CN115819582A

CN115819582A - Antibody or antigen-binding fragment of PVRIG and application thereof

Info

Publication number: CN115819582A
Application number: CN202111599172.1A
Authority: CN
Inventors: 田志刚; 李洋洋; 肖卫华; 孙汭; 孙昊昱
Original assignee: Hefei Tiangang Immune Drugs Co ltd
Current assignee: Hefei Tiangang Immune Drugs Co ltd
Priority date: 2021-12-24
Filing date: 2021-12-24
Publication date: 2023-03-21

Abstract

The invention relates to an antibody of PVRIG or an antigen binding fragment thereof and application thereof. The antibody or antigen-binding fragment thereof comprises: a CDR sequence selected from at least one of: light chain variable region CDR sequences: SEQ ID NO:1 to 3, SEQ ID NO:7 to 9, SEQ ID NO:13 to 15, SEQ ID NO:19 to 21; heavy chain variable region CDR sequences: SEQ ID NO:4 to 6, SEQ ID NO:10 to 12, SEQ ID NO:16 to 18, SEQ ID NO:22 to 24. The antibody of the invention can specifically target and bind to PVRIG receptor, and block the combination of CD112 and PVRIG.

Description

Antibody or antigen-binding fragment of PVRIG and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to an antibody or an antigen binding fragment thereof of PVRIG and application thereof, and more particularly relates to an antibody or an antigen binding fragment thereof capable of specifically recognizing PVRIG, a nucleic acid molecule, an expression vector, a recombinant cell, a pharmaceutical composition, pharmaceutical application, a kit for detecting PVRIG and application thereof.

Background

Poliovirus receptor-associated immunoglobulin domains (PVRIG, also known as CD 112R) play an important role in reversing cell depletion and enhancing NK cell activation. PVRIG belongs to the nectin and nectin-like families, family members also include TIGIT, DNAM-1 (also known as CD 226), and CD96.PVRIG is expressed on NK and T cell surfaces, and expression is further up-regulated on the surface of activated T cells. The intracellular segment of PVRIG contains an ITIM motif, and the ligand CD112 (also known as PVRL 2) of the PVRIG is expressed on the surfaces of antigen presenting cells and various tumor cells, and can transmit inhibitory signals to NK cells and T cells after being combined with the PVRIG, so that the activation of the T cells and the NK cells is inhibited. CD112 is also a ligand for CD226, which can activate T cells and NK cells. CD112 has a higher affinity for PVRIG than CD226, so under normal conditions CD112 inhibits NK and T cell activation primarily by binding PVRIG.

Therefore, there is a need to develop a highly specific antibody against PVRIG.

Disclosure of Invention

The present invention has been accomplished based on the following problems and findings of the inventors:

tumor immunotherapy is a great breakthrough in the field of tumor therapy in recent years, wherein immune checkpoint inhibitors represented by PD-1/PD-L1 have good effects in clinical application. At present, PVRIG is a very important emerging immune checkpoint. For cancer patients, the surface of various tumor cells can up-regulate and express CD112, and after the CD112 is combined with PVRIG on the surface of immune cells, the cell activity of T cells and NK cells is inhibited, and the immune escape is realized by inhibiting the anti-tumor function of the cells; the PVRIG antibody can block a PVRIG/CD112 signal channel by being combined with PVRIG, recover the immune killing function of T cells and NK cells, can play a good treatment effect and has a good clinical application prospect. Moreover, research shows that the antibody treatment of the PVRIG can obviously inhibit the growth of tumors, and the combination of the antibody treatment with other immune checkpoint inhibitors can achieve better curative effect. At present, a humanized antibody targeting PVRIG is undergoing a clinical phase I experiment, and has achieved better curative effect.

Based on the above, through a large number of experiments, the inventors have obtained a variety of antibodies capable of specifically recognizing PVRIG, and the antibodies of the present invention can specifically target and bind to PVRIG receptor, thereby blocking the binding of CD112 and PVRIG, enhancing the killing effect of lymphocytes (especially T cells and NK cells), especially for cancer patients, after the PVRIG antibody binds to PVRIG, enhancing the killing effect of lymphocytes, and improving the therapeutic effect of the existing antibody drugs.

In a first aspect of the invention, the invention provides an antibody or antigen-binding fragment thereof capable of specifically recognizing PVRIG. According to an embodiment of the invention, the antibody or antigen-binding fragment thereof comprises: a CDR sequence selected from at least one of: light chain variable region CDR sequences: SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 19. SEQ ID NO:20 and SEQ ID NO:21; heavy chain variable region CDR sequences: SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 22. SEQ ID NO:23 and SEQ ID NO:24.

SSVNY(SEQ ID NO:1)。

DTS(SEQ ID NO:2)。

QQWSSNPYT(SEQ ID NO:3)。

GYSFTGYN(SEQ ID NO:4)。

IHFYTGAT(SEQ ID NO:5)。

AIMRWAVDY(SEQ ID NO:6)。

QDVSTS(SEQ ID NO:7)。

STS(SEQ ID NO:8)。

QQHYSTPWT(SEQ ID NO:9)。

GYSFTGYT(SEQ ID NO:10)。

ITPYNNDT(SEQ ID NO:11)。

ARWVYGNYVGWFAY(SEQ ID NO:12)。

ENIYSN(SEQ ID NO:13)。

GAS(SEQ ID NO:14)。

QHFWGTPYT(SEQ ID NO:15)。

GYTFTDYN(SEQ ID NO:16)。

IYPYNGGS(SEQ ID NO:17)。

AREEVRRDEALDF(SEQ ID NO:18)。

QSLLNSGTQKNY(SEQ ID NO:19)。

GTS(SEQ ID NO:20)。

QNDHSYPYT(SEQ ID NO:21)。

GYNFTSYW(SEQ ID NO:22)。

IYPGNYSP(SEQ ID NO:23)。

ASGYFDY(SEQ ID NO:24)。

according to the embodiment of the invention, the antibody can specifically target and bind to a PVRIG receptor and block the binding of PVRIG and CD 112.

According to an embodiment of the present invention, the above antibody or antigen-binding fragment may further comprise at least one of the following additional technical features:

according to an embodiment of the invention, the antibody comprises: light chain variable region CDR1, CDR2 and CDR3 sequences shown in the amino acid sequences of SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3; or light chain variable region CDR1, CDR2, CDR3 sequences as shown in the amino acid sequences of SEQ ID NO 7, 8 and 9, respectively; or light chain variable region CDR1, CDR2, CDR3 sequences as set forth in the amino acid sequences of SEQ ID Nos. 13, 14 and 15, respectively; alternatively, the light chain variable region CDR1, CDR2, CDR3 sequences are set forth as the amino acid sequences of SEQ ID NOs 19, 20 and 21, respectively.

According to an embodiment of the invention, the antibody comprises: heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in the amino acid sequences of SEQ ID Nos. 4, 5 and 6, respectively; or the heavy chain variable region CDR1, CDR2 and CDR3 sequences shown in the amino acid sequences of SEQ ID NO 10, 11 and 12 respectively; or the heavy chain variable region CDR1, CDR2 and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO 16, 17 and 18 respectively; or the heavy chain variable region CDR1, CDR2 and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO. 22, 23 and 24, respectively.

According to an embodiment of the invention, the antibody or antigen binding fragment thereof specifically recognizes the extracellular region of PVRIG.

According to an embodiment of the invention, the antibody or antigen-binding fragment thereof comprises: at least one of a heavy chain framework region sequence and a light chain framework region sequence; wherein at least a portion of at least one of the heavy chain framework region sequence and the light chain framework region sequence is derived from at least one of a murine antibody, a human antibody, a primate antibody, or a mutant thereof.

According to an embodiment of the invention, the antibody has the amino acid sequence as shown in SEQ ID NO:25 to 28.

EVQLQQSGPDLVKTGASVKISCKAVGYSFTGYNVHWVKQSHDKSLEWIGYIHFYTGATDYNQKFKDKATFTVDASSNTAYMQFNSLTSEDSAVYYCAIMRWAVDYWGQGTSVTVSS(SEQ ID NO:25).

EVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSPGKNLEWIGLITPYNNDTSYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARWVYGNYVGWFAYWGQGTLVTVSA(SEQ ID NO:26)。

EVQLQQSGPELVKPGASVRITCKASGYTFTDYNIHWVKQSHGKSLEWIGYIYPYNGGSGYNQKFKSQATLTVDTSSTTAYMEIRSLTSEDSAVYYCAREEVRRDEALDFWGQGTSVTVSS(SEQ ID NO:27)。

QVQLQQPGAELVKPGTSVKLSCKASGYNFTSYWINWVKLRPGQGLEWIGDIYPGNYSPNYNEKFLNKATLTTDTASSTAYMQLNNLASEDSAVYYCASGYFDYWGQGTTLIVSA(SEQ ID NO:28)。

According to an embodiment of the invention, the antibody has the amino acid sequence as shown in SEQ ID NO:29 to 32 in a light chain variable region.

QIVLTQSPTIMSASPGETVTMTCSVSSSVNYIYWYQQKSGASPKRWIYDTSKLASGVPSRFSGSGSGASYSLTISNMEAEDAATYYCQQWSSNPYTFGGGTKLEIK(SEQ ID NO:29)。

DIVMTQSHKFMSTSVGDRVNITCKASQDVSTSVTWYQQKPGQSPKLLIYSTSYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPWTFGGGTKLEIK(SEQ ID NO:30)。

DIQMTQSPASLSVSVGETVNITCRVSENIYSNLAWYQQKQGKSPQLLVYGASNLPDGVPSRFSGSGSGTQYSLKINTLQSEDFGTYYCQHFWGTPYTFGGGTKLEIK(SEQ ID NO:31)。

DIVMTQSPSSLSVSAGEEVIMSCKSSQSLLNSGTQKNYLAWYQQKPGQPPKLLIYGTSTRESGVPDRFTGSGSGTDFTLTINNVQAEDLAIYYCQNDHSYPYTFGGGTKLEIK(SEQ ID NO:32)。

According to an embodiment of the present invention, the antibody has a heavy chain variable region having an amino acid sequence shown as SEQ ID NO:25 and a heavy chain variable region having an amino acid sequence shown as SEQ ID NO:29, or a light chain variable region of the amino acid sequence set forth in seq id No. 29.

According to an embodiment of the present invention, the antibody has a heavy chain variable region having an amino acid sequence shown as SEQ ID NO:26 and a heavy chain variable region having an amino acid sequence shown as SEQ ID NO:30 in a light chain variable region.

According to an embodiment of the present invention, the antibody has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:27 and a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:31, or a light chain variable region of the amino acid sequence set forth in seq id No. 31.

According to an embodiment of the present invention, the antibody has a heavy chain variable region having an amino acid sequence shown as SEQ ID NO 28 and a heavy chain variable region having an amino acid sequence shown as SEQ ID NO:32, or a light chain variable region of the amino acid sequence set forth in seq id no.

According to an embodiment of the invention, the antibody comprises at least one of a heavy chain constant region and a light chain constant region, at least a portion of the at least one of a heavy chain constant region and a light chain constant region being derived from at least one of a murine antibody, a human antibody, a primate antibody, or a mutant thereof.

According to an embodiment of the invention, the light chain constant region and the heavy chain constant region are both from a murine IgG antibody or a mutant thereof.

According to an embodiment of the invention, the antibody has the amino acid sequence as shown in SEQ ID NO:33 to 36 and a light chain having the amino acid sequence as set forth in any one of SEQ ID NOs: 37 to 40, or a pharmaceutically acceptable salt thereof.

EVQLQQSGPDLVKTGASVKISCKAVGYSFTGYNVHWVKQSHDKSLEWIGYIHFYTGATDYNQKFKDKATFTVDASSNTAYMQFNSLTSEDSAVYYCAIMRWAVDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(SEQ ID NO:33)。

EVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSPGKNLEWIGLITPYNNDTSYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARWVYGNYVGWFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(SEQ ID NO:34)。

EVQLQQSGPELVKPGASVRITCKASGYTFTDYNIHWVKQSHGKSLEWIGYIYPYNGGSGYNQKFKSQATLTVDTSSTTAYMEIRSLTSEDSAVYYCAREEVRRDEALDFWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(SEQ ID NO:35)。

QVQLQQPGAELVKPGTSVKLSCKASGYNFTSYWINWVKLRPGQGLEWIGDIYPGNYSPNYNEKFLNKATLTTDTASSTAYMQLNNLASEDSAVYYCASGYFDYWGQGTTLIVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(SEQ ID NO:36)。

QIVLTQSPTIMSASPGETVTMTCSVSSSVNYIYWYQQKSGASPKRWIYDTSKLASGVPSRFSGSGSGASYSLTISNMEAEDAATYYCQQWSSNPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ ID NO:37)。

DIVMTQSHKFMSTSVGDRVNITCKASQDVSTSVTWYQQKPGQSPKLLIYSTSYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ ID NO:38)。

DIQMTQSPASLSVSVGETVNITCRVSENIYSNLAWYQQKQGKSPQLLVYGASNLPDGVPSRFSGSGSGTQYSLKINTLQSEDFGTYYCQHFWGTPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ ID NO:39)。

DIVMTQSPSSLSVSAGEEVIMSCKSSQSLLNSGTQKNYLAWYQQKPGQPPKLLIYGTSTRESGVPDRFTGSGSGTDFTLTINNVQAEDLAIYYCQNDHSYPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ ID NO:40)。

According to an embodiment of the invention, the antibody is a single chain antibody, a multimeric antibody, a CDR-grafted antibody or a small molecule antibody.

According to an embodiment of the invention, the antibody is a single chain antibody having the amino acid sequence of SEQ ID NO:41 to 48.

QIVLTQSPTIMSASPGETVTMTCSVSSSVNYIYWYQQKSGASPKRWIYDTSKLASGVPSRFSGSGSGASYSLTISNMEAEDAATYYCQQWSSNPYTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLQQSGPDLVKTGASVKISCKAVGYSFTGYNVHWVKQSHDKSLEWIGYIHFYTGATDYNQKFKDKATFTVDASSNTAYMQFNSLTSEDSAVYYCAIMRWAVDYWGQGTSVTVSS(SEQ ID NO:41)。

DIVMTQSHKFMSTSVGDRVNITCKASQDVSTSVTWYQQKPGQSPKLLIYSTSYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPWTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSPGKNLEWIGLITPYNNDTSYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARWVYGNYVGWFAYWGQGTLVTVSA(SEQ ID NO:42)。

DIQMTQSPASLSVSVGETVNITCRVSENIYSNLAWYQQKQGKSPQLLVYGASNLPDGVPSRFSGSGSGTQYSLKINTLQSEDFGTYYCQHFWGTPYTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLQQSGPELVKPGASVRITCKASGYTFTDYNIHWVKQSHGKSLEWIGYIYPYNGGSGYNQKFKSQATLTVDTSSTTAYMEIRSLTSEDSAVYYCAREEVRRDEALDFWGQGTSVTVSS(SEQ ID NO:43)。

DIVMTQSPSSLSVSAGEEVIMSCKSSQSLLNSGTQKNYLAWYQQKPGQPPKLLIYGTSTRESGVPDRFTGSGSGTDFTLTINNVQAEDLAIYYCQNDHSYPYTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQPGAELVKPGTSVKLSCKASGYNFTSYWINWVKLRPGQGLEWIGDIYPGNYSPNYNEKFLNKATLTTDTASSTAYMQLNNLASEDSAVYYCASGYFDYWGQGTTLIVSA(SEQ ID NO:44)。

EVQLQQSGPDLVKTGASVKISCKAVGYSFTGYNVHWVKQSHDKSLEWIGYIHFYTGATDYNQKFKDKATFTVDASSNTAYMQFNSLTSEDSAVYYCAIMRWAVDYWGQGTSVTVSSGGGSGGGGSGGGGSQIVLTQSPTIMSASPGETVTMTCSVSSSVNYIYWYQQKSGASPKRWIYDTSKLASGVPSRFSGSGSGASYSLTISNMEAEDAATYYCQQWSSNPYTFGGGTKLEIK(SEQ ID NO:45)。

EVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSPGKNLEWIGLITPYNNDTSYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARWVYGNYVGWFAYWGQGTLVTVSAGGGGSGGGGSGGGGSDIVMTQSHKFMSTSVGDRVNITCKASQDVSTSVTWYQQKPGQSPKLLIYSTSYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPWTFGGGTKLEIK(SEQ ID NO:46)。

EVQLQQSGPELVKPGASVRITCKASGYTFTDYNIHWVKQSHGKSLEWIGYIYPYNGGSGYNQKFKSQATLTVDTSSTTAYMEIRSLTSEDSAVYYCAREEVRRDEALDFWGQGTSVTVSSGGGGSGGGGSGGGGSDIQMTQSPASLSVSVGETVNITCRVSENIYSNLAWYQQKQGKSPQLLVYGASNLPDGVPSRFSGSGSGTQYSLKINTLQSEDFGTYYCQHFWGTPYTFGGGTKLEIK(SEQ ID NO:47)。

QVQLQQPGAELVKPGTSVKLSCKASGYNFTSYWINWVKLRPGQGLEWIGDIYPGNYSPNYNEKFLNKATLTTDTASSTAYMQLNNLASEDSAVYYCASGYFDYWGQGTTLIVSAGGGGSGGGGSGGGGSDIVMTQSPSSLSVSAGEEVIMSCKSSQSLLNSGTQKNYLAWYQQKPGQPPKLLIYGTSTRESGVPDRFTGSGSGTDFTLTINNVQAEDLAIYYCQNDHSYPYTFGGGTKLEIK(SEQ ID NO:48)。

According to embodiments of the invention, the antigen binding fragment includes a Fab fragment, (Fab) ₂ At least one of a fragment, scFv-Fc fusion protein, scFv-Fv fusion protein, fv fragment, and minimal recognition unit.

In a second aspect of the invention, a nucleic acid molecule is presented. According to an embodiment of the invention, the nucleic acid molecule encodes an antibody or antigen-binding fragment thereof according to the first aspect of the invention. The antibody or antigen binding fragment encoded by the nucleic acid molecule according to the embodiment of the invention can specifically target and bind to PVRIG, and has high antigen binding activity.

According to an embodiment of the invention, the nucleic acid molecule is DNA.

In a third aspect of the invention, the invention features an expression vector. According to an embodiment of the invention, a nucleic acid molecule according to the second aspect of the invention is carried. The expression of the antibody or antigen-binding fragment thereof specifically recognizing PVRIG as described above can be efficiently achieved under the mediation of a regulatory system after the expression vector according to the embodiment of the present invention is introduced into a suitable recipient cell, so that the antibody or antigen-binding fragment can be obtained in a large amount.

According to an embodiment of the invention, the expression vector is a eukaryotic expression vector or a virus, preferably the virus is a lentivirus.

In a fourth aspect of the invention, a recombinant cell is provided. According to an embodiment of the invention, the recombinant cell: carrying a nucleic acid molecule according to the second aspect of the invention; or, expressing the antibody or antigen-binding fragment thereof of the first aspect of the invention. The recombinant cells according to embodiments of the invention can be used for in vitro expression and mass production of the aforementioned antibodies or antigen-binding fragments thereof that specifically recognize PVRIG.

According to an embodiment of the present invention, the recombinant cell is obtained by introducing the expression vector of the third aspect of the present invention into a host cell.

According to an embodiment of the invention, the recombinant cell is a eukaryotic cell.

According to an embodiment of the invention, the recombinant cell is a mammalian cell.

In a fifth aspect of the invention, a pharmaceutical composition is provided. According to an embodiment of the invention, the pharmaceutical composition comprises: an antibody or antigen-binding fragment thereof according to the first aspect of the invention; a nucleic acid molecule according to the second aspect of the invention; an expression vector according to the third aspect of the present invention; alternatively, a recombinant cell according to the fourth aspect of the invention. According to the use of the embodiment of the present invention, the pharmaceutical composition may be used for preventing and/or treating cancers, such as melanoma, lung cancer, head and neck cancer, and the like.

In a sixth aspect of the invention, the invention provides the use of an antibody or antigen-binding fragment thereof, a nucleic acid molecule, an expression vector, a recombinant cell or a pharmaceutical composition as hereinbefore described in the manufacture of a medicament. According to an embodiment of the invention, the medicament is for the prevention and/or treatment of cancer. According to the embodiment of the invention, the antibody can specifically and targetedly bind to a PVRIG receptor, so that the binding of CD112 and PVRIG is blocked, the killing effect of lymphocytes (especially T cells and NK cells) is enhanced, especially for cancer patients, after the PVRIG antibody is combined with PVRIG, the killing effect of the lymphocytes can be enhanced, and the curative effect of the existing antibody drug is improved.

According to an embodiment of the invention, the medicament is for the prevention and/or treatment of non-small cell lung cancer, papillary thyroid cancer, glioblastoma multiforme, colorectal cancer, lung cancer, head and neck cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, liver cancer, cholangiocarcinoma or sarcoma, acute myelogenous leukemia, large cell neuroendocrine cancer, neuroblastoma, prostate cancer, neuroblastoma, pancreatic cancer, melanoma, head and neck squamous cell carcinoma or gastric cancer.

In a seventh aspect of the invention, the invention provides a kit for detecting PVRIG. According to an embodiment of the invention, the kit comprises an antibody according to any one of the first aspect of the invention. The PVRIG antibody can be specifically and targetedly combined with PVRIG, and the kit according to the embodiment of the invention can realize specific detection of PVRIG, for example, when the antibody is combined with a fluorescent group, a fluorescent detection device can be adopted to realize positioning or real-time detection of PVRIG.

In an eighth aspect of the invention, the invention provides the use of an antibody or antigen-binding fragment thereof, a nucleic acid molecule, an expression vector or a recombinant cell as hereinbefore described in the manufacture of a kit. According to an embodiment of the invention, the kit is for detecting PVRIG.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 is an ELISA assay for the binding of PVRIG antibodies of example 2 of the present invention to recombinant human PVRIG-human IgG1Fc fusion protein;

FIG. 2 is a FACS detection of the binding of PVRIG antibodies of example 3 of the present invention to cell surface human PVRIG protein;

FIG. 3 is an ELISA assay for PVRIG antibodies of example 4 of the present invention that blocks the binding of PVRIG to its ligand, CD112, in vitro;

FIG. 4 is a FACS assay of PVRIG antibodies of example 5 of the invention blocking the binding of PVRIG to its ligand CD112 in vitro;

FIG. 5 is a graph of the detection of the binding of PVRIG antibodies of example 6 of the present invention to cell surface cynomolgus monkey PVRIG protein;

FIG. 6 is a graph showing the detection of the killing effect of NK-92 cells on tumor cells by PVRIG antibody in example 7 of the present invention.

Detailed Description

The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention.

It should be noted that the terms "first" and "second" 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 defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Further, in the description of the present invention, "a plurality" means two or more unless otherwise specified.

Antibodies

Herein, the term "antibody" is an immunoglobulin molecule capable of binding to a specific antigen. Comprises two light chains with light molecular weight and two heavy chains with heavy molecular weight, wherein the heavy chains (H chains) and the light chains (L chains) are connected by disulfide bonds to form a tetrapeptide chain molecule. Among them, the amino-terminal (N-terminal) amino acid sequence of the peptide chain varies widely and is called variable region (V region), and the carboxy-terminal (C-terminal) is relatively stable and varies little and is called constant region (C region). The V regions of the L chain and H chain are referred to as VL and VH, respectively.

Certain regions in the variable region, which have a higher degree of variation in amino acid composition and arrangement order, are called Hypervariable regions (HVRs), which are the sites where antigens and antibodies bind and are therefore also called complementarity-determining regions (CDRs). There are three CDRs on both the heavy chain variable region and the light chain variable region.

The invention utilizes the recombinant human PVRIG-human IgG1Fc fusion protein containing human PVRIG extracellular region gene to immunize BALB/c female mice, and four strains of murine antibodies capable of specifically binding human PVRIG protein are screened by a hybridoma fusion technology. The antibody fragment can be specifically targeted and combined with a PVRIG receptor, blocks the combination of PVRIG and CD112, enhances the killing effect of lymphocytes (especially T cells and NK cells), particularly for cancer patients, can enhance the killing effect of the lymphocytes after the PVRIG antibody is combined with the PVRIG, and improves the curative effect of the existing antibody drugs.

In some embodiments, the invention provides an antibody or antigen-binding fragment thereof capable of specifically recognizing PVRIG, comprising: a CDR sequence selected from at least one of: light chain variable region CDR sequences: SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. the amino acid sequence of SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. the amino acid sequence of SEQ ID NO: 13. the amino acid sequence of SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 19. SEQ ID NO:20 and SEQ ID NO:21; heavy chain variable region CDR sequences: SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 22. SEQ ID NO:23 and SEQ ID NO:24. in other embodiments, the antibodies or antigen-binding fragments provided herein have conservative amino acid substitutions as compared to the heavy and light chains described above. "antigen-binding fragment" refers to an antibody fragment that retains the ability to specifically bind to an antigen (ROR 2), examples of which include, but are not limited to, at least one of Fv fragments, disulfide stabilized Fv fragments (dsFv), fab fragments, (Fab) 2, scFv-Fc fusion proteins, scFv-Fv fusion proteins, fv-Fc fusion proteins, multispecific antibodies formed from antigen-binding fragments, single domain antibodies, VHH nanobodies, domain antibodies, bivalent domain antibodies, or minimal recognition units. "conservative amino acid substitution" refers to the replacement of an amino acid by another amino acid residue that is biologically, chemically, or structurally similar. Biologically similar means that the substitution does not destroy the biological activity of the PVRIG antibody or the PVRIG antigen. Structurally similar refers to amino acids having side chains of similar length, such as alanine, glycine, or serine, or side chains of similar size. Chemical similarity refers to amino acids that are identically charged or are both hydrophilic or hydrophobic. For example, the hydrophobic residues isoleucine, valine, leucine or methionine are substituted for one another. Or substitution of polar amino acids such as lysine with arginine, aspartic acid with glutamic acid, asparagine with glutamine, threonine with serine, and the like.

In some embodiments, the invention provides an antibody or antigen binding fragment having the amino acid sequence set forth in SEQ ID NO:25 to 28 and a heavy chain variable region having an amino acid sequence as set forth in any one of SEQ ID NOs: 29 to 32, or a light chain variable region of an amino acid sequence as set forth in any one of claims 29 to 32. The inventor can obtain the CDR regions (shown as SEQ ID NO: 4-6, SEQ ID NO: 10-12, SEQ ID NO: 16-18, SEQ ID NO: 22-24) of the heavy chain variable region sequence and the CDR regions (shown as SEQ ID NO: 1-3, SEQ ID NO: 7-9, SEQ ID NO: 13-15, SEQ ID NO: 19-21) of the light chain variable region sequence of the antibody through an antibody sequence comparison database (NCBI, IMGT) or related software. In other embodiments, the heavy chain variable region sequence of the antibody or antigen-binding fragment is identical to SEQ ID NO:25 to 28, or a conservative amino acid substitution. In some embodiments, the light chain variable region sequence of the antibody or antigen-binding fragment is identical to SEQ ID NO:29 to 32, which have conservative amino acid substitutions. Of course, these conservative amino acid substitutions do not result in a change in the biological function of the antibody or antigen-binding fragment. In some embodiments, these conservative amino acid substitutions may occur at amino acids other than the CDR regions in the heavy chain variable region and the light chain variable region.

In some preferred embodiments, the invention provides a PVRIG antibody having the amino acid sequence set forth in SEQ ID NO:33 to 36 and a light chain having an amino acid sequence as set forth in any one of SEQ ID NOs: 37 to 40, or a pharmaceutically acceptable salt thereof.

In some preferred embodiments, the present invention provides a PVRIG single chain antibody having the amino acid sequence of SEQ ID NO:41 to 48.

Nucleic acid molecule, expression vector, recombinant cell

In the process of preparing or obtaining these antibodies or antigen-binding fragments thereof, nucleic acid molecules expressing these antibodies or antigen-binding fragments thereof can be used to link with different vectors and then express in different cells to obtain the corresponding antibodies or antigen-binding fragments thereof.

To this end, the invention also provides an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof described above.

In some preferred embodiments, the nucleic acid molecule is species-optimized for easier expression in mammalian cells.

The present invention also provides an expression vector comprising the isolated nucleic acid molecule described above. When the isolated polynucleotide is ligated to a vector, the polynucleotide may be ligated to control elements on the vector directly or indirectly, so long as the control elements are capable of controlling the translation, expression, etc. of the polynucleotide. Of course, these control elements may be derived directly from the vector itself, or may be exogenous, i.e., not derived from the vector itself. Of course, the polynucleotide may be operably linked to a control element.

"operably linked" herein refers to the attachment of a foreign gene to a vector such that control elements within the vector, such as transcriptional and translational control sequences and the like, are capable of performing their intended function of regulating the transcription and translation of the foreign gene. Of course, the polynucleotides encoding the heavy and light chains of the antibody may be inserted into separate vectors, usually into the same vector. Commonly used vectors may be, for example, plasmids, phages and the like.

The invention also provides a recombinant cell which contains the expression vector. The expression vector may be introduced into mammalian cells, constructed to obtain recombinant cells, and then used to express the antibody or antigen-binding fragment provided by the present invention. The recombinant cell is cultured to obtain the corresponding antibody. These usable mammalian cells may be, for example, CHO cells or the like.

Pharmaceutical composition, kit and pharmaceutical application and application in preparation of kit

The invention also provides a pharmaceutical composition, which comprises the antibody or the antigen-binding fragment thereof, and also can comprise the nucleic acid molecule, the expression vector and the recombinant cell.

The PVRIG antibodies provided herein can be incorporated into a pharmaceutical composition suitable for administration to a subject. Typically, these pharmaceutical compositions comprise PVRIG antibodies provided herein.

In some embodiments, the pharmaceutical compositions further comprise a pharmaceutically acceptable carrier, including any solvent, solid excipient, diluent, binder, disintegrant, or other liquid excipient, dispersant, flavoring or suspending agent, surfactant, isotonic agent, thickening agent, emulsifier, preservative, solid binder, glidant or lubricant, and the like, suitable for the particular target dosage form. Except insofar as any conventional adjuvant is incompatible with the compounds of the invention, e.g., any adverse biological effect produced or interaction in a deleterious manner with any other component of a pharmaceutically acceptable composition, their use is contemplated by the present invention. For example, the antibodies of the invention can be incorporated into pharmaceutical compositions suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These pharmaceutical compositions can be prepared in various forms. Such as liquid, semi-solid, and solid dosage forms, and the like, including, but not limited to, liquid solutions (e.g., injection solutions and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. Typical pharmaceutical compositions are in the form of injection solutions or infusion solutions. The antibody may be administered by intravenous infusion or injection or intramuscular or subcutaneous injection.

Of course, the PVRIG antibodies herein can also be made part of a kit or other diagnostic reagent as desired. According to an embodiment of the present invention, the present invention also provides a kit comprising the above PVRIG antibody. The kit provided by the invention can be used for immunoblotting, immunoprecipitation and the like, and relates to a kit and the like for detection by utilizing the specific binding performance of the PVRIG antigen and the antibody. These kits may comprise any one or more of the following: an antagonist, a PVRIG antibody, or a drug reference material; a protein purification column; an immunoglobulin affinity purification buffer; an assay diluent for the cells; instructions or literature, etc. PVRIG antibodies can be used for different types of diagnostic tests, e.g. to detect a wide variety of diseases or the presence of drugs, toxins or other proteins, etc. in vitro or in vivo. PVRIG can be tested, for example, by testing the serum or blood of the subject.

The PVRIG antibody and/or the medicament comprising the PVRIG antibody of the invention can prevent and/or treat cancer or tumor, which can be any unregulated cell growth. Specifically, non-small cell lung cancer, papillary thyroid cancer, glioblastoma multiforme, colorectal cancer, lung cancer, head and neck cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, liver cancer, cholangiocarcinoma or sarcoma, acute myelogenous leukemia, large cell neuroendocrine cancer, neuroblastoma, prostate cancer, neuroblastoma, pancreatic cancer, melanoma, head and neck squamous cell carcinoma or gastric cancer, and the like may be mentioned.

When the PVRIG antibody provided by the present invention is used for treating the above-mentioned diseases, the PVRIG antibody provided by the present invention may be provided to a subject. To this end, the present invention provides a method for treating the above-mentioned diseases, comprising administering to a subject in need thereof an antibody or antigen-binding fragment thereof provided by the present invention.

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1: preparation of anti-human PVRIG monoclonal antibody

In this example, monoclonal antibodies against PVRIG were prepared by hybridoma cell fusion technique as follows:

(1) Preparation of recombinant fusion proteins

Artificially synthesizing a human PVRIG full-length gene, amplifying by using a PCR technology to obtain a human PVRIG extracellular region coding gene, introducing a gene segment shown in SEQ ID NO:51 into the C end of the human PVRIG extracellular region coding gene, cloning into a pLVX-IRES-zsGreen vector (Youbio. Cn /) through packaging lentivirus, infecting HEK293T cell line (293T cell for short) to obtain 293T cells over-expressing human PVRIG-human IgG1Fc fusion Protein, culturing the 293T cells, collecting cell culture supernatant, and purifying by a Protein G affinity chromatography (GE Healthcare) method to obtain the recombinant Protein with the purity of more than 90%. Mass spectrum identification shows that the peptide segment of the obtained recombinant protein is matched with a PVRIG sequence, and the recombinant protein is proved to be recombinant human PVRIG-human IgG1Fc fusion protein.

Wherein the content of the first and second substances,

human PVRIG full-length gene sequence:

ATGAGAACAGAGGCACAGGTGCCGGCCCTGCAGCCCCCAGAACCTGGACTGGAGGGGGCCATGGGGCACCGGACCCTGGTCCTGCCCTGGGTGCTGCTGACCTTGTGTGTCACTGCGGGGACCCCGGAGGTGTGGGTTCAAGTTCGGATGGAGGCCACCGAGCTCTCGTCCTTCACCATCCGTTGTGGGTTCCTGGGGTCTGGCTCCATCTCCCTGGTGACTGTGAGCTGGGGGGGCCCCAACGGTGCTGGGGGGACCACGCTGGCTGTGTTGCACCCAGAACGTGGCATCCGGCAATGGGCCCCTGCTCGCCAGGCCCGCTGGGAAACCCAGAGCAGCATCTCTCTCATCCTGGAAGGCTCTGGGGCCAGCAGCCCCTGCGCCAACACCACCTTCTGCTGCAAGTTTGCGTCCTTCCCTGAGGGCTCCTGGGAGGCCTGTGGGAGCCTCCCGCCCAGCTCAGACCCAGGGCTCTCTGCCCCGCCGACTCCTGCCCCCATTCTGCGGGCAGACCTGGCCGGGATCTTGGGGGTCTCAGGAGTCCTCCTCTTTGGCTGTGTCTACCTCCTTCATCTGCTGCGCCGACATAAGCACCGCCCTGCCCCTAGGCTCCAGCCGTCCCGCACCAGCCCCCAGGCACCGAGAGCACGAGCATGGGCACCAAGCCAGGCCTCCCAGGCTGCTCTTCACGTCCCTTATGCCACTATCAACACCAGCTGCCGCCCAGCTACTTTGGACACAGCTCACCCCCATGGGGGGCCGTCCTGGTGGGCGTCACTCCCCACCCACGCTGCACACCGGCCCCAGGGCCCTGCCGCCTGGGCCTCCACACCCATCCCTGCACGTGGCAGCTTTGTCTCTGTTGAGAATGGACTCTACGCTCAGGCAGGGGAGAGGCCTCCTCACACTGGTCCCGGCCTCACTCTTTTCCCTGACCCTCGGGGGCCCAGGGCCATGGAAGGACCCTTAGGAGTTCGATGA(SEQ ID NO:49)；

the gene sequence of the human PVRIG extracellular region codes:

ACCCCGGAGGTGTGGGTTCAAGTTCGGATGGAGGCCACCGAGCTCTCGTCCTTCACCATCCGTTGTGGGTTCCTGGGGTCTGGCTCCATCTCCCTGGTGACTGTGAGCTGGGGGGGCCCCAACGGTGCTGGGGGGACCACGCTGGCTGTGTTGCACCCAGAACGTGGCATCCGGCAATGGGCCCCTGCTCGCCAGGCCCGCTGGGAAACCCAGAGCAGCATCTCTCTCATCCTGGAAGGCTCTGGGGCCAGCAGCCCCTGCGCCAACACCACCTTCTGCTGCAAGTTTGCGTCCTTCCCTGAGGGCTCCTGGGAGGCCTGTGGGAGCCTCCCGCCCAGCTCAGACCCAGGGCTCTCTGCCCCGCCGACTCCTGCCCCCATTCTGCGGGCAGAC(SEQ ID NO:50)；

human IgG1Fc coding gene sequence:

CCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCCGCCCCCGAGCTGCTGGGCGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCCCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCAGGGAGCCCCAGGTGTACACCCTGCCCCCCAGCAGGGACGAGCTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGCAAG(SEQ ID NO:51)；

the amino acid sequence of the extracellular region of the human PVRIG protein in the recombinant human PVRIG-human IgG1Fc fusion protein is as follows: <xnotran> TPEVWVQVRMEATELSSFTIRCGFLGSGSISLVTVSWGGPNGAGGTTLAVLHPERGIRQWAPARQARWETQSSISLILEGSGASSPCANTTFCCKFASFPEGSWEACGSLPPSSDPGLSAPPTPAPILRAD (SEQ ID NO: 52); </xnotran>

Amino acid sequence of human IgG1Fc protein in recombinant human PVRIG-human IgG1Fc fusion protein: <xnotran> PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 53). </xnotran>

(2) Animal immunization, hybridoma cell fusion and clone screening

Fully mixing the recombinant human PVRIG-human IgG1Fc fusion protein with an equal volume of Complete Freund's Adjuvant (CFA), immunizing BALB/c female mice (purchased from Shanghai Rice and with the weight of about 20 g) with the age of 8-10 weeks for the first time, and injecting 40-60 mu g of the recombinant human PVRIG-human IgG1Fc fusion protein into the abdominal cavity of each mouse; after immunization every 2 weeks, the same dose of recombinant human PVRIG-human IgG1Fc fusion protein was mixed with an equal volume of incomplete Freund's adjuvant, and after 5 total immunizations, plasma was screened by ELISA to obtain mice with anti-human PVRIG immunoglobulin, and finally 40-60. Mu.g of recombinant human PVRIG-human IgG1Fc fusion protein was injected for boosting. On the third day after the boost, splenocytes were isolated and fused with murine myeloma SP2/0 cells (ATCC accession number CRL-1581) using standard techniques, and the fused hybridoma cells were screened by ELISA for binding and blocking functions to obtain murine mAbs mAb001, mAb002, mAb003 and mAb004.

Wherein, the amino acid sequence of the mouse monoclonal antibody mAb001 is a polypeptide containing SEQ ID NO:33 and a light chain having the amino acid sequence shown as SEQ ID NO: 37;

the amino acid sequence of the mouse monoclonal antibody mAb002 is SEQ ID NO:34 and a light chain having the amino acid sequence shown as SEQ ID NO: 38;

the amino acid sequence of the murine monoclonal antibody mAb003 is a polypeptide comprising the amino acid sequence of SEQ ID NO:35 and a light chain having the amino acid sequence shown as SEQ ID NO: 39;

the amino acid sequence of the murine monoclonal antibody mAb004 is a polypeptide containing SEQ ID NO:36 and a light chain having the amino acid sequence shown as SEQ ID NO:40, or a light chain of the amino acid sequence set forth in seq id no.

Based on the murine mabs mAb001, mAb002, mAb003 and mAb004 obtained by screening in example 1, the following examples are the binding or blocking experiments of the four murine mabs with PVRIG, respectively.

Example 2: ELISA experiment of PVRIG antibody combined with human PVRIG recombinant protein in vitro

The recombinant human PVRIG-human IgG1Fc fusion protein was diluted to 0.5. Mu.g/ml with PBS (phosphate buffered saline), 96-well ELISA plates were coated in a volume of 100. Mu.l/well, overnight at 4 ℃, washed 3 times with PBST (0.05% Tween 20-PBS), blocked with 1% BSA, incubated at 37 ℃ for 2 hours, washed 3 times with PBST, and four murine mAbs (i.e., mAb001, mAb002, mAb003, and mAb 004) diluted in multiples were added as experimental groups, the murine mAbs were set at 8 concentrations (100. Mu.g/ml, 10. Mu.g/ml, 1. Mu.g/ml, 0.1. Mu.g/ml, 0.01. Mu.g/ml, 0.001. Mu.g/ml, 0.0001. Mu.g/ml, 0.00001. Mu.g/ml, respectively), and mouse IgG1 was used as a negative control, and the volume of mouse IgG1 was added at 100. Mu.l/well, and incubated at 37 ℃ for 1 hour. The experimental and control groups were washed 3 times with PBST, respectively, and 100. Mu.l of goat anti-mouse IgG antibody labeled with horseradish peroxidase (HRP) (purchased from Southern Biotech, 1: 5000 dilution) was added to each well and incubated at 37 ℃ for 1 hour. Adding 100 μ l TMB substrate solution into each well, developing in dark for 5-10 min, adding 100 μ l stop solution (2M H2SO 4) into each well, detecting with enzyme reader immediately after stopping, and reading light absorption value (OD 450 nm) ₄₅₀ ) The results are shown in FIG. 1.

The ELISA results in fig. 1 show that murine mabs mAb001, mAb002, mAb003 and mAb004 specifically bind to human PVRIG protein by binding to the extracellular region of PVRIG.

Example 3: FACS experiment of in vitro binding of PVRIG antibodies to human PVRIG

The 293T-human PVRIG cell line was harvested, and after washing with PBS, cell counting was performed. Then subpackaged in a plurality of 1.5ml Ep tubes, and each 1.5ml Ep tube is put into a 2X 10 ⁵ Four murine monoclonal antibodies were added to each cell as an experimental group, and the concentration of each murine monoclonal antibody was set to 8 concentrations (100. Mu.g/ml, 10. Mu.g/ml, 1. Mu.g/ml, 0.1. Mu.g/ml, 0.01. Mu.g/ml, respectively)0.001. Mu.g/ml, 0.0001. Mu.g/ml, 0.00001. Mu.g/ml) and mouse IgG1 was used as a negative control, incubated at 4 ℃ for 30 minutes, followed by washing with PBS, and centrifuged at 3500rpm for 5 minutes to collect cells. The cell pellet was resuspended in 100. Mu.l of PBS and Alexa Fluor 647 fluorescein-labeled goat anti-mouse IgG antibody (purchased from Biolegend) was added, incubated at 4 ℃ for 30 minutes in the dark, followed by addition of PBS for two washes, centrifugation at 3500rpm for 5 minutes, and cells were collected. The cell pellet was resuspended in 200. Mu.l PBS and examined using a flow cytometer (BD FACS Celesta), and the analytical statistics are shown in FIG. 2.

The results in FIG. 2 show that murine mAbs mAb001, mAb002, mAb003 and mAb004 are all capable of specifically binding to human PVRIG proteins on the cell membrane surface.

Example 4: ELISA assay for PVRIG antibody in vitro blocking of PVRIG binding to CD112

In this example, recombinant human PVRIG-human IgG1Fc fusion protein was diluted to 0.5. Mu.g/ml in PBS, 96-well ELISA plates were coated in a volume of 100. Mu.l/well, overnight at 4 ℃, washed 3 times with PBST (0.05% Tween 20-PBS), blocked with 1% BSA, and incubated for 2 hours at 37 ℃. After 3 washes with PBST, 40ng/ml of biotin-labeled CD 112-human Fc fusion protein and four murine mAbs diluted in multiples were added, set at 8 concentrations (100. Mu.g/ml, 10. Mu.g/ml, 1. Mu.g/ml, 0.1. Mu.g/ml, 0.01. Mu.g/ml, 0.001. Mu.g/ml, 0.0001. Mu.g/ml, 0.00001. Mu.g/ml, respectively), and mouse IgG1 was used as a negative control, the volume of mouse IgG1 added was 100. Mu.l/well, and incubated at 37 ℃ for 1 hour. PBST was washed 3 times, 100. Mu.l of horseradish peroxidase (HRP) -labeled streptavidin (purchased from Southern Biotech, 1: 5000 dilution) was added per well, and incubated at 37 ℃ for 1 hour. After cleaning, 100 mul of TMB substrate solution is added into each hole, color development is carried out for 5-10 minutes in a dark place, 100 mul of stop solution (2M H2SO 4) is added into each hole, detection is carried out by a microplate reader immediately after termination, and the light absorption value (OD 450) with the wavelength of 450nm is read, and the result is shown in figure 3.

The results show that murine mAb001, mAb002, mAb003, and mAb004 all blocked PVRIG and CD112 binding very effectively.

Example 5: FACS experiment with PVRIG antibodies blocking PVRIG and CD112 binding in vitro

The 293T-human CD112 cell line was harvested, and after washing with PBS and resuspension, cell counting was performed. Then subpackaged in a plurality of 1.5ml Ep tubes, and each 1.5ml Ep tube is put into 2 multiplied by 10 ⁵ The cells were subjected to an experimental group of four kinds of murine mAbs each set to 8 concentrations (100. Mu.g/ml, 10. Mu.g/ml, 1. Mu.g/ml, 0.1. Mu.g/ml, 0.01. Mu.g/ml, 0.001. Mu.g/ml, 0.0001. Mu.g/ml, 0.00001. Mu.g/ml) and 1. Mu.g/ml of human PVRIG-human IgG1Fc fusion protein as negative controls, incubated at 4 ℃ for 30 minutes, followed by washing with PBS and centrifugation at 3500rpm for 5 minutes to collect the cells. The cell pellet was resuspended in 100. Mu.l of PBS and Alexa Fluor 647 fluorescein-labeled mouse anti-human IgG-Fc antibody (purchased from Biolegend) was added, incubated at 4 ℃ for 30 minutes in the dark, followed by two washes with PBS and centrifugation at 3500rpm for 5 minutes to collect cells. The cell pellet was resuspended in 200. Mu.l PBS and examined using a flow cytometer (BD FACS Celesta), and the analytical statistics are shown in FIG. 4.

The results show that the murine mAbs mAb001, mAb002, mAb003 and mAb004 can effectively block the binding of human PVRIG to its ligand CD 112.

Example 6: PVRIG antibody binding in vitro to cynomolgus monkey PVRIG assay

The 293T-Macaca fascicularis PVRIG cell line was harvested, and cell counting was performed after washing the resuspended cells with PBS. Then subpackaged in a plurality of 1.5ml Ep tubes, and each 1.5ml Ep tube is put into 2 multiplied by 10 ⁵ As an experimental group, four kinds of murine mAbs were added, each of which was set at 8 concentrations (100. Mu.g/ml, 10. Mu.g/ml, 1. Mu.g/ml, 0.1. Mu.g/ml, 0.01. Mu.g/ml, 0.001. Mu.g/ml, 0.0001. Mu.g/ml, 0.00001. Mu.g/ml, respectively), and mouse IgG1 was used as a negative control, incubated at 4 ℃ for 30 minutes, washed with PBS, and centrifuged at 3500rpm for 5 minutes to collect cells. The cell pellet was resuspended in 100. Mu.l of PBS and Alexa Fluor 647 fluorescein-labeled goat anti-mouse IgG antibody (purchased from Biolegend) was added, incubated at 4 ℃ for 30 minutes in the dark, followed by two washes with PBS and centrifugation at 3500rpm for 5 minutes to collect the cells. The cell pellet was resuspended in 200. Mu.l PBS and examined using a flow cytometer (BD FACS Celesta), and the analytical statistics are shown in FIG. 5.

The results show that the murine mAbs mAb001, mAb002, mAb003 and mAb004 can be specifically bound to the PVRIG protein of cynomolgus macaque on the surface of the cell membrane.

Example 7: NK cell killing assay for PVRIG antibody

The NK-92 cell line is used for the killing experiment under the action of the antibody and is used for detecting the cell activity of the antibody.

NK-92 cell lines (model ATCC, CRL-2407) were collected by centrifugation, and using NK-92 cells as effector cells, the cell membranes of NK-92 cells expressed PVRIG receptor, and using CellTrace Violet (purchased from Thermo Scientific) labeled HCT-15 (model ATCC, CCL-225) colon cancer cell line as target cells, according to 1:2, in a 96-well round bottom plate, 10. Mu.g/ml of mouse IgG1 as a control antibody (mouse IgG 1) and mouse mAbs mAb001, mAb002, mAb003 or mAb004 were added to the culture system, and centrifuged at 250 Xg for 4 minutes, followed by incubation at 37 ℃ for 4 hours. Killed tumor cells were labeled with 7-amino actinomycin D (7-AAD) and detected using a flow cytometer (BD FACS Celesta), and the analytical statistics are shown in FIG. 6.

The results show that compared with the control group, the murine mAbs mAb001, mAb002, mAb003 and mAb004 can promote the killing of the tumor cells by NK-92 cells, and specifically, the four murine mAbs are combined with PVRIG receptors to block the combination of CD112 on the surface of the tumor cells and the PVRIG receptors, so that the killing of the tumor cells by the NK-92 cells is increased.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof capable of specifically recognizing PVRIG, comprising:

a CDR sequence selected from at least one of:

light chain variable region CDR sequences: the amino acid sequence of SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 19. the amino acid sequence of SEQ ID NO:20 and SEQ ID NO:21;

heavy chain variable region CDR sequences: SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 22. SEQ ID NO:23 and SEQ ID NO:24.

2. the antibody or antigen-binding fragment thereof of claim 1, wherein the antibody comprises:

light chain variable region CDR1, CDR2 and CDR3 sequences shown in the amino acid sequences of SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3; or

Light chain variable region CDR1, CDR2, CDR3 sequences as shown in the amino acid sequences of SEQ ID Nos. 7, 8 and 9, respectively; or

Light chain variable region CDR1, CDR2 and CDR3 sequences as shown in amino acid sequences of SEQ ID No. 13, 14 and 15; or

Light chain variable region CDR1, CDR2, CDR3 sequences as set forth in the amino acid sequences of SEQ ID NOS 19, 20 and 21, respectively.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody comprises:

the heavy chain variable region CDR1, CDR2 and CDR3 sequences as shown in the amino acid sequences of SEQ ID No. 4, 5 and 6; or

Heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in the amino acid sequences of SEQ ID NO 10, 11 and 12, respectively; or

The heavy chain variable region CDR1, CDR2 and CDR3 sequences as shown in the amino acid sequences of SEQ ID No. 16, 17 and 18; or

The heavy chain variable region CDR1, CDR2 and CDR3 sequences are shown as the amino acid sequences of SEQ ID NO 22, 23 and 24 respectively.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof specifically recognizes the extracellular region of PVRIG.

5. The antibody or antigen-binding fragment thereof according to claim 1, comprising:

at least one of a heavy chain framework region sequence and a light chain framework region sequence;

wherein at least a portion of at least one of the heavy chain framework region sequence and the light chain framework region sequence is derived from at least one of a murine antibody, a human antibody, a primate antibody, or a mutant thereof.

6. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody has the amino acid sequence set forth in SEQ ID NO:25 to 28 of the amino acid sequence of any one of seq id no;

optionally, the antibody has the amino acid sequence as set forth in SEQ ID NO: a light chain variable region of the amino acid sequence set forth in any one of claims 29 to 32;

optionally, the antibody has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:25 and a heavy chain variable region as set forth in SEQ ID NO:29, the light chain variable region of an amino acid sequence set forth in seq id no;

optionally, the antibody has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO 26 and a heavy chain variable region as set forth in SEQ ID NO: 30;

optionally, the antibody has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:27 and a heavy chain variable region as set forth in SEQ ID NO: 31;

optionally, the antibody has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO 28 and a heavy chain variable region as set forth in SEQ ID NO:32, or a light chain variable region of the amino acid sequence set forth in seq id no.

7. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody comprises at least one of a heavy chain constant region and a light chain constant region, wherein at least a portion of the at least one of a heavy chain constant region and a light chain constant region is derived from at least one of a murine antibody, a human antibody, a primate antibody, or a mutant thereof;

optionally, the light chain constant region and the heavy chain constant region are both from a murine IgG antibody or a mutant thereof.

8. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody has the amino acid sequence set forth in SEQ ID NO:33 to 36 and a light chain having the amino acid sequence as set forth in any one of SEQ ID NOs: 37 to 40, or a pharmaceutically acceptable salt thereof.

9. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is a single chain antibody, a multimeric antibody, a CDR-grafted antibody or a small molecule antibody;

optionally, the antibody is a single chain antibody having the amino acid sequence of SEQ ID NO:41 to 48.

10. The antibody or antigen-binding fragment thereof of claim 1, wherein the antigen-binding fragment comprises a Fab fragment, a (Fab) ₂ At least one of a fragment, scFv-Fc fusion protein, scFv-Fv fusion protein, fv fragment, and minimal recognition unit.

11. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 10.

12. The nucleic acid molecule of claim 11, wherein said nucleic acid molecule is DNA.

13. An expression vector carrying the nucleic acid molecule of claim 11 or 12.

14. The expression vector of claim 13, wherein the expression vector is a eukaryotic expression vector or a virus, preferably wherein the virus is a lentivirus.

15. A recombinant cell, wherein the recombinant cell:

carrying the nucleic acid molecule of claim 11 or 12; or the like, or a combination thereof,

expressing the antibody or antigen-binding fragment thereof of any one of claims 1 to 10.

16. The recombinant cell according to claim 15, wherein the recombinant cell is obtained by introducing the expression vector of claim 13 or 14 into a host cell;

optionally, the recombinant cell is a eukaryotic cell;

preferably, the recombinant cell is a mammalian cell.

17. A pharmaceutical composition comprising:

the antibody or antigen-binding fragment thereof of any one of claims 1 to 10;

the nucleic acid molecule of claim 11 or 12;

the expression vector of claim 13 or 14; or

The recombinant cell of claim 15 or 16.

18. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, the nucleic acid molecule according to claim 11 or 12, the expression vector according to claim 13 or 14, the recombinant cell according to claim 15 or 16, or the pharmaceutical composition according to claim 17 for the preparation of a medicament for the prevention and/or treatment of cancer.

19. Use according to claim 25, wherein the medicament is for the prevention and/or treatment of non-small cell lung cancer, papillary thyroid cancer, glioblastoma multiforme, colorectal cancer, lung cancer, head and neck cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, liver cancer, cholangiocarcinoma or sarcoma, acute myeloid leukemia, large cell neuroendocrine cancer, neuroblastoma, prostate cancer, neuroblastoma, pancreatic cancer, melanoma, head and neck squamous cell carcinoma or gastric cancer.

20. A kit for detecting PVRIG comprising an antibody of any one of claims 1 to 10.

21. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 10, the nucleic acid molecule of claim 11 or 12, the expression vector of claim 13 or 14, or the recombinant cell of claim 15 or 16 for the preparation of a kit for the detection of PVRIG.