CN117343185A

CN117343185A - Anti-canine PD-1 antibody and application thereof

Info

Publication number: CN117343185A
Application number: CN202311658628.6A
Authority: CN
Inventors: 罗昊澍; 谭泽民; 杨友桥; 段军叶; 吴冰春
Original assignee: Zhejiang Weijiexin Biotechnology Co ltd; Beijing Weijiexin Biotechnology Co ltd
Current assignee: Zhejiang Weijiexin Biotechnology Co ltd; Beijing Weijiexin Biotechnology Co ltd
Priority date: 2023-12-06
Filing date: 2023-12-06
Publication date: 2024-01-05
Anticipated expiration: 2043-12-06
Also published as: CN117343185B

Abstract

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

Description

Anti-canine PD-1 antibody and application thereof

Technical Field

The invention relates to the technical field of antibodies, in particular to an anti-canine PD-1 antibody and application thereof.

Background

Tumors are a common, frequently occurring disease in humans and animals, wherein canine malignancies have become a leading cause of death in dogs. The relevant study data showed that one of every 4 dogs was diagnosed with tumor, with a proportion of dogs older than 10 suffering from tumor being more than 50%. The current clinical treatment means for canine tumors mainly comprise surgery, chemotherapy and radiotherapy. Common chemotherapeutic drugs include cyclophosphamide, doxorubicin, vincristine, prednisolone and the like, which have the defects of poor tumor specificity, large toxic and side effects and the like. Immunotherapy is an emerging treatment means, and can be effective and has little side effect when being used by patients with poor chemotherapy response, and research and application of the immunotherapy have made a major breakthrough in human tumors, so that the immunotherapy is popularized to animal tumors.

Programmed cell death receptor 1 (PD-1), a type I transmembrane glycoprotein of about 55 kDa, belongs to the CD28 superfamily of receptors and is expressed primarily on the surface of T cells, B lymphocytes and activated macrophages. There are two ligands for PD-1 proteins: PD-L1 and PD-L2. Under normal physiological conditions, the combination of PD-1 and PD-L1/PD-L2 can inhibit the activation of T cells, thereby protecting the organism from being attacked by an autoimmune system; however, many solid tumors, as well as some hematological tumors, including melanoma, breast cancer, various digestive system tumors, lymphoma, leukemia, and other cancer cells also express PD-L1 in large amounts. PD-L1 on tumor cell membrane is combined with PD-1 on T cell to inhibit activation of T cell, so as to avoid the recognition and attack of immune system and realize immune escape of tumor cell. Simultaneous studies have found that expression of PD-L1 on tumor cells correlates with poor prognosis for multiple tumor types. Thus, blocking the binding of PD-1 and PD-L1 is a concept for tumor immunotherapy.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a canine PD-1 antibody with high affinity and high activity, and the purpose of treating canine tumor diseases is achieved by blocking the combination of canine PD-1 and a ligand thereof.

In order to achieve the aim of the invention, the invention adopts the following technical scheme:

in a first aspect of the invention, there is provided a CDR sequence of an antibody or antigen binding fragment thereof capable of binding to canine PD-1.

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

According to an embodiment of the invention, the antibody or antigen binding fragment thereof capable of binding to canine PD-1 comprises a CDR sequence or an amino acid sequence having at least 85% identity thereto selected from at least one of the following: the CDR sequences of the heavy chain variable region are selected from SEQ ID NO 2-4, 8-11 and 14, and the CDR sequences of the light chain variable region are selected from SEQ ID NO 5-7, 12-13 and 15.

It is noted that in the above description, the protection ranges are all CDR sequences that are possible within the above sequence range defined by the variable region: the CDRs 1-3 of the heavy and light chain variable regions may be obtained from different coding systems of the prior art, including, but not limited to, kabat, chothia, IMGT, north, abM, contact, etc., and are within the scope of the present application as determined by other known or unknown coding systems. In addition, in different databases (e.g., different commercial, non-commercial, scientific, etc. databases for predicting CDR positions, including but not limited to abYsis database, IMGT database, TABS database, smart bud BIO database, etc.), coding systems including but not limited to Kabat, chothia, IMGT, north, abM, contact, etc. are used, and the predicted CDR sequences are also within the scope of the present application. The CDRs predicted by different databases by using the same coding system for the same antibody variable region sequence have certain differences and are also within the protection scope of the invention.

Further, 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, which is of the sequence: a heavy chain variable region shown in SEQ ID NO. 16, a light chain variable region shown in SEQ ID NO. 17;

or, a heavy chain variable region shown in SEQ ID NO. 18, a light chain variable region shown in SEQ ID NO. 21;

or, a heavy chain variable region shown in SEQ ID NO. 18, a light chain variable region shown in SEQ ID NO. 22;

or, a heavy chain variable region shown in SEQ ID NO. 18, a light chain variable region shown in SEQ ID NO. 23;

or, a heavy chain variable region shown in SEQ ID NO. 19, a light chain variable region shown in SEQ ID NO. 21;

or, a heavy chain variable region shown in SEQ ID NO. 19, a light chain variable region shown in SEQ ID NO. 22;

or, a heavy chain variable region shown in SEQ ID NO. 19, a light chain variable region shown in SEQ ID NO. 23;

or, a heavy chain variable region shown in SEQ ID NO. 20, a light chain variable region shown in SEQ ID NO. 21;

or, a heavy chain variable region shown in SEQ ID NO. 20, a light chain variable region shown in SEQ ID NO. 22;

or, a heavy chain variable region shown in SEQ ID NO. 20, a light chain variable region shown in SEQ ID NO. 23;

or an amino acid sequence having at least 85% identity thereto.

Further, the present invention provides an antibody or antigen binding fragment thereof capable of binding to canine PD-1. According to an embodiment of the 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 sequences of the heavy chain variable region are selected from the group consisting of SEQ ID NOS.2-4, 8-11, 14 and the CDR sequences of the light chain variable region are selected from the group consisting of SEQ ID NOS.5-7, 12-13, 15, or amino acid sequences having at least 85% identity thereto.

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

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

According to an embodiment of the invention, the antibody or antigen binding fragment thereof capable of binding to canine PD-1 comprises heavy chain variable region CDRs and amino acid sequences capable of achieving more than 85% identity: heavy chain variable region CDR1, CDR2, CDR3 sequences (obtained according to the Kabat coding system) shown in the amino acid sequences of SEQ ID NO:2, 3 and 4, respectively; heavy chain variable region CDR1, CDR2, CDR3 sequences (obtained according to the Chothia coding system) shown in the amino acid sequences of SEQ ID NO: 8, 9 and 4, respectively; heavy chain variable region CDR1, CDR2, CDR3 sequences (obtained according to the IMGT coding system) as shown in the amino acid sequences of SEQ ID NO 14, 10 and 11, respectively.

According to an embodiment of the 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 capable of greater than 85% identity: light chain variable region CDR1, CDR2, CDR3 sequences (obtained according to the Kabat or Chothia coding system) as shown in the amino acid sequences of SEQ ID NO: 5, 6 and 7, respectively; the sequences of the light chain variable regions CDR1, CDR2 and CDR3 shown in the amino acid sequences of SEQ ID NO. 12, 13 and 7 respectively, and the sequences of the light chain variable regions CDR1, CDR2 and CDR3 shown in the amino acid sequences of SEQ ID NO. 12, 15 and 7 respectively (the sequences are obtained according to an IMGT coding system but are obtained by adopting different database analysis).

Specifically, the antibody or antigen binding fragment thereof capable of binding to canine PD-1 comprises:

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

The above sequences each comprise an amino acid sequence capable of achieving 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity.

In a second aspect of the invention there is provided a variable region sequence of an antibody or antigen binding fragment thereof capable of binding to canine PD-1.

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

further, the one antibody or antigen binding fragment thereof capable of binding to canine PD-1 may be:

a heavy chain variable region as shown in SEQ ID NO. 16 and a light chain variable region as shown in SEQ ID NO. 17;

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. 21;

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;

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;

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;

or, a heavy chain variable region as set forth in SEQ ID NO. 19, and a light chain variable region as set forth in SEQ ID NO. 22;

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;

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;

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;

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;

or an amino acid sequence having at least 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity thereto.

In a third aspect of the invention there is provided a constant region of an antibody or antigen binding fragment thereof capable of binding to canine PD-1.

The heavy chain constant region of the antibody or antigen binding fragment thereof capable of binding to canine PD-1 can be canine IgG A, igG B, igG C, igG D or mutant sequences ensuring the same function, further preferably an IgG B mutant sequence, and the specific heavy chain constant region domain amino acid sequence can be SEQ ID NO: shown at 24; the light chain constant region used in the antibody may be a canine kappa chain, lambda chain domain constant region sequence, further preferably a canine kappa chain, the amino acid sequence of which is SEQ ID NO: 25.

However, the inventive concept of the present invention is not limited to the constant region shown in the above exemplary sequence, as long as the design of the constant region capable of achieving the effects of prevention, treatment, detection, etc. based on the PD-1 target of the present invention is within the scope of the present invention.

In a fourth aspect the invention provides a full length sequence of an antibody or antigen binding fragment thereof capable of binding to canine PD-1.

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, the full length heavy chain sequence is selected from any one of SEQ ID NO. 26, 28-30, and the full length light chain is selected from any one of SEQ ID NO. 27, 31-33;

the affinity constant KD of the antibody or antigen binding fragment thereof capable of binding to canine PD-1 and canine PD-1 ranges from 8.76E-10 to 3.83E-09M;

further, the full length of the heavy chain of the antibody or antigen binding fragment capable of binding to canine PD-1 is shown as SEQ ID NO. 26, and the full length of the light chain is shown as SEQ ID NO. 27;

or the full length of the heavy chain is shown as SEQ ID NO. 28, and the full length of the light chain is shown as SEQ ID NO. 31;

or the full length of the heavy chain is shown as SEQ ID NO. 28, and the full length of the light chain is shown as SEQ ID NO. 32;

or the full length of the heavy chain is shown as SEQ ID NO. 28, and the full length of the light chain is shown as SEQ ID NO. 33;

or the full length of the heavy chain is shown as SEQ ID NO. 29, and the full length of the light chain is shown as SEQ ID NO. 31;

or the full length of the heavy chain is shown as SEQ ID NO. 29, and the full length of the light chain is shown as SEQ ID NO. 32;

Or the full length of the heavy chain is shown as SEQ ID NO. 29, and the full length of the light chain is shown as SEQ ID NO. 33;

or the full length of the heavy chain is shown as SEQ ID NO. 30, and the full length of the light chain is shown as SEQ ID NO. 31;

or the full length of the heavy chain is shown as SEQ ID NO. 30, and the full length of the light chain is shown as SEQ ID NO. 32;

or the full length of the heavy chain is shown as SEQ ID NO. 30, the full length of the light chain is shown as SEQ ID NO. 33,

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

In a fifth aspect the invention provides a type of antibody or antigen binding fragment thereof capable of binding to canine PD-1.

The one antibody or antigen binding fragment thereof capable of binding to canine PD-1 includes at least one selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a multimeric antibody, and a CDR-grafted antibody;

optionally, the one antibody or antigen binding fragment capable of binding to canine PD-1 comprises at least one selected from the group consisting of a single chain antibody, fab antibody, fv antibody, single chain antibody, single domain antibody, and minimal recognition unit; in the case of the antigen-binding fragment, the number of CDR sequences is not limited to 6, but may be less than or more than 6, and the CDR sequences may be limited to those capable of affinity binding to the antigen of interest.

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

In a sixth aspect, the invention provides a nucleic acid element.

The element may be a nucleic acid molecule encoding one of the antibodies or antigen binding fragments thereof described above that is capable of binding to canine PD-1. Such nucleic acid molecules include, but are not limited to, DNA, RNA; preferably, the nucleic acid molecule is DNA. However, the scope of the present invention encompasses all nucleic acid and nucleotide sequences capable of expressing the aforementioned amino acid sequences, including nucleic acid sequences deduced from various codon preferences, and those skilled in the art can deduce other nucleotide sequences capable of expressing the aforementioned amino acid sequences using conventional techniques in the art, and the following sequences are merely examples:

specifically, the heavy chain variable region sequence 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. 49, 51-53, and the light chain variable region sequence 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. 50, 54-56.

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

the antibody or antigen binding fragment thereof capable of binding to canine PD-1 can be a DNA coding sequence with a heavy chain variable region shown as SEQ ID NO. 51, and a light chain variable region nucleotide sequence shown as SEQ ID NO. 54;

the antibody or antigen binding fragment thereof capable of binding to canine PD-1 can be a DNA coding sequence with a heavy chain variable region shown as SEQ ID NO. 51, and a light chain variable region nucleotide sequence shown as SEQ ID NO. 55;

the antibody or antigen binding fragment thereof capable of binding to canine PD-1 can be a DNA coding sequence with a heavy chain variable region shown as SEQ ID NO. 51, and a light chain variable region nucleotide sequence shown as SEQ ID NO. 56;

the antibody or antigen binding fragment thereof capable of binding to canine PD-1 can be a DNA coding sequence with a heavy chain variable region shown as SEQ ID NO. 52, and a light chain variable region nucleotide sequence shown as SEQ ID NO. 54;

The antibody or antigen binding fragment thereof capable of binding to canine PD-1 can be a DNA coding sequence with a heavy chain variable region shown as SEQ ID NO. 52, and a light chain variable region nucleotide sequence shown as SEQ ID NO. 55;

the antibody or antigen binding fragment thereof capable of binding to canine PD-1 can be a DNA coding sequence with a heavy chain variable region shown as SEQ ID NO. 52, and a light chain variable region nucleotide sequence shown as SEQ ID NO. 56;

the antibody or antigen binding fragment thereof capable of binding to canine PD-1 can be a DNA coding sequence with a heavy chain variable region shown as SEQ ID NO. 53, and a DNA coding sequence with a light chain variable region shown as SEQ ID NO. 54;

the antibody or antigen binding fragment thereof capable of binding to canine PD-1 can be a DNA coding sequence with a heavy chain variable region shown as SEQ ID NO. 53, and a DNA coding sequence with a light chain variable region shown as SEQ ID NO. 55;

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

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.

Specifically, the full-length DNA sequence of the heavy chain of the antibody or antigen binding fragment thereof capable of binding to canine PD-1 can be represented by any one of SEQ ID NO 59 and 61-63, and the full-length DNA sequence of the light chain of the antibody can be represented by any one of SEQ ID NO 60 and 64-66.

Specifically, the antibody or antigen binding fragment thereof capable of binding to canine PD-1 may be: the full-length DNA sequence of the heavy chain is shown as SEQ ID NO. 59, and the full-length DNA sequence of the light chain is shown as SEQ ID NO. 60;

or, the full-length DNA sequence of the heavy chain is shown as SEQ ID NO. 61, and the full-length DNA sequence of the light chain is shown as SEQ ID NO. 64;

or, the full-length DNA sequence of the heavy chain is shown as SEQ ID NO. 61, and the full-length DNA sequence of the light chain is shown as SEQ ID NO. 65;

or, the full-length DNA sequence of the heavy chain is shown as SEQ ID NO. 61, and the full-length DNA sequence of the light chain is shown as SEQ ID NO. 66;

or, the full-length DNA sequence of the heavy chain is shown as SEQ ID NO. 62, and the full-length DNA sequence of the light chain is shown as SEQ ID NO. 64;

Or, the full-length DNA sequence of the heavy chain is shown as SEQ ID NO. 62, and the full-length DNA sequence of the light chain is shown as SEQ ID NO. 65;

or, the full-length DNA sequence of the heavy chain is shown as SEQ ID NO. 62, and the full-length DNA sequence of the light chain is shown as SEQ ID NO. 66;

or, the full-length DNA sequence of the heavy chain is shown as SEQ ID NO. 63, and the full-length DNA sequence of the light chain is shown as SEQ ID NO. 64;

or, the full-length DNA sequence of the heavy chain is shown as SEQ ID NO. 63, and the full-length DNA sequence of the light chain is shown as SEQ ID NO. 65;

alternatively, the heavy chain full-length DNA sequence is shown as SEQ ID NO. 63 and the light chain full-length DNA sequence is shown as SEQ ID NO. 66.

Specifically, SEQ ID NO:2-15, the DNA coding sequence corresponding in sequence may be SEQ ID NO: 35-48.

The element may be a recombinant vector, including but not limited to a cloning vector, an expression vector, a shuttle vector, a viral vector (e.g., lentiviral vector, adeno-associated viral vector, poxvirus-associated vector, etc.), carrying a nucleic acid molecule as described above, 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 and pcDNA3.4.

The element may be a recombinant cell carrying the nucleic acid molecule described above, or a recombinant vector expressing an antibody or antigen-binding fragment thereof described above 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, and the recombinant cell is preferably a mammalian cell. Including but not limited to chinese hamster ovary cells (CHOK 1), HEK293 cells, and the like.

A seventh aspect of the invention relates to a pharmaceutical composition and use.

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

Optionally, the pharmaceutical composition further comprises pharmaceutically acceptable excipients and excipients.

The pharmaceutical composition may be prepared as a combination or kit comprising as a first active ingredient the aforementioned antibody or antigen-binding fragment thereof capable of binding to canine PD-1 or pharmaceutical composition; and optionally, a second active ingredient for combined use, the first and second active ingredients may be the same or different, and the second active ingredient may include, but is not limited to, palatine, cyclophosphamide, doxorubicin, vincristine, prednisolone, and the like.

The anti-canine PD-1 antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector, recombinant cell, pharmaceutical composition, combination drug or kit for use in preventing and/or treating tumor, or for use in preparing a medicament, or for use in preparing a kit for detecting PD-1 protein in biological matrix; the tumor is mammalian tumor, including human, monkey, mouse (rat, mouse), horse, cow, sheep, pig, dog, cat, etc., and more preferably canine tumor.

The tumor species include, but are not limited to, "tumors," but refer to a class of diseases characterized by the development of abnormal cells that proliferate uncontrolled and have the ability to infiltrate and destroy normal body tissue. Exemplary tumors include, but are not limited to, squamous carcinoma (SCC); papillomas; fiber papilloma; intra-osseous cancer; invasive nasal cancer; malignant Melanoma (MM); fibrosarcoma (FSA); fibroids; rhabdomyosarcoma; hemangiosarcoma (HAS); granulocytoma; mixed mesophyll tumors; neurofibrosarcoma; undifferentiated sarcoma; myxosarcoma; chondrosarcoma (CSA); phyllostachys osteosarcoma (MLO); osteosarcoma (OSA); infectious neoplasms (TVT); mast cell neoplasms (MCTs); lymphomas (LSA); enameloblastoma; enameloblastoma; dental epithelial calcification tumor; dental tumor; cellulose mucinous tumor; cementoma; dental fibroma; acantha gum tumor; ossified gum tumor; fibrogingival tumor; basal cell neoplasm; cerumen adenoma; viral papilloma, salivary gland tumor, nasal cavity lymphoma; cerumen adenocarcinoma (CGC); benign eosinophilic adenoma (rhabdomyoma); cartilage tumor; osteoma; osteochondrioma; tracheal cancer; smooth myoma; benign laryngeal tumors (e.g., rhabdomyomas or eosinophilic adenomas); mediastinal tumors (e.g., thymoma and mediastinal lymphoma); malignant chest wall tumor (sarcoma); benign chest wall tumors (osteomas and chondromas); primary lung tumor; secondary (metastatic lung tumor); heart tumors (e.g., angiosarcoma; mesothelioma; myxoma; ectopic thyroid carcinoma); esophageal cancer; gastric tumors (e.g., gastric adenocarcinoma and lymphoma); gastric leiomyosarcoma; gastric leiomyoma; gastric pulp cytoma; gastrointestinal mast cell neoplasms; a non-lymphoid intestinal tumor; intestinal lymphoma; intestinal adenocarcinoma; an extramedullary plasma cell tumor; carcinoid; adeno-cancerous polyps; large intestine adenocarcinoma; colorectal leiomyomas and leiomyosarcomas; anus Zhou Xianliu; anus Zhou Xianai; anal sac adenocarcinoma; liver tumors (e.g., liver sarcomas, liver cancer or carcinoid); hepatocellular carcinoma (HCC); hepatocellular adenoma; hepatoblastoma; biliary tract cancer; bile duct adenocarcinoma; gall bladder tumor; pancreatic adenocarcinoma; a breast tumor; malignant breast tumors (breast cancer); malignant mixed tumor; breast sarcoma; exoskeletal sarcoma of mammary gland; uterine tumors; vaginal and vulvar tumors; ovarian tumors (e.g., adenocyst, granulosa cell tumor); genital cord stromal tumors (e.g., follicular cell tumor and luteoma); germ cell neoplasms (asexual cell neoplasms, teratomas or teratocarcinomas); testicular tumors (e.g., backing cell tumors, seminomas, and stromal cell tumors); histiocytic reticuloendothelioma, transitional Cell Carcinoma (TCC); prostate tumor; renal tumors (e.g., renal carcinoma, fibrosarcoma, and angiosarcoma); ureteral tumors; bladder tumor; a tumor of the urethra; cutaneous hemangioma; hyponychium squamous carcinoma; basal cell neoplasms (including basal cell carcinoma, basal cell epithelial holes, and tomb-like neoplasms); cerumen gland tumor; intraepithelial neoplasia (keratoacanthoma); hair follicle tumors (hair blastoma, hair epithelium tumor, and nail bed cell tumor); skin plasma cell tumors; cutaneous lymphomas; sebaceous adenoma; sweat gland tumor; epitheliophilic Lymphoma (ELSA); skin tissue cell tumor; cutaneous neuroendocrine (merkel cell) neoplasms; skin smooth muscle tumor; soft tissue or spindle cell sarcoma (STS); lymphangiosarcoma; synovial cell sarcoma; histiocytosarcoma (HS); osteosarcoma of four limbs; osteosarcoma of axial bone; extraosseous osteosarcoma; superficial osteosarcoma; many small She Gu chondrosarcoma (MLO); multiple exochondral osteowarts (MCE); lymphocytic Leukemia (CLL); non-lymphocytic leukemia (granulocytic leukemia); chronic Myelogenous Leukemia (CML); acute Myelogenous Leukemia (AML); spleen Angiosarcoma (HSA); spleen sarcoma (nonlymphoid, nonvascular); glioma; meningioma; gangliocytomas (pheochromocytomas and non-pheochromoparagangliomas); ganglion glioma; spinal cord tumor (OSA, CSA, FSA, HSA, vascular endothelial tumor, myeloma); peripheral schwannomas (PNSTs); neuroepithelial tumors (ependymoma, medulloblastoma, nephroblastoma, and spinal cord blastoma); intramedullary spinal column tumor; epidural sarcoma; epidural sarcoma; a sheath tumor; peripheral nervous system tumors (e.g., neuroblastomas, medulloblastomas, retinoblastomas); eye risk tumor; meibomian adenoma; myoblasts; a third eye risk tumor; conjunctival tumors; extrabulbar (cornea and sclera) tumors; orbit (the space surrounding the eyeball) and retrobulbar tumor; thyroid tumor; parathyroid tumor; APUD cell tumor (gum precursor uptake decarboxylated cell tumor); insulinomas; glucagon tumor; gastrinomas; adrenal cortex tumor; pheochromocytoma; pituitary tumor. Melanoma, mast cell tumor, colon cancer are preferred.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1. the anti-PD-1 chimeric antibody and the caninized antibody provided by the invention have good affinity, wherein 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.

2. The anti-PD-1 chimeric antibody and the caninized 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.

3. The anti-PD-1 chimeric antibody and the caninized antibody can inhibit the growth of mouse MC38 tumors in vivo, and have better anti-tumor curative effect on canine tumors, which is of great significance in developing efficient cancer therapeutic drugs.

Drawings

The accompanying drawings, which are included to provide a further understanding of embodiments of the invention and are incorporated in and constitute a part of this application, illustrate embodiments of the invention. In the drawings:

FIG. 1 is a graph showing the results of flow cytometry detection 3334 of competitive binding between a chimeric antibody and canine PD-1/PD-L1 (A is a flow chart; and B is an APC positive cell rate quantification chart).

FIG. 2 is a graph showing the results of an in vitro activity assay-luciferase reporter method for 3334 chimeric antibody and canine antibody (A is 3334 chimeric antibody and 3334-H1K1/H1K2/H1K3-canIgG B canine antibody group; B is 3334 chimeric antibody and 3334-H2K1/H2K2/H2K3-canIgG B canine antibody group; C is 3334 chimeric antibody and 3334-H3K1/H3K2/H3K3-canIgG B canine antibody group).

FIG. 3 is a graph showing the results of in vitro activity detection-T cell activation experiments for 3334 chimeric antibodies and caninized antibodies.

FIG. 4 is a graph showing the results of the effects of 3334 chimeric antibody, 3334-H1K2-canIgG B caninised antibody on mouse body weight.

FIG. 5 is a graph showing the effect of the 3334 chimeric antibody and the 3334-H1K2-canIgG B caninized antibody on the weight change rate of mice.

FIG. 6 is a graph showing the effect of 3334 chimeric antibody, 3334-H1K2-canIgG B caninised antibody on tumor volume in mice.

Detailed Description

The following examples are illustrative of the invention and are not intended to limit the scope of the invention.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The present invention will now be described in more detail by way of examples with reference to the accompanying drawings, which are not intended to limit the invention thereto, but are illustrative only.

In order that the present disclosure may be more clearly understood, some terms are first defined. The terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. Further, in the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.

In this document, the terms "comprise" or "include" are used in an open-ended fashion, i.e., to include what is indicated by the present invention, but not to exclude other aspects.

In this document, the terms "optionally," "optional," or "optionally" generally refer to the subsequently described event or condition may, but need not, occur, and the description includes instances in which the event or condition occurs, as well as instances in which the event or condition does not.

Herein, the term "antibody" generally refers to an antibody that recognizes one or more epitopes, including, but not limited to, monoclonal antibodies, polyclonal antibodies, and CDR-grafted antibodies, and is an immunoglobulin molecule capable of binding to a specific antigen. Comprising two light chains of relatively light molecular weight and two heavy chains of relatively heavy molecular weight, the heavy (H) and light (L) chains being linked by disulfide bonds to form a tetrapeptide chain molecule. Among them, the amino-terminal (N-terminal) amino acid sequence of the peptide chain varies greatly, called variable region (V region), and the carboxyl-terminal (C-terminal) is relatively stable, and varies little, called constant region (C region). The V chains of the L chain and H chain are referred to as VL and VH, respectively. Certain regions in the variable region have a higher degree of variation in amino acid composition and order, called hypervariable regions (Hypervariable region, HVR), which are the sites of antigen and antibody binding and are therefore also known as complementarity-determining region (CDRs). The heavy chain variable region and the light chain variable region each have three CDRs.

In this context, the term "Fab antibody" generally refers to an antibody comprising only Fab molecules, consisting of VH and CH1 of the heavy chain and the complete light chain, linked by a disulfide bond between the light and heavy chains.

As used herein, the term "Fv antibody" generally refers to an antibody consisting of only the light chain variable region (VL) and the heavy chain variable region (VH) joined by a non-covalent bond, which is the smallest functional fragment of an antibody that retains the intact antigen-binding site.

The terms "single domain antibody", "nanobody" and "VHH antibody" are used interchangeably herein and are initially described as the antigen binding immunoglobulin (variable) domain of a "heavy chain antibody" (i.e. "antibody lacking a light chain") (mers-masterman C, atarhouchT, muyldermans S, robinson G, mers C, songa EB, bendahman N, hamers R.: "Naturallyoccurring antibodies devoid of light chains"; nature 363, 446-448 (1993)) comprising only the heavy chain variable region (VH) and conventional CH2 and CH3 regions, which specifically bind to antigen through the heavy chain variable region.

Herein, the term "single chain antibody" generally refers to an antibody in which a heavy chain variable region (VH) and a light chain variable region (VL) are linked by a linker peptide.

As used herein, the terms "minimal recognition unit" and "MRU" both refer to antibodies consisting of only one CDR, which have a molecular weight that is very small, accounting for only about 1% of the total antibody.

In this context, the terms "identity", "homology" or "similar phase" are used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences when compared to the amino acid sequence or nucleic acid sequence of a reference sequence, using conventional methods, e.g., see Ausubel et al, et al (1995), current Protocols in Molecular Biology, chapter 19 (Greene Publishing and Wiley-Interscience, new York); and ALIGN programs (Dayhoff (1978), atlas of Protein Sequence and Structure 5: support.3 (National Biomedical Research Foundation, washington, D.C.), there are many algorithms for alignment and determination of sequence identity, including homology alignment algorithms of needle et al (1970) J.mol.biol.48:443, computer programs using these algorithms are also available and include, but are not limited to, ALIGN or software of application.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.70:173-187 (1997), and BLASTP, BLASTN, and BLASTX algorithms (see Altsul et al (1990) J.mol.biol.215:403-410), and include, but are not limited to, ALtsn or software of application program, inc. Appl.460-5:35, and software of Abelson.35:266, and Flex. Table 35, F.35, and Flex. Table, F.35, and Flex. Strand so on.

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

As used herein, "binds to PD-1", "specifically binds to PD-1"Antibodies or "anti-PD-1 antibodies" refer to antibodies having a KD of 1.0X10 ^-8 mol/L or less. Binding affinity is determined using a standard binding assay, such as biological membrane interferometry (Gator), using the PD-1 extracellular domain.

In this context, the term "expression vector" generally refers to a nucleic acid molecule capable of insertion into a suitable host for self-replication, which transfers the inserted nucleic acid molecule into and/or between host cells. The expression vector may include a vector mainly used for inserting DNA or RNA into cells, a vector mainly used for replicating DNA or RNA, and a vector mainly used for expression of transcription and/or translation of DNA or RNA. The expression vector also includes vectors having a plurality of the above functions. The expression vector may be a polynucleotide capable of transcription and translation into a polypeptide when introduced into a suitable host cell. Typically, the expression vector will produce the desired expression product by culturing a suitable host cell containing the expression vector. The expression vector carries the aforementioned nucleic acid molecule. In the case of attaching the above-mentioned nucleic acid molecule to a vector, the nucleic acid molecule may be directly or indirectly attached to a control element on the vector, as long as the control element is capable of controlling translation, expression, etc. of the nucleic acid molecule. Of course, these control elements may be directly from the carrier itself or may be exogenous, i.e. not from the carrier itself. Of course, the nucleic acid molecule may be operably linked to a control element. "operably linked" herein refers to the linkage of a foreign gene to a vector such that control elements within the vector, such as transcription control sequences and translation control sequences, and the like, are capable of performing their intended functions of regulating transcription and translation of the foreign gene. The usual vectors may be, for example, plasmids, phages and the like. After the expression vector according to some embodiments of the present invention is introduced into a suitable recipient cell, the expression of the antibody or antigen-binding fragment can be effectively achieved under the mediation of a regulatory system, thereby achieving the in vitro mass acquisition of the antibody or antigen-binding fragment.

As used herein, the term "recombinant cell" generally refers to a cell that has been modified or recombined with genetic material of a host cell using genetic engineering techniques or cell fusion techniques to obtain a unique trait that is stably inherited. Wherein the term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector may be introduced. The term "transformed" or "transfected" as used herein refers to the introduction of a nucleic acid (e.g., 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 invention and can be used for expression and/or secretion of a target protein.

The term "pharmaceutical composition" as used herein generally refers to unit dosage forms and may be prepared by any of the methods well known in the pharmaceutical arts. 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 intimately bringing into association the active compound with liquid carriers, finely divided solid carriers or both.

As used herein, the term "pharmaceutically acceptable excipients" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for the particular dosage form of interest. In addition to the extent to which any conventional adjuvant is incompatible with the compounds of the present invention, such as any adverse biological effects produced or interactions with any other component of the pharmaceutically acceptable composition in a deleterious manner, their use is also contemplated by the present invention.

In this context, the term "administering" refers to introducing a predetermined amount of a substance into a subject by some suitable means. The antibody or antigen binding fragment or pharmaceutical composition of the invention may be administered by any common route, provided that it reaches the desired tissue. Various modes of administration are contemplated, including peritoneal, intravenous drip, intramuscular injection, subcutaneous injection, and the like, but the invention is not limited to these illustrated modes of administration. The subject may be a mammal, preferably a canine.

In this context, the term "treatment" refers to the use to obtain a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or symptoms thereof, and/or may be therapeutic in terms of partially or completely curing the disease and/or adverse effects caused by the disease. As used herein, "treating" encompasses diseases in mammals, particularly dogs, including: (a) Preventing the occurrence of a disease or disorder in an individual susceptible to the disease but not yet diagnosed with the disease; (b) inhibiting disease, e.g., arresting disease progression; or (c) alleviating a disease, e.g., alleviating symptoms associated with a disease. As used herein, "treating" or "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, alleviate, ameliorate, reduce or inhibit a disease in the individual, including, but not limited to, administration of a drug comprising a compound described herein to an individual in need thereof.

EXAMPLE 1 preparation and identification of mouse anti-canine PD-1 antibodies

1. Antigen preparation

Searching canine PD-1 protein (UniProtKB: A0A024FCJ 9) in a UniProt library and a GenBank library, artificially synthesizing a corresponding nucleotide sequence with a His tag, constructing a pcDNA3.4 vector, transiently transferring into HEK293 cells, collecting cell supernatant after 5 days of transfection, carrying out affinity chromatography on the supernatant by a Ni column, and eluting to obtain the purified canine PD-1 recombinant protein.

2. Immunization of mice

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

3. Spleen RNA extraction and reverse transcription

Extracting RNA from spleen tissue of mice by Trizol method, identifying RNA integrity by denaturing agarose gel electrophoresis, and determining RNA concentration and OD by using ultra-micro spectrophotometer ₂₆₀ /OD ₂₈₀ . RNA of acceptable quality was selected and reverse transcribed according to the instructions of reverse transcriptase Superscript II reverse transcriptase (Thermo Scientific) to obtain cDNA.

4. Antibody library construction and phage display

Amplifying heavy chain and light chain of antibody from cDNA to respectively construct scFv antibody library and Fab antibody library, and making them possess uniform library volume Up to 1X 10 ⁸ CFU/mL. The antibody library is electrically transferred into TG1 escherichia coli, then helper phage M13KO7 is added to infect the TG1 escherichia coli, after night culture, phage are precipitated by using PEG/NaCl, and the phage display antibody library is prepared.

5. Phage display antibody library screening

And (3) adopting an immune tube and a magnetic bead screening instrument to respectively carry out solid-phase screening, solid-phase and liquid-phase cross screening on the phage display antibody library. Wherein the solid phase screening uses canine PD-1 recombinant protein to coat an immune tube, and the liquid phase screening uses biotinylated canine PD-1 recombinant protein to coat Dynabeads magnetic beads. After three rounds of screening, the enriched Phage collection was identified using a phase ELISA and the monoclonal isolated from the collection.

6. Construction and expression purification of antibodies

DNA sequencing is carried out on Phage ELISA positive monoclonal phagemid, and the murine antibody variable region with the correct reading frame is selected from the sequencing result for gene synthesis, thus obtaining the active murine antibody. The amino acid sequences of the heavy and light chain variable region Complementarity Determining Regions (CDRs) 1, 2, 3 of the murine antibody were obtained using Kabat, chothia, IMGT encoding in the abYsis and IMGT databases, respectively.

The murine antibody heavy chain variable region (SEQ ID NO: 16) was then fused to the heavy chain constant region (including CH1, CH2, CH 3) from the canine IgG B mutant, and the light chain variable region (SEQ ID NO: 17) was fused to the canine kappa chain constant region (CL), inserted into the pcDNA3.4 vector, and a full length chimeric antibody expression plasmid was constructed. Transiently transfecting and expressing a chimeric antibody in HEK293 cells, and carrying out affinity chromatography on a culture supernatant by recombinant protein A to obtain a purified chimeric antibody with the number of 3334, wherein the whole length of a heavy chain of the chimeric antibody is SEQ ID NO:26, the full length of the light chain is SEQ ID NO: shown at 27.

Example 2 screening of antibodies blocking canine PD-1 from ligand binding

Constructing the full length of the canine PD-L1 (UniProtKB: E2RKZ 5), transfecting CHO-K1 cells, screening to obtain monoclonal cell strains with high expression (hereinafter referred to as canine PD-L1/CHO-K1 cells), and detecting the capability of the canine PD-1 antibody to block the combination of the canine PD-1 and the canine PD-L1/CHO-K1 cells by a flow cytometry.

Biotin-labeled canine PD-1 recombinant protein was incubated with different concentrations of antibody for 5 min, then added to PD-L1/CHO-K1 cells, incubated at 4 ℃ for 60 min, rinsed twice with cold 1% fbs-containing PBS, then added with streptavidin-APC fluorescent secondary antibody, incubated at 4 ℃ for 30 min, rinsed twice with cold 1% fbs-containing PBS, resuspended in 200 mL 1% fbs-containing PBS, and analyzed for APC-labeled cell fraction using a flow cytometer (Beckman Coulter, cytoFLEX). The lower the APC positive cell rate reflects the binding capacity of the biotin-labeled canine PD-1 recombinant protein to PD-L1/CHO-K1 cells, indicating that the greater the ability of the antibody to block the binding of the biotin-labeled canine PD-1 recombinant protein to its ligand.

As the results in fig. 1 show, APC positive cell rate gradually decreased as 3334 chimeric antibody concentration increased, indicating that 3334 chimeric antibody was able to block canine PD-1 binding to PD-L1.

EXAMPLE 3 construction of caninized antibodies

CDR grafting and back mutation based on framework region homology, the 3334 chimeric antibody was caninized. First, a canine heavy chain variable region variant (SEQ ID NOS: 18-20) and a canine light chain variable region variant (SEQ ID NOS: 21-23) were constructed. The variable region of a caninized antibody is freely combined by the canine and canine variable region heavy chains of the same antibody, e.g., heavy chain 3334-H1 (SEQ ID NO: 18) may be combined with light chain 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 canine antibody consists of a constant domain of a canine IgG B mutant and a canine kappa constant region domain, for example, 3334-H1K1-canIgG B is a constant region of a canine IgG B mutant, which has a canine variable region heavy chain H1, a canine variable region light chain K1 and a canine IgG B mutant, and the specific sequence combination names are shown in Table 1, the constant regions of the antibodies are respectively the constant region domains of a canine IgG B mutant heavy chain, and the amino acid sequence is SEQ ID NO:24, the canine kappa chain constant region domain has the amino acid sequence of SEQ ID NO: 25.

Table 1 3334 Table of sequence numbering of chimeric and caninized antibodies

Example 4 affinity assay constructed of chimeric and caninized antibodies

The affinity of 3334 chimeric antibodies and 9 3334 caninized antibodies was determined using a Gator instrument based on the biofilm interference (Bio-Layer Interferometry, BLI) technique. Antibodies were first biotin-labeled (thermo fisher, 21343), and biotinylated detection antibodies were diluted to 100 nM and added to a black 96-well plate as stationary phase; the analytes (canine PD-1-His) were then serially diluted (200, 100, 50, 25, 12.5, 6.25, 3.13, nM) and added to a black 96-well plate; finally, the affinity of the antibodies for canine PD-1 was determined using streptavidin probe (Gator ™ Streptavidin Probes) following the equilibration, stationary phase, equilibration, analyte, equilibration shift procedure.

As a result, as shown in Table 2, the affinity of the 3334 chimeric antibody was 8.76E-10, and the 3334 canine antibody was slightly weaker than the 3334 chimeric antibody, but the change was not significant.

Table 2 affinity data for 3334 chimeric antibodies and 3334 caninized antibodies

Note that: e is scientific counting method, is multiplied by 10, and the numerical value behind E is the power of 10.

Example 5 in vitro Activity assay of chimeric antibodies and caninized antibodies

1. Detection of in vitro biological Activity of novel antibodies Using canine PD-1/PD-L1 luciferase reporter Gene cell lines

Canine_PD-L1 aAPC CHO-K1 and Canine_PD-1 Reporter Jurkat Canine PD-1/PD-L1 luciferase Reporter monoclonal cell line cell lines based on Canine PD-1/PD-L1 signaling pathway were constructed, respectively, for evaluation of the ability of antibodies to affect the PD-1/PD-L1 signaling pathway, as well as for 3334 chimeric antibodies and 9 caninized antibodies as detection antibodies.

First, target cells, canine_PD-L1 aAPC CHO-K1 Cell Line, were plated in 96 well Cell plates 16-24 h ahead of 1E4 cells/well density. Then, 50. Mu.L of 2E6 cells/mL effector cell Canine_PD-1 Reporter Jurkat Cell Line was incubated with 50. Mu.L of the series of 3334 chimeric and caninized antibodies, respectively, for 1 h. Then, the medium of the target cells in the 96-well cell plate was removed, and the mixed solution of the effector cells and the antibody was added to continue incubation 16 h. Finally, 50. Mu.L of one-glo enzyme reaction substrate (Promega, E6120) is added to the suspension cells in the supernatant, and after incubation for 15 min at room temperature in a dark place, fluorescence signals are detected on the machine. Calculation of EC using Prism software ₅₀ 。

As shown in Table 3 and FIG. 2, both 3334 chimeric antibody and 9 canine antibodies were effective in blocking PD-1/PD-L1 signaling pathway, EC ₅₀ The value is 0.36-1.23 mug/mL.

Table 3 3334 chimeric antibodies and caninized antibodies in vitro cell biological Activity data

2. Further testing 3334 chimeric and caninized antibodies for in vitro biological Activity Using T cell activation experiments

Healthy adult beagle dogs were selected, whole blood was collected, and canine PBMC were extracted using Ficoll (GE Healthcare, 17-1440-02) density gradient centrifugation. Canine PBMCs were added to 96-well cell plates at 4e5 cells/well, 10 ng/mL of staphylococcus aureus enterotoxin B (SEB, axin Technology, BT 202) were added as superantigens for stimulation, while negative controls (canIgG B), 3334 chimeric antibody and caninized antibody were added separately, incubated 72 h in a carbon dioxide incubator at 37 ℃ and cell supernatants were collected by centrifugation. The supernatant was assayed for cIFN- γ (R & D, DY 781B) and cIL-2 (R & D, DY 1815) content, respectively, using ELISA assay kit.

The results are shown in FIG. 3, where both 3334 chimeric antibody and 9 caninized antibodies increased secretion of T cells cIFN-gamma and cIL-2 at in vitro levels, and were significantly different from the negative control. 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.

EXAMPLE 6 evaluation of in vivo efficacy of 3334 chimeric antibody and 3334-H1K2-canIgG B antibody

Early verification showed that canine PD-1 could cross-bind to human PD-L1 (hPD-L1). Therefore, the anti-tumor effect of 3334 chimeric antibody and 3334-H1K2-canIgG B was further evaluated by using the detection of a mouse model of a female dog PD-1C 57BL/6 transgenic mouse transplanted subcutaneously with a colon cancer MC38/hPD-L1 recombinant cell strain.

MC38/hPD-L1 cells in exponential growth phase were collected, resuspended to 1E7/mL using PBS buffer, and 0.1 mL of MC38/hPD-L1 cells were inoculated subcutaneously into the right back of canine PD-1C 57BL/6 transgenic mice, respectively. All animals were weighed and tumor volume was measured with vernier calipers until tumor growth to an average volume of 102.10 mm ³ At this time, the tumors were randomly grouped and administered according to their tumor size. The experimental groups were a negative control group (vehicle control), a 3334 chimeric antibody group and a 3334-H1K2-canIgG B antibody group, each of which was 6 mice. The dosing was started on the day of grouping (day 0), 3 mg/kg of the corresponding antibody was intravenously injected into the tail of the mice in the antibody group, and the mice in the negative control group were intravenously injected with the same volume of vehicle at a dosing frequency of 3 times per week for 3 weeks. Following dosing, the animals were observed daily for activity, feeding and drinking, weight gain and loss, eyes, hair and other abnormalities, and the body 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 ) (wherein a represents a long diameter and b represents a short diameter). The experiment was ended 9 times after dosing.

The anti-tumor effect of 3334 antibodies 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 i-th day dosing group and Ci represents the average tumor volume of the i-th day negative control group.

The results are shown in fig. 4-6, and fig. 4 and 5 show that the weight of mice in each group increases during the administration period, the weight change among the groups is not significantly different, and the unexpected death phenomenon of the mice does not occur during the treatment process, which indicates that the administration groups have good tolerance and no significant toxic reaction; figure 6 shows the tumor growth of each group, wherein the tumor growth rate was significantly slowed in the 3334 antibody group and 3334-H1K2-canIgG B antibody group mice compared to the negative control group.

Statistical results of T/C% and TGI% after the last dose are shown in Table 4, mice from the 3334 chimeric antibody and 3334-H1K2-canIgG B antibody groups survived on day 19 with average tumor volumes of 1333.08 mm, respectively ³ And 1044.91 mm ³ TGI was 55.54% and 65.15%, respectively. There were statistically significant and very significant differences, respectively, compared to the negative control group.

Table 4 3334 chimeric antibody and 3334-H1K2 canIgG B antibody inhibit mouse MC38 tumor growth data

EXAMPLE 7 evaluation of efficacy of chimeric antibody 3334 and 3334-H1K2-canIgG B antibody in target animals

This example demonstrates the therapeutic effect of 3334 and 3334-H1K2-canIgG B on canine malignancy.

40 diagnosed tumor dogs (including 20 melanoma and 20 mast cell tumor) were screened from Beijing certain pet hospitals by adopting a single arm test design, and the curative effect study of 3334 chimeric antibody and 3334-H1K2-canIgG B antibody for treating malignant tumor dogs was carried out respectively. The experimental canine breeds included: the average ages of the Xuenari, labrador, jipustule, golden hair beagle and the like are 12 years (10-15 years), and the Xuenari, labrador, jipustule, golden hair beagle and the like are all advanced malignant tumor patients.

All dogs with disease were divided into two groups, 3334 chimeric antibody group and 3334-H1K2-canIgG B group, each group comprising 10 dogs with melanoma and 10 dogs with mast cell tumor. All dogs with lesions were dosed with corresponding 3334 chimeric antibody and 3334-H1K2-canIgG B antibody (diluted with physiological saline for injection) intravenously at a dose of 5 mg/kg body weight, administered once every two weeks, 8 consecutive times, and the test was ended after the last week of administration. Tumor burden was assessed by gross examination and computer tomography, tumor size was measured every two weeks to assess efficacy, and Objective Remission Rate (ORR), complete remission rate (CR), and partial remission rate (PR) were compared. Where ORR = fully and partially relieved diseased dogs/total diseased dogs x 100%; CR = all diseased dogs with detectable tumor disappearance/total diseased dogs x 100%; PR = sum of maximum diameters of target lesions reduced by at least 30% diseased dogs/total diseased dogs x 100%.

The primary clinical treatment effect is shown in table 5 below: the 3334 chimeric antibody and 3334-H1K2-canIgG B have better anti-tumor curative effect; and 3334-H1K2-canIgG B has better therapeutic effect than 3334 chimeric antibody.

Tables 5 3334 and 3334-H1K2-canIgG B clinical anti-tumor efficacy data

The result shows that the antibody obtained by screening the invention has high affinity and high activity, and the aim of treating canine tumor diseases is achieved by blocking canine PD-1 and ligand binding thereof.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims

1. An anti-PD-1 antibody or antigen-binding fragment capable of binding to canine PD-1, said antibody or antigen-binding fragment comprising CDRs 1, 2, 3 of the heavy chain variable region, and/or CDRs 1, 2, 3 of the light chain variable region; the heavy chain variable region is selected from SEQ ID NO. 16 and 18-20, and the light chain variable region is selected from SEQ ID NO. 17 and 21-23.

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

or, a heavy chain variable region shown in SEQ ID NO. 20, a light chain variable region shown in SEQ ID NO. 23.

3. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises a CDR sequence or an amino acid sequence having at least 85% identity thereto selected from at least one of the following: the CDR sequences of the heavy chain variable region are selected from SEQ ID NO 2-4, 8-11 and 14, and the CDR sequences of the light chain variable region are selected from SEQ ID NO 5-7, 12-13 and 15.

4. The antibody or antigen-binding fragment of claim 1, wherein the heavy chain constant region of the antibody or antigen-binding fragment is a canine IgG B mutant having an amino acid sequence as set forth 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.

5. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises a full length heavy chain and/or a full length light chain, wherein the antibody or antigen-binding fragment uses a full length heavy chain sequence selected from any one of SEQ ID NOs 26, 28-30, and wherein the antibody or antigen-binding fragment uses a full length light chain selected from any one of SEQ ID NOs 27, 31-33;

further, the heavy chain full length of the antibody or antigen binding fragment is shown as SEQ ID NO. 26, and the light chain full length is shown as SEQ ID NO. 27;

or the full length of the heavy chain is shown as SEQ ID NO. 30, and the full length of the light chain is shown as SEQ ID NO. 33.

6. A nucleic acid molecule encoding the antibody or antigen binding fragment thereof of any one of claims 1-5, wherein the nucleic acid molecule is DNA or RNA.

7. An expression vector, wherein the nucleotide sequence of the expression vector comprises the nucleic acid molecule of claim 6.

8. A recombinant cell obtained by introducing the expression vector of claim 7 into a host cell.

9. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1-5, or the nucleic acid molecule of claim 6, or the expression vector of claim 7, or the recombinant cell of claim 8.

10. Use of the antibody or antigen binding fragment of any one of claims 1-5, or the nucleic acid molecule of claim 6, or the expression vector of claim 7, or the recombinant cell of claim 8, or the pharmaceutical composition of claim 9, for the prevention and/or treatment of a mammalian tumor; or in the preparation of medicaments for preventing and/or treating tumors of mammals; or for detecting PD-1 proteins.