reaches 1×10 8 CFU / mL. Electroporate the antibody library into TG1 Escherichia coli, then add helper phage M13KO7 to infect TG1 Escherichia coli. After overnight culture, precipitate the phage using PEG / NaCl to prepare the phage - displayed antibody library.

[0140] 5. Screening of Phage - Displayed Antibody Library

[0141] Using an immunotube and a magnetic bead screener, solid-phase screening and solid-phase and liquid-phase cross-screening were performed on the phage display antibody library, respectively. For solid-phase screening, the immunotube was coated with canine PD-1 recombinant protein, and for liquid-phase screening, Dynabeads magnetic beads were coated with biotinylated canine PD-1 recombinant protein. After three rounds of screening, Phage ELISA was used to identify the enriched phage population and the monoclonal antibodies isolated from the population.

[0142] 6. Construction, Expression and Purification of Antibodies

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

[0144] Then, the murine antibody heavy chain variable region (SEQ ID NO: 16) was fused with the heavy chain constant region (including CH1, CH2, and CH3) of the canine IgG B mutant, and the light chain variable region (SEQ ID NO: 17) was fused with the canine kappa chain constant region (CL), and 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, and the purified chimeric antibody was obtained by recombinant protein A affinity chromatography. The chimeric antibody was numbered 3334. The full length of the heavy chain of the chimeric antibody is shown as SEQ ID NO: 26, and the full length of the light chain is shown as SEQ ID NO: 27.

[0145] Example 2 Screening of Antibodies Blocking the Binding of Canine PD-1 to Its Ligand

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

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

[0148] As Figure 1 The results showed that as the concentration of the 3334 chimeric antibody increased, the APC positive cell rate gradually decreased, indicating that the 3334 chimeric antibody could block the binding of canine PD-1 to PD-L1.

[0149] Example 3 Construction of a Canine Antibody

[0150] Based on CDR grafting and back mutation of framework region homology, the 3334 chimeric antibody was canineized. First, canineized heavy chain variable region variants (SEQ ID NOs: 18 - 20) and canineized light chain variable region variants (SEQ ID NOs: 21 - 23) were constructed. The variable regions of the canineized antibodies were freely combined from the canineized variable region heavy chain and canineized variable region light chain of 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 as 3334-H1K1, 3334-H1K2, and 3334-H1K3 respectively. The constant regions of the canineized antibodies consisted of the constant domain of the canine IgG B mutant and the canine kappa constant region domain. For example, 3334-H1K1-canIgG B had a canineized variable region heavy chain H1, a canineized variable region light chain K1, a constant region of the canine IgG B mutant, and a canine kappa constant region. The specific sequence combination names are shown in Table 1. The constant regions of the antibodies shown were the constant domain of the heavy chain of the canine IgG B mutant, with the amino acid sequence shown in SEQ ID NO: 24, and the constant region domain of the canine kappa chain, with the amino acid sequence shown in SEQ ID NO: 25.

[0151] Table 1 Sequence Number Table of 3334 Chimeric and Canineized Antibodies

[0152]

[0153] Example 4 Affinity Determination of Chimeric Antibodies and Caninized Antibodies

[0154] The affinity of 3334 chimeric antibody and 9 3334 caninized antibodies was determined using a Gator instrument based on the Bio-Layer Interferometry (BLI) technique. First, the antibodies were biotinylated (ThermoFisher, 21343), and the biotinylated detection antibodies were diluted to 100 nM 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, 3.13 nM) and added to the black 96-well plate. Finally, the streptavidin probe (Gator TM Streptavidin Probes) was used to measure the affinity of the antibody for Canine PD-1 according to the moving program of equilibration, stationary phase, equilibration, analyte, equilibration.

[0155] The results are shown in Table 2. The affinity of the 3334 chimeric antibody was 8.76E-10. The 3334 caninized antibody was slightly weaker than the 3334 chimeric antibody, but the change was not obvious.

[0156] Table 2 Affinity Data of 3334 Chimeric Antibody and 3334 Caninized Antibody

[0157]

[0158] Note: E is the scientific notation, which is ×10, and the value after E is the power of 10.

[0159] Example 5 In Vitro Activity Determination of Chimeric Antibodies and Caninized Antibodies

[0160] 1. Detection of the in vitro biological activity of new antibodies using a Canine PD-1 / PD-L1 luciferase reporter gene cell line

[0161] Monoclonal cell lines of Canine_PD-L1 aAPC CHO-K1 and Canine_PD-1Reporter Jurkat based on the Canine PD-1 / PD-L1 signaling pathway were constructed respectively to evaluate the ability of the antibodies to affect the PD-1 / PD-L1 signaling pathway. The detection antibodies were 3334 chimeric antibody and 9 caninized antibodies.

[0162] First, seed the target cells Canine_PD-L1 aAPC CHO-K1 Cell Line in a 96-well cell culture plate at a density of 1E4 cells / well 16 - 24 h in advance. Then, incubate 50 μL of the effector cells Canine_PD-1Reporter Jurkat Cell Line at 2E6 cells / mL with 50 μL of a series of concentrations of the chimeric antibody 3334 and the humanized antibody for 1 h. After that, remove the culture medium of the target cells in the 96-well cell culture plate, add the above mixture of effector cells and antibody, and continue to incubate for 16 h. Finally, take the suspended cells in the supernatant, add 50 μL of one-glo enzyme reaction substrate (Promega, E6120), incubate at room temperature in the dark for 15 min, and then detect the fluorescence signal on the machine. Use Prism software to calculate the EC 50 .

[0163] As shown in Table 3 and Figure 2 , both the chimeric antibody 3334 and 9 humanized antibodies can effectively block the PD-1 / PD-L1 signaling pathway, and the EC 50 values are in the range of 0.36 - 1.23 μg / mL.

[0164] Table 3 In vitro cell biological activity data of chimeric antibody 3334 and humanized antibodies

[0165] Anti-canine PD-1 antibody <![CDATA[EC 50 (μg / mL)]]> 3334-canIgG B 0.36 3334-H1K1-canIgG B 0.72 3334-H1K2-canIgG B 0.37 3334-H1K3-canIgG B 0.59 3334-H2K1-canIgG B 1.07 3334-H2K2-canIgG B 0.42 3334-H2K3-canIgG B 1.06 3334-H3K1-canIgG B 1.23 3334-H3K2-canIgG B 0.55 3334-H3K3-canIgG B 0.75

[0166] 2. Further detect the in vitro biological activities of the chimeric antibody 3334 and the humanized antibodies using the T cell activation assay

[0167] Select healthy adult beagle dogs, collect whole blood, and use Ficoll (GE Healthcare, 17 - 1440 - 02) density gradient centrifugation to extract canine PBMC. Add the canine PBMC to a 96-well cell culture plate at a density of 4E5 cells / well, add 10 ng / mL of staphylococcal enterotoxin B (SEB, Toxin Technology, BT202) as a superantigen for stimulation, and simultaneously add a negative control (canIgG B), the chimeric antibody 3334, and the humanized antibodies, and culture in a 37 °C carbon dioxide incubator for 72 h. Centrifuge to collect the cell supernatant. Use an ELISA detection kit to detect the contents of cIFN-γ (R&D, DY781B) and cIL-2 (R&D, DY1815) in the supernatant respectively.

[0168] The results are as Figure 3As shown, at the in vitro level, both the 3334 chimeric antibody and 9 canineized antibodies can increase the secretion of T cell cIFN-γ and cIL-2, and the difference is significant compared with the negative control. This indicates that the 3334 antibody can effectively block the canine T cell activation inhibition pathway (cPD-1 / cPD-L1) in vitro, thereby enhancing T cell activation and having biological activity.

[0169] Example 6 In Vivo Pharmacodynamic Evaluation of 3334 Chimeric Antibody and 3334-H1K2-canIgG B Antibody

[0170] Previous verification showed that canine PD-1 can cross-bind with human PD-L1 (hPD-L1). Therefore, a female canine PD-1 C57BL / 6 transgenic mouse animal model transplanted subcutaneously with the colon cancer MC38 / hPD-L1 recombinant cell line was used for detection to further evaluate the antitumor effects of the 3334 chimeric antibody and 3334-H1K2-canIgG B.

[0171] Collect MC38 / hPD-L1 cells in the exponential growth phase, resuspend them with PBS buffer to 1E7 / mL, and subcutaneously inoculate 0.1 mL of MC38 / hPD-L1 cells on the right dorsal side of female canine PD-1 C57BL / 6 transgenic mice. Weigh all the animals and measure the tumor volume with a vernier caliper. When the tumor grows to an average volume of 102.10 mm 3 , randomly group the animals according to the tumor size and administer drugs. The experimental groups are the negative control group (vehicle control), the 3334 chimeric antibody group, and the 3334-H1K2-canIgG B antibody group, with 6 mice in each group. Start administering drugs on the day of grouping (day 0). Inject the corresponding antibody at a dose of 3 mg / kg into the tail vein of the mice in the antibody groups, and inject an equal volume of vehicle into the tail vein of the mice in the negative control group. The administration frequency is 3 times a week for a total of 3 weeks. After drug administration, observe the activity, food intake, water intake, weight gain and loss, eyes, hair, and other abnormal conditions of the experimental animals every day, and measure the body weight and tumor size of the mice three times a week. The tumor volume calculation formula: Tumor volume (mm 3 ) = 1 / 2 × (a × b 2 ) (where a represents the long diameter and b represents the short diameter). End the experiment after administering drugs 9 times.

[0172] Use the absolute tumor growth rate (T / C%) and absolute tumor inhibition rate (TGI%) to evaluate the antitumor effects of the 3334 antibody. T / C% = Ti / Ci × 100%, TGI% = (Ci - Ti) / Ci × 100%, where Ti represents the average tumor volume of the drug administration group on the i-th day, and Ci represents the average tumor volume of the negative control group on the i-th day.

[0173] The results are as Figures 4-6 shown, Figure 4 andFigure 5 It was shown that the body weights of mice in each group increased during drug administration, and there were no significant differences in body weight changes among groups. At the same time, no accidental deaths of mice occurred during the treatment process, indicating that each drug administration group had good tolerance and no significant toxic reactions. Figure 6 The tumor growth conditions of each group were shown. Compared with the negative control group, the tumor growth rates of mice in the 3334 antibody group and the 3334-H1K2-canIgG B antibody group were significantly slowed down.

[0174] The statistical results of T / C% and TGI% after the last drug administration are shown in Table 4. Mice in the 3334 chimeric antibody and 3334-H1K2-canIgG B antibody groups survived on the 19th day, and the average tumor volumes were 1333.08 mm 3 and 1044.91 mm 3 , and the TGIs were 55.54% and 65.15% respectively. Statistically, there were significant and highly significant differences compared with the negative control group.

[0175] Table 4 Data of 3334 chimeric antibody and 3334-H1K2-canIgG B antibody inhibiting the growth of MC38 tumors in mice

[0176]

[0177] Example 7 In Vivo Pharmacodynamic Evaluation of 3334 Chimeric Antibody and 3334-H1K2-canIgG B Antibody in Target Animals

[0178] This example was used to illustrate the therapeutic effects of 3334 and 3334-H1K2-canIgG B on canine malignant tumors.

[0179] A single-arm trial design was adopted. Forty tumor-bearing dogs (including 20 melanoma cases and 20 mast cell tumor cases) seeking medical treatment were screened from a pet hospital in Beijing, and the efficacy studies of 3334 chimeric antibody and 3334-H1K2-canIgG B antibody in treating tumor-bearing dogs were carried out respectively. The breeds of experimental dogs included: Schnauzer, Labrador Retriever, Chihuahua, Golden Retriever, etc. The average age was 12 years old (10 - 15 years old), and all were advanced malignant tumor-bearing dogs.

[0180] All diseased dogs were divided into two groups, namely 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 tumor. For all diseased dogs, the corresponding 3334 chimeric antibody and 3334-H1K2-canIgG B antibody (diluted with physiological saline for injection) were intravenously infused at a dose of 5 mg / kg body weight, administered once every two weeks for 8 consecutive times, and the experiment ended one week after the last administration. The tumor burden was evaluated by gross examination and computed tomography, and the tumor size was measured once every two weeks to evaluate the efficacy. The objective response rate (ORR), complete response rate (CR), and partial response rate (PR) were compared. Among them, ORR = (number of diseased dogs with complete response and partial response / total number of diseased dogs) × 100%; CR = (number of diseased dogs with all detectable tumors disappeared / total number of diseased dogs) × 100%; PR = (number of diseased dogs with the sum of the maximum diameters of target lesions reduced by at least 30% / total number of diseased dogs) × 100%.

[0181] The preliminary clinical treatment effects are shown in Table 5 below: Both the 3334 chimeric antibody and 3334-H1K2-canIgG B have good anti-tumor efficacy; and the treatment effect of 3334-H1K2-canIgG B is better than that of the 3334 chimeric antibody.

[0182] Table 5 Clinical anti-tumor efficacy data of 3334 and 3334-H1K2-canIgG B

[0183]

[0184] From the above results, it can be seen that the antibody screened by the present invention has high affinity and high activity, and can achieve the purpose of treating canine tumor diseases by blocking the binding of canine PD-1 and its ligand.

[0185] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. An anti-PD-1 antibody or antigen-binding fragment, wherein, it is capable of binding to canine PD-1, and 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 those shown in SEQ ID NO: 16, 18-20, and the light chain variable region is selected from those shown in SEQ ID NO: 17, 21-23.

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

3. The antibody or antigen-binding fragment according to claim 1, wherein, the antibody or antigen-binding fragment 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 sequences of the heavy chain variable region are selected from those shown in SEQ ID NO: 2-4, 8-11, 14, and the CDR sequences of the light chain variable region are selected from those shown in SEQ ID NO: 5-7, 12-13, 15.

4. The antibody or antigen-binding fragment according to claim 1, wherein, the heavy chain constant region of the antibody or antigen-binding fragment is a canine IgG B mutant, and the amino acid sequence is as 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 as shown in SEQ ID NO:

25.

5. The antibody or antigen-binding fragment according to claim 1, wherein, The antibody or antigen-binding fragment thereof comprises a full-length heavy chain and / or a full-length light chain. The full-length heavy chain sequence used for the antibody or antigen-binding fragment thereof is selected from any one of SEQ ID NO: 26, 28-30, and the full-length light chain used for the antibody or antigen-binding fragment thereof is selected from any one of SEQ ID NO: 27, 31-33; Furthermore, the full-length heavy chain of the antibody or antigen-binding fragment thereof is as shown in SEQ ID NO: 26, and the full-length light chain is as shown in SEQ ID NO: 27; 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; 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; 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; 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; 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; 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; or, the full-length heavy chain is as shown in SEQ ID NO: 30, and the full-length light chain is as shown in SEQ ID NO: 31; or, the full-length heavy chain is as shown in SEQ ID NO: 30, and the full-length light chain is as shown in SEQ ID NO: 32; or, the full-length heavy chain is as shown in SEQ ID NO: 30, and the full-length light chain is as shown in SEQ ID NO:

33.

6. 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 1-5, and the nucleic acid molecule is DNA or RNA.

7. An expression vector, characterized in that the nucleotide sequence of the expression vector comprises the nucleic acid molecule according to claim 6.

8. A recombinant cell, characterized in that the recombinant cell is obtained by introducing the expression vector according to claim 7 into a host cell.

9. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the antibody or antigen-binding fragment thereof according to any one of claims 1-5, or the nucleic acid molecule according to claim 6, or the expression vector according to claim 7, or the recombinant cell according to claim 8.

10. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-5, or the nucleic acid molecule according to claim 6, or the expression vector according to claim 7, or the recombinant cell according to claim 8, or the pharmaceutical composition according to claim 9, in the prevention and / or treatment of mammalian tumors; or in the use for preparing a drug for the prevention and / or treatment of mammalian tumors; or in the use for detecting PD-1 protein.

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