PDL1 antibody and application thereof

Abstract

The invention relates to an antibody capable of specifically binding PDL1 or an antigen binding fragment of the antibody and application of the antibody or the antigen binding fragment in preparation of drugs. The invention also provides a nucleic acid molecule for coding the antibody or the antigen binding fragment thereof, an expression vector, a recombinant cell, a method for preparing the antibody or the antigen binding fragment thereof, and a pharmaceutical composition containing the antibody or the antigen binding fragment thereof.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, specifically, the present invention relates to antibodies against PDL1 and applications thereof, more specifically, the present invention relates to antibodies or antigen-binding fragments thereof, nucleic acid molecules, expression vectors, recombinant cells, pharmaceutical compositions, pharmaceutical uses, kits and mouse B cells that can specifically bind to PDL1. Background Art

[0002] Programmed death 1 ligand 1 (PDL1 or PD-L1), also known as CD274 or B7H1, is a member of the B7 family and a type I transmembrane glycoprotein with IgV and IgC domains in the extracellular region and 30 amino acid residues in the intracellular region. It is expressed on immune cells such as B cells, T cells, macrophages, dendritic cells, and mast cells. It is also expressed on non-immune cells such as pancreatic islet cells, liver Kupffer cells, vascular endothelial cells, and some epithelial cells. It is also upregulated in cancer cells or tumor microenvironment immune cells of various malignant solid tumors and hematological tumors. High expression of PDL1 in tumor cells is associated with poor prognosis of cancer patients. Unlike other B7 family members, PDL1 has the function of negatively regulating immune response. PDL1 is the ligand of PD1.

[0003] Programmed death 1 (PD1 or PD-1), also known as CD279, is a member of the CD28 family and a type I transmembrane glycoprotein of the immunoglobulin superfamily. It has a total length of 288 amino acid residues, a single IgV-like domain at the N-terminus, a stem structure of about 20 amino acid residues that separates the IgV structure from the cell membrane, and a transmembrane region and an intracellular region at the C-terminus. PD1 is expressed on the surface of B cells, dendritic cells, macrophages, natural killer cells, and activated T lymphocytes.

[0004] Under normal circumstances, PDL1 interacts with PD1 or activates the PD1 signaling pathway. PD1 can inhibit the function of T lymphocytes and promote the function of Treg cells, thereby inhibiting autoimmune responses and preventing the occurrence of autoimmune diseases. However, in the occurrence of tumors, PD1 binds to PDL1 expressed by tumor cells, transmits immunosuppressive signals, inhibits the proliferation and activity of T cells, and promotes tumor immune escape.

[0005] Anti-PDL1 antibodies can block PDL1 binding to PD1, inhibit PDL1 / PD1 signaling, and activate immune responses that can not only fight cancer cell growth, but also fight viral infection and proliferation in the human body.

[0006] Therefore, although PDL1 antibody tumor immunotherapy has achieved certain results in recent years, due to the heterogeneity of different PDL1 antibody amino acid sequences, especially the CDR region sequences, their characteristics and tumor treatment effects are also different. Scientific researchers still need to continue to develop and improve them to provide more medication options for patients with cancer, infection and other diseases. Summary of the invention

[0007] In the first aspect of the present invention, the present invention provides an isolated antibody or antigen-binding fragment thereof. According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof contains a CDR sequence selected from at least one of the following or an amino acid sequence having at least 95% identity therewith:

[0008] Heavy chain variable region CDR sequence: SEQ ID NO: 1-13; light chain variable region CDR sequence: SEQ ID NO: 14-27.

[0009] EYTLH (SEQ ID NO: 1).

[0010] GIHPNYGDSTYTQKFKG (SEQ ID NO: 2).

[0011] GHGFAHYVMDY (SEQ ID NO: 3).

[0012] SGYWN (SEQ ID NO:4).

[0013] YISYTGSTYYNPSLKS (SEQ ID NO: 5).

[0014] SANWGRGAWFAY (SEQ ID NO: 6).

[0015] GGDDWLLPWFTF (SEQ ID NO: 7).

[0016] SYVMS (SEQ ID NO: 8).

[0017] TISSGGSYTYYSDSVKG (SEQ ID NO:9).

[0018] YGSSHYYFDN (SEQ ID NO: 10).

[0019] GYTMS (SEQ ID NO: 11).

[0020] TISSGGSYTYYPDSVKG (SEQ ID NO: 12).

[0021] DGDTSFYFDY (SEQ ID NO: 13).

[0022] RASQSLSHYLH (SEQ ID NO: 14).

[0023] YASQSIS (SEQ ID NO: 15).

[0024] QNGHSFPPT (SEQ ID NO: 16).

[0025] RASKSISKYLA (SEQ ID NO: 17).

[0026] SGSTLQS (SEQ ID NO: 18).

[0027] QHHNEYPYT (SEQ ID NO: 19).

[0028] RASKSISKYLV (SEQ ID NO: 20).

[0029] QQYNEYPWT (SEQ ID NO: 21).

[0030] SASSSVSYMY (SEQ ID NO: 22).

[0031] DTTNLAS (SEQ ID NO: 23).

[0032] QQWSSHPLT (SEQ ID NO: 24).

[0033] SASSSVSYVY (SEQ ID NO: 25).

[0034] DTSNLAS (SEQ ID NO: 26).

[0035] QQWSSYPLT (SEQ ID NO: 27).

[0036] The above-mentioned antibody or antigen-binding fragment thereof according to the embodiment of the present invention can specifically target and bind to PDL1, and block the binding between PD1 and PDL1.

[0037] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof comprises:

[0038] When defined by the Kabat numbering system,

[0039] The heavy chain variable region CDR1, CDR2, CDR3 sequences are shown in SEQ ID NOs: 1, 2 and 3, respectively, or amino acid sequences that are at least 95% identical to SEQ ID NOs: 1, 2 and 3; or

[0040] The heavy chain variable region CDR1, CDR2, CDR3 sequences shown in SEQ ID NOs: 4, 5 and 6, respectively, or amino acid sequences having at least 95% identity to SEQ ID NOs: 4, 5 and 6; or

[0041] The heavy chain variable region CDR1, CDR2, CDR3 sequences are shown in SEQ ID NOs: 4, 5 and 7, respectively, or amino acid sequences that are at least 95% identical to SEQ ID NOs: 4, 5 and 7; or

[0042] The heavy chain variable region CDR1, CDR2, CDR3 sequences shown in SEQ ID NOs: 8, 9 and 10, respectively, or amino acid sequences having at least 95% identity to SEQ ID NOs: 8, 9 and 10; or

[0043] The heavy chain variable region CDR1, CDR2, CDR3 sequences are shown in SEQ ID NOs: 11, 12 and 13, respectively, or amino acid sequences that are at least 95% identical to SEQ ID NOs: 11, 12 and 13.

[0044] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof comprises:

[0045] When defined by the Kabat numbering system,

[0046] The light chain variable region CDR1, CDR2, CDR3 sequences are shown in SEQ ID NOs: 14, 15 and 16, respectively, or amino acid sequences that are at least 95% identical to SEQ ID NOs: 14, 15 and 16; or

[0047] The light chain variable region CDR1, CDR2, CDR3 sequences are shown in SEQ ID NOs: 17, 18 and 19, respectively, or amino acid sequences that are at least 95% identical to SEQ ID NOs: 17, 18 and 19; or

[0048] The light chain variable region CDR1, CDR2, CDR3 sequences are shown in SEQ ID NOs: 20, 18 and 21, respectively, or amino acid sequences that are at least 95% identical to SEQ ID NOs: 20, 18 and 21; or

[0049] The light chain variable region CDR1, CDR2, CDR3 sequences are shown in SEQ ID NOs: 22, 23 and 24, respectively, or amino acid sequences that are at least 95% identical to SEQ ID NOs: 22, 23 and 24; or

[0050] The light chain variable region CDR1, CDR2, CDR3 sequences are shown in SEQ ID NOs: 25, 26 and 27, respectively, or amino acid sequences that are at least 95% identical to SEQ ID NOs: 25, 26 and 27.

[0051] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof specifically binds to PDL1 or PDL1-Fc fusion protein.

[0052] In some embodiments, the antibody or its antigen-binding fragment specifically binds to human PDL1 or its PDL1-Fc fusion protein; in some embodiments, the antibody or its antigen-binding fragment specifically binds to monkey PDL1 or its PDL1-Fc fusion protein.

[0053] According to an embodiment of the present invention, the antibody or its antigen-binding fragment contains at least one of a heavy chain framework region sequence and a light chain framework region sequence, and at least a portion of at least one of the heavy chain framework region sequence and the light chain framework region sequence is from a rodent, a human, a chicken, a camel, an ostrich, a sheep, a cow, a non-human primate or a shark.

[0054] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof contains a heavy chain variable region, and the heavy chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NOs: 28 to 32 or an amino acid sequence that has at least 95% identity with any one of SEQ ID NOs: 28 to 32.

[0055] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof contains a light chain variable region, and the light chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NOs: 33 to 37 or an amino acid sequence that has at least 95% identity with any one of SEQ ID NOs: 33 to 37.

[0056] EVQLQQSGPELVKPGASVKVSCKTSGYSFTEYTLHWVKQSHGKSLEWIGGIHPNYG DSTYTQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARGHGFAHYVMDYWGQGTSVTVSS (SEQ ID NO: 28).

[0057] EVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWNWIRKFPGNKLEYMGYISYTGST YYNPSLKSRISVTRDTSKNQYYLQLNSVTTEDTATYYCARSANWGRGAWFAYWGQGTL VTVSA (SEQ ID NO: 29).

[0058] EVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWNWIRKFPGNKLEYMGYISYTGST YYNPSLKSRISITRDTSKNQYFLQLNSVTTEDTSTYYCARGGDDWLLPWFTFWGQGTLV TVSA(SEQ ID NO:30)。

[0059] EVQLVESGGGLVKPGGSLKLSCAASGITFSSYVMSWVRQTPEKRLEWVATISSGGSY TYYSDSVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCAKYGSSHYYFDNWGQGTTL TVSS(SEQ ID NO:31)。

[0060] DVKLVESGGGLVKPGGSLKLSCAASGFTFSGYTMSWVRQTPEKRLEWVATISSGGS YTYYPDSVKGRFTISRDSAKNTLYLQMSSLKSEDTAMYYCTRDGDTSFYFDYWGQGITL TVSS(SEQ ID NO:32)。

[0061] DIVMTQSPATLSVIPGDRVSLSCRASQSLSHYLHWYQKKSRESPRLLIKYASQSISGIP SRFSGRGSGSDFTLTINSVEPEDVGVYYCQNGHSFPPTFGAGTKLEL(SEQ ID NO:33)。

[0062] DVQMIQTPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNKLLIYSGSTLQSGIP SRFSGSGSGTDFTLTISSLEPEDFAMYYCQHHNEYPYTFGGGTKLEI(SEQ ID NO:34)。

[0063] DIQITQSPSYLAASPGETITINCRASKSISKYLVWYQEKPGKTNKVLIYSGSTLQSGIPS RFSGSGSGTDFTLTISSLEPEDFAMYYCQQYNEYPWTFGGGTKLKS(SEQ ID NO:35)。

[0064] QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGYSPRLLIYDTTNLASGV PVRFSASGSGTSYSLTISRMEAEDAATYYCQQWSSHPLTFGAGTKLEL (SEQ ID NO: 36).

[0065] QIVLTQSPAIMSASPGEKVTMTCSASSSVSYVYWYQQKPGSSPRLLIYDTSNLASGVP VRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPLTFGAGTKLEI (SEQ ID NO: 37).

[0066] According to an embodiment of the present invention, the antibody contains at least one of a heavy chain constant region and a light chain constant region, and at least a portion of at least one of the heavy chain constant region and the light chain constant region is from a rodent, a human, a chicken, a camel, an ostrich, a sheep, a cow, a non-human primate or a shark.

[0067] According to an embodiment of the present invention, the heavy chain constant region of the antibody is of IgG1 subtype, IgG2 subtype, IgG3 subtype or IgG4 subtype.

[0068] According to an embodiment of the present invention, the light chain constant region of the antibody is of κ subtype or λ subtype.

[0069] In some embodiments, the heavy chain constant region of the antibody is of the IgG2 subtype, and the light chain constant region is of the κ subtype.

[0070] According to an embodiment of the present invention, the light chain constant region and the heavy chain constant region of the antibody are both from a murine antibody or a mutant thereof.

[0071] According to an embodiment of the present invention, the light chain constant region and the heavy chain constant region of the antibody are both derived from murine IgG2.

[0072] According to an embodiment of the present invention, the full-length sequence of the antibody constant region is shown in SEQ ID NO:48 or 49.

[0073] AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEV HTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQ ID NO:48).

[0074] KRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSW TDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 49).

[0075] The full-length sequence of the antibody constant region shown in SEQ ID NO: 48 includes the IgG2 heavy chain CH1 region, hinge region and Fc region, wherein the IgG2 heavy chain CH1 region sequence is AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDL YTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKI, the hinge region sequence is EPRGPTIKPCP, the Fc region sequence is PCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSAL PIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK.

[0076] The full-length sequence of the antibody constant region shown in SEQ ID NO: 49 is the Kappa light chain constant region.

[0077] According to an embodiment of the present invention, the antibody has a heavy chain having an amino acid sequence shown in any one of SEQ ID NOs: 38 to 42 and a light chain having an amino acid sequence shown in any one of SEQ ID NOs: 43 to 47.

[0078] EVQLQQSGPELVKPGASVKVSCKTSGYSFTEYTLHWVKQSHGKSLEWIGGIHPNYGDSTYTQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARGHGFAHYVMDYWGQG TSVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPC PPCKCPAPNLLGGPSVFIFPPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTIS KPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQ ID NO:38).

[0079] EVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWNWIRKFPGNKLEYMGYISYTGSTYYNPSLKSRISVTRDTSKNQYYLQLNSVTTEDTATYYCARSANWGRGAWFAYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQ ID NO:39)。

[0080] EVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWNWIRKFPGNKLEYMGYISYTGSTYYNPSLKSRISITRDTSKNQYFLQLNSVTTEDTSTYYCARGGDDWLLPWFTFWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQ ID NO:40)。

[0081] EVQLVESGGGLVKPGGSLKLSCAASGITFSSYVMSWVRQTPEKRLEWVATISSGGSYTYYSDSVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCAKYGSSHYYFDNWGQGTTLTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQ ID NO:41)。

[0082] DVKLVESGGGLVKPGGSLKLSCAASGFTFSGYTMSWVRQTPEKRLEWVATISSGGSYTYYPDSVKGRFTISRDSAKNTLYLQMSSLKSEDTAMYYCTRDGDTSFYFDYWGQGITLTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQ ID NO:42)。

[0083] DIVMTQSPATLSVIPGDRVSLSCRASQSLSHYLHWYQKKSRESPRLLIKYASQSISGIPSRFSGRGSGSDFTLTINSVEPEDVGVYYCQNGHSFPPTFGAGTLKELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO:43).

[0084] DVQMIQTPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQHHNEYPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO:44).

[0085] DIQITQSPSYLAASPGETITINCRASKSISKYLVWYQEKPGKTNKVLIYSGSTLQSGIPSRFSGSGDTDFTLTISSLEPEDFAMYYCQQYNEYPWTFGGGTKLKSKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO:45).

[0086] QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGYSPRLLIYDTTNLASGVPVRFSASGSGTSYSLTISRMEAEDAATYYCQQWSSHPLTFGAGTLKELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO:46).

[0087] QIVLTQSPAIMSASPGEKVTMTCSASSSVSYVYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPLTFGAGTKLEIKRA DAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ IDNO:47).

[0088] According to an embodiment of the present invention, the antibody is a monoclonal antibody.

[0089] According to an embodiment of the present invention, the antibody or its antigen-binding fragment is at least one of a whole antibody, a single-chain antibody, a multimeric antibody, a CDR-grafted antibody, a fusion protein, a multivalent antibody, a Fab fragment, a Fv fragment, a single domain antibody or a minimum recognition unit.

[0090] Wherein, in the present application, the above-mentioned single-chain antibody includes a heavy chain variable region with an amino acid sequence shown in any one of SEQ ID NOs: 28 to 32 and a light chain variable region with an amino acid sequence shown in any one of SEQ ID NOs: 33 to 37, wherein the C-terminus of the heavy chain variable region is connected to the N-terminus of the light chain variable region through a connecting peptide linker, which can be expressed as VH-Link-VL (VL represents the light chain variable region, VH represents the heavy chain variable region, and Link represents the connecting chain connecting VL and VH), or the C-terminus of the light chain variable region is connected to the N-terminus of the heavy chain variable region through a connecting peptide linker, which can be expressed as VL-Link-VH (VL represents the light chain variable region, VH represents the heavy chain variable region, and Link represents the connecting chain connecting VL and VH).

[0091] According to an embodiment of the present invention, the multivalent antibody is a homologous homovalent antibody or a heterologous multivalent antibody.

[0092] According to an embodiment of the present invention, the fusion protein is a scFv-Fc fusion protein or a scFv-Fv fusion protein.

[0093] In a second aspect of the present invention, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the above-mentioned antibody or antigen-binding fragment thereof. The antibody or antigen-binding fragment encoded by the nucleic acid molecule according to an embodiment of the present invention can specifically target and bind to PDL1, blocking the binding of PDL1 and PD1.

[0094] According to an embodiment of the present invention, the above nucleic acid molecule may further include at least one of the following additional technical features:

[0095] According to an embodiment of the present invention, the nucleic acid molecule is DNA.

[0096] In the third aspect of the present invention, the present invention proposes an expression vector. According to an embodiment of the present invention, the expression vector carries the aforementioned nucleic acid molecule. After the expression vector according to the embodiment of the present invention is introduced into a suitable recipient cell, the aforementioned antibody or antigen-binding fragment thereof that specifically binds to PDL1 can be effectively expressed under the mediation of a regulatory system, thereby achieving a large amount of in vitro acquisition of the antibody or antigen-binding fragment.

[0097] According to an embodiment of the present invention, the above expression vector may further include at least one of the following additional technical features:

[0098] According to an embodiment of the present invention, the expression vector is a eukaryotic expression vector, thereby achieving the expression of the above-mentioned antibody or antigen-binding fragment thereof that specifically binds to PDL1 in eukaryotic cells, such as CHO cells.

[0099] In some embodiments, the expression vector is a eukaryotic expression vector, such as a vector comprising common eukaryotic expression promoter elements, including CMV promoter, SV40 promoter, and the like.

[0100] According to an embodiment of the present invention, the expression vector is a prokaryotic expression vector.

[0101] In a fourth aspect of the present invention, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell carries the aforementioned nucleic acid molecule, or expresses the aforementioned antibody or antigen-binding fragment thereof. The recombinant cell according to an embodiment of the present invention can be used for in vitro expression and mass production of the aforementioned antibody or antigen-binding fragment that specifically binds to PDL1.

[0102] According to an embodiment of the present invention, the above-mentioned recombinant cell may further include at least one of the following additional technical features:

[0103] According to an embodiment of the present invention, the recombinant cell is obtained by introducing the aforementioned expression vector into a host cell.

[0104] According to an embodiment of the present invention, the expression vector is introduced into the host cell by electrotransduction.

[0105] According to an embodiment of the present invention, the recombinant cell is a eukaryotic cell.

[0106] According to an embodiment of the present invention, the recombinant cell is a mammalian cell.

[0107] In a fifth aspect of the present invention, the present invention provides a method for preparing the above-mentioned antibody or antigen-binding fragment thereof. According to an embodiment of the present invention, the method comprises the following steps:

[0108] (1) expressing the antibody or antigen-binding fragment thereof in the recombinant cell; and

[0109] (2) isolating the antibody or antigen-binding fragment thereof from the recombinant cell or cell culture.

[0110] In a sixth aspect of the present invention, the present invention provides a bispecific molecule, an immunoconjugate, a chimeric antigen receptor, an engineered T cell receptor or an oncolytic virus. According to an embodiment of the present invention, the bispecific molecule, immunoconjugate, chimeric antigen receptor, engineered T cell receptor or oncolytic virus contains the aforementioned antibody or antigen-binding fragment thereof.

[0111] In the seventh aspect of the present invention, the present invention proposes a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition contains any of the above-mentioned antibodies or antigen-binding fragments thereof, any of the above-mentioned nucleic acid molecules, any of the above-mentioned expression vectors, or any of the above-mentioned recombinant cells. The antibodies or expressed antibodies contained in the pharmaceutical composition according to the embodiment of the present invention can not only specifically target and bind to the PDL1 protein, effectively blocking the binding of PDL1 and the human recombinant protein PD1, but also can effectively bind to human T cells, effectively stimulate T cells in vitro to secrete IL2 and IFN-γ, and effectively reverse the inhibitory effect of Treg cells on CD4+T and CD8+T cells in vitro.

[0112] In the eighth aspect of the present invention, the present invention proposes the use of any of the above-mentioned antibodies or antigen-binding fragments thereof, any of the above-mentioned nucleic acid molecules, any of the above-mentioned expression vectors or the above-mentioned recombinant cells, and any of the above-mentioned pharmaceutical compositions in the preparation of drugs, which are used for treating or preventing tumors, infections and enhancing immunity.

[0113] According to an embodiment of the present invention, the above-mentioned tumor is a blood tumor or a solid tumor, selected from lymphoma, leukemia, multiple myeloma, melanoma, colon adenocarcinoma, pancreatic cancer, colon cancer, gastrointestinal cancer, prostate cancer, bladder cancer, kidney cancer, ovarian cancer, cervical cancer, breast cancer, lung cancer, renal cell carcinoma, nasopharyngeal carcinoma or any combination thereof.

[0114] According to an embodiment of the present invention, the above-mentioned infection is selected from any infection caused by any pathogenic microorganisms such as viruses, bacteria, fungi, parasites, etc. Non-limiting examples of the virus include hepatitis A, B, C virus, human immunodeficiency virus, human papilloma virus or herpes virus, etc.; non-limiting examples of the bacteria include chlamydia, mycobacterium, staphylococcus, streptococcus, pneumococcus, meningococcus, etc.; non-limiting examples of the fungi include Trichophyton, Epidermophyton, Microsporum, Candida albicans, Cryptococcus neoformans, etc.; non-limiting examples of the parasite include Plasmodium, Schistosoma, Leishmania donovani, filaria, hookworm, etc.

[0115] In a ninth aspect of the present invention, the present invention provides a kit. According to an embodiment of the present invention, the kit comprises any of the above-mentioned antibodies or antigen-binding fragments thereof, any of the above-mentioned nucleic acid molecules, any of the above-mentioned expression vectors, any of the above-mentioned recombinant cells, or any of the above-mentioned pharmaceutical compositions.

[0116] In certain preferred embodiments, the antibody or Fab of the present invention is provided with a detectable label. In a preferred embodiment, the kit further comprises a second antibody that specifically recognizes the antibody or Fab of the present invention. Preferably, the second antibody further comprises a detectable label.

[0117] In the tenth aspect of the present invention, the present invention proposes the use of the aforementioned antibody, the aforementioned nucleic acid molecule, the aforementioned expression vector or the aforementioned recombinant cell in the preparation of a kit for diagnosing or treating PDL1-mediated related diseases.

[0118] In some embodiments, the kit is used to detect the presence or level of PDL1 in a sample. Preferably, the PDL1 is human PDL1.

[0119] In the eleventh aspect of the present invention, the present invention provides a mouse B cell.

[0120] The mouse B cells according to the embodiments of the present invention can be used to secrete mouse PDL1 antibodies or prepare PDL1 monoclonal antibodies.

[0121] In the process of describing the present invention, the relevant terms in this document are explained and illustrated. These explanations and illustrations are only for the convenience of understanding of the scheme and cannot be regarded as limitations on the protection scheme of the present invention.

[0122] Antibody

[0123] As used herein, the term "antibody" refers to an immunoglobulin molecule that is capable of binding to a specific antigen. It generally includes two light chains with a relatively low molecular weight and two heavy chains with a relatively high molecular weight. The heavy chain (H chain) and the light chain (L chain) are connected by disulfide bonds to form a tetrapeptide chain molecule. Among them, the amino acid sequence at the amino end (N end) of the peptide chain varies greatly, which is called the variable region (V region), while the carboxyl end (C end) is relatively stable and varies little, which is called the constant region (C region). The V regions of the L chain and the H chain are called VL and VH, respectively.

[0124] In the variable region, certain regions have a higher degree of variation in amino acid composition and arrangement order, called hypervariable regions (HVRs), which are the locations where antigens and antibodies bind, and are therefore also called complementarity-determining regions (CDRs). There are three CDR regions on both the heavy chain variable region and the light chain variable region. The CDR region sequence can be defined by the IMGT, Kabat, Chothia and AbM methods or the amino acid residues in the variable region identified by any CDR region sequence determination method known in the art. In certain embodiments, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention are preferably determined by the Kabat, Chothia or IMGT numbering system.

[0125] The term "Fc region" or "Fc" refers to the C-terminal region of an immunoglobulin heavy chain, which contains at least a portion of the hinge region, the CH2 domain, and the CH3 domain, which mediate the binding of the immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or binding to the first component of the classical complement system (e.g., C1q), and includes native sequence Fc regions and variant Fc regions.

[0126] The terms "full-length antibody" and "intact antibody" are used interchangeably and refer to an antibody in substantially complete form, as distinct from an antigen-binding fragment. Specifically, intact antibodies include those having heavy and light chains (including an Fc region). The constant region may be a native sequence constant region (e.g., a human native sequence constant region) or an amino acid sequence variant thereof. In some cases, an intact antibody may have one or more effector functions.

[0127] The term "antibody fragment" or "antigen-binding fragment" refers to antibody fragments and antibody analogs that retain the ability to specifically bind to an antigen (e.g., PD1), which generally include at least part of the antigen-binding region or variable region of the parental antibody. Antigen-binding fragments include, but are not limited to: Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, Fd fragments, complementary determining region (CDR) fragments, disulfide bond-stabilized proteins (dsFv), etc.; linear antibodies (Linear Antibody), single-chain antibodies (e.g., ScFv single antibodies) (technology from Genmab), bivalent single-chain antibodies, single-chain phage antibodies, single-domain antibodies (Single Domain Antibody) (e.g., VH domain antibodies), domain antibodies (technology from Ablynx); multispecific antibodies formed by antibody fragments (e.g., three-chain antibodies, four-chain antibodies, etc.); and engineered antibodies such as chimeric antibodies (e.g., humanized mouse antibodies), heteroconjugate antibodies, etc. These antigen-binding fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0128] The term "single-chain antibody (or ScFv antibody)" refers to antibody fragments comprising the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain.

[0129] The term "single domain antibody" is obtained by genetic engineering methods, and there are mainly three categories. The first category is the heavy chain variable region obtained from camelid HCAb, which is a single folding unit, retains complete antigen binding activity, and is the smallest natural antibody fragment. The second category is the heavy chain variable region obtained from cartilaginous fish IgNAR such as sharks, represented by VNAR. The third category is the heavy chain or light chain variable region obtained from human or mouse monoclonal antibodies, which retains antigen binding activity, but the affinity and solubility are greatly reduced.

[0130] The term "monoclonal antibody (mAb)" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies contained in the population are identical except for a few possible naturally occurring mutations, and display a single binding specificity and affinity for a particular epitope. The modifier "monoclonal" indicates the property of an antibody obtained from a substantially uniform group of antibodies, without the need for producing the antibody by a specific method. Monoclonal antibodies are produced by methods known to those skilled in the art, such as by fusing myeloma cells with immune spleen cells to prepare hybrid antibody-producing cells. They are synthesized by culturing hybridomas without contaminating any other antibodies. Monoclonal antibodies can also be obtained by recombinant techniques such as recombinant technology, phage display technology, synthetic technology, or other existing technologies.

[0131] The term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its antigen.

[0132] The present invention uses the full-length sequence of human recombinant PDL1 to obtain a highly specific and high-affinity anti-PDL1 Fab (antigen-binding fragment) antibody fragment through multiple immunizations. The antibody fragment can specifically bind to PDL1, thereby inhibiting PDL1 / PD1 signal transduction.

[0133] In some embodiments, the present invention provides an antibody or antigen-binding fragment that can specifically bind to PDL1, the antibody containing at least one of the following CDR sequences or amino acid sequences having at least 95% identity therewith: heavy chain variable region CDR sequence: SEQ ID NO: 1-13; light chain variable region CDR sequence: SEQ ID NO: 14-27. In some embodiments, the present invention also provides an antibody or antigen-binding fragment, the antibody or antigen-binding fragment having a heavy chain variable region with an amino acid sequence as shown in any one of SEQ ID NO: 28-32 or a light chain variable region with an amino acid sequence as shown in any one of SEQ ID NO: 33-37. In other embodiments, the antibody or antigen-binding fragment provided by the present invention has conservative amino acid substitutions compared with the above sequences. The term "conservative amino acid substitution" refers to the substitution of an amino acid with 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 PDL1 antibody or binding to the PDL1 protein. Structurally similar means that the amino acids have side chains of similar lengths, such as alanine, glycine or serine, or have side chains of similar size. Chemical similarity refers to amino acids having the same charge or being hydrophilic or hydrophobic. For example, hydrophobic residues such as isoleucine, valine, leucine or methionine are substituted for each other. Or polar amino acids are substituted for each other, such as arginine for lysine, glutamic acid for aspartic acid, glutamine for asparagine, serine for threonine, etc. In some specific embodiments, these conservative amino acid substitutions can occur in amino acids other than the CDR regions in the heavy chain variable region and the light chain variable region.

[0134] In some preferred embodiments, the present invention provides an anti-PDL1 antibody having a heavy chain with an amino acid sequence shown in any one of SEQ ID NOs: 38 to 42 and a light chain with an amino acid sequence shown in any one of SEQ ID NOs: 43 to 47.

[0135] The term "identity" is used to refer to the degree of sequence similarity between two polypeptides or between two nucleic acids. When a certain position in the two compared sequences is occupied by the same base or amino acid monomer subunit (for example, a certain position in each of the two DNA molecules is occupied by adenine, or a certain position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if 6 out of 10 positions of the two sequences match, then the two sequences have 60% identity.

[0136] The term "PDL1-Fc fusion protein" refers to a protein product co-expressed by two genes, the PDL1 extracellular region obtained by DNA recombination and the human antibody Fc gene. The PDL1 extracellular region refers to the part of the PDL1 protein expressed outside the cell membrane.

[0137] Nucleic acid molecules, expression vectors, recombinant cells

[0138] The term "nucleic acid molecule" includes DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, and may be a cDNA.

[0139] The term "vector" refers to any recombinant polynucleotide construct that can be used for the purpose of transformation, i.e., introducing heterologous DNA into a host cell, and can be a plasmid (e.g., Plasmid-X plasmid), a virus (e.g., replication-defective retrovirus, adenovirus and adeno-associated virus), a phage, etc.

[0140] The term "expression vector" refers to a nucleic acid molecule that is capable of replicating and expressing a gene of interest when transformed, transfected or transduced into a host cell. The expression vector comprises one or more phenotypic selection markers and an origin of replication to ensure maintenance of the vector and to provide amplification in the host if necessary. The expression vector further comprises a promoter to drive expression of the polypeptide in the cell.

[0141] The term "recombinant cell" refers to a cell into which an expression vector has been introduced.

[0142] In the process of preparing or obtaining these antibodies, nucleic acid molecules expressing these antibodies can be connected to different vectors and then expressed in different cells to obtain the corresponding antibodies.

[0143] To this end, the present invention also provides an isolated nucleic acid molecule, which encodes the antibody or antigen-binding fragment described above.

[0144] The present invention also provides an expression vector, which comprises the above-mentioned isolated nucleic acid molecule. When the above-mentioned isolated polynucleotide is connected to the vector, the polynucleotide can be directly or indirectly connected to the control components on the vector, as long as these control components can control the translation and expression of the polynucleotide. Of course, these control components can come directly from the vector itself, or they can be exogenous, that is, not from the vector itself. Of course, the polynucleotide and the control components can be operably connected. Herein, "operably connected" means connecting the exogenous gene to the vector so that the control components in the vector, such as transcription control sequences and translation control sequences, etc., can play their expected functions of regulating the transcription and translation of the exogenous gene. Of course, the polynucleotides used to encode the heavy chain and light chain of the antibody can be inserted into different vectors independently, and it is common to insert them into the same vector.

[0145] The present invention also provides a recombinant cell, which contains the expression vector. The expression vector can be introduced into mammalian cells to construct recombinant cells, and then these recombinant cells are used to express the antibody or antigen-binding fragment provided by the present invention. The corresponding antibody can be obtained by culturing the recombinant cells. These available mammalian cells can be, for example, CHO cells.

[0146] Pharmaceutical composition, kit and pharmaceutical use and use in preparing a kit.

[0147] The present invention also provides a pharmaceutical composition, which comprises the above-mentioned antibody or antigen-binding fragment and a pharmaceutically acceptable carrier.

[0148] The anti-PDL1 antibodies provided herein can be incorporated into pharmaceutical compositions suitable for administration to a subject. Typically, these pharmaceutical compositions include the anti-PDL1 antibodies provided herein and a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carriers" may include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and delayed absorption agents that are physiologically compatible, and the like. Specific examples may be one or more of water, saline, phosphate-buffered saline, glucose, glycerol, ethanol, and the like, and combinations thereof. In many cases, isotonic agents, such as sugars, polyols (such as mannitol, sorbitol), or sodium chloride, are included in the pharmaceutical composition. Of course, pharmaceutically acceptable carriers may also include trace amounts of auxiliary substances, such as wetting agents or emulsifiers, preservatives, or buffers, to extend the shelf life or efficacy of the antibody.

[0149] For example, the antibodies of the present 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. For example, liquid, semisolid and solid dosage forms, including but not limited to liquid solutions (e.g., injection solutions and infusion solutions), dispersants or suspensions, tablets, pills, powders, liposomes and suppositories. Typical pharmaceutical compositions are in the form of injection solutions or infusion solutions. The antibodies can be administered by intravenous infusion or injection or intramuscular or subcutaneous injection.

[0150] Of course, the anti-PDL1 antibody herein can also be made into a kit or part of other diagnostic reagents as needed. According to an embodiment of the present invention, the present invention also provides a kit, which includes the above-mentioned PDL1 antibody. The kit provided by the present invention can be used, for example, for immunoblotting, immunoprecipitation, etc., which involve using the specific binding properties of antigens and antibodies to detect kits, etc.

[0151] When using the anti-PDL1 antibody provided by the present invention to treat a PDL1-mediated disease, the anti-PDL1 antibody or its antigen-binding fragment provided by the present invention can be provided to a subject. To this end, the present invention provides a method for treating the above-mentioned disease, comprising administering the antibody or its antigen-binding fragment or the above-mentioned pharmaceutical composition provided by the present invention to a subject in need. BRIEF DESCRIPTION OF THE DRAWINGS

[0152] Figure 1 This is a graph showing the experimental results of detecting the binding of the test antibody to the human PDL1 high-expressing stably transfected CHO cell line by flow cytometry according to an embodiment of the present invention;

[0153] Figure 2 This is a graph showing the experimental results of detecting the binding of the test antibody to the monkey-derived PDL1 high-expressing stably transfected CHO cell line by flow cytometry according to an embodiment of the present invention;

[0154] Figure 3 is a graph showing the experimental results of detecting the binding of a test antibody to activated T cells by flow cytometry according to an embodiment of the present invention;

[0155] Figure 4 is a graph showing the experimental results of detecting the binding of the test antibody to the human ovarian cancer cell line ES-2 by flow cytometry according to an embodiment of the present invention;

[0156] Figure 5 This is a graph showing the experimental results of detecting the competition between the test antibody and the human recombinant PD1 protein for binding to PDL1 by flow cytometry according to an embodiment of the present invention;

[0157] Figure 6This is a graph showing the experimental results of detecting the effect of the test antibody on the secretion of IL2 by T cells through a mixed lymphocyte reaction experiment according to an embodiment of the present invention;

[0158] Figure 7 This is a graph showing the experimental results of detecting the effect of the test antibody on the secretion of IFN-γ by T cells through a mixed lymphocyte reaction experiment according to an embodiment of the present invention;

[0159] Figure 8 This is the effect of the test antibody according to the embodiment of the present invention on the production of IL2 by human PBMC stimulated by SEA in vitro. DETAILED DESCRIPTION

[0160] Below will be explained in conjunction with embodiment scheme of the present invention.The example of described embodiment is shown in the accompanying drawings, wherein the same or similar reference numerals represent the same or similar components or components with the same or similar functions from beginning to end.Unindicated specific technology or conditions in the embodiment, according to the technology or conditions described in the document in this area or according to the product specification sheet.Reagents used or instruments are not indicated by manufacturers, and are conventional products that can be obtained commercially.It will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention.

[0161] Example 1 Process of obtaining antibody sequences by hybridoma method

[0162] The human recombinant protein PDL1 was obtained through the global public database UniProt (Q9NZQ7, 19-290AA), and the sequence was SEQ ID NO:50.

[0163] FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQ AEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET(SEQ ID NO:50).

[0164] The human recombinant protein PDL1 and complete Freund's adjuvant or incomplete Freund's adjuvant are mixed into an emulsion and dispersedly injected subcutaneously into Balb / c mice. After 2-4 immunizations, the binding titer of the mouse serum to the human recombinant protein PDL1 is determined by ELISA. When the titer reaches the standard, the mouse spleen can be obtained, and the mouse spleen is fully ground to obtain a single cell suspension, which is fused with myeloma cells by PEG to form hybridoma cells. The supernatant of hybridoma cells cultured for 7-14 days is detected by ELISA, and positive cells that meet the standards are selected for continued culture. The supernatant of the above ELISA-positive cells is obtained for the second time, and the binding of the antibody in the supernatant to the stable CHO cell line with high expression of PDL1 is detected by the FACs method. The cells that meet the standards are subcloned, and the cell mRNA is obtained after harvesting the cells, and reverse transcribed into cDNA. The antibody sequence in the hybridoma cell line is obtained and sequenced using commonly used antibody fishing sequence primers. At the same time, the subcloned cells can be cultured at 5×10 5 ~1×10 6 The number of cells was inoculated into the peritoneal cavity of mice, and the ascites in the peritoneal cavity was collected after 7-10 days. The mouse monoclonal antibody with a purity of more than 90% was obtained through purification preparation with Protein A medium and used for detection and evaluation of other methods. According to the above method, a large sample of hybridoma cells was screened to obtain hybridoma cell lines numbered HEC401, HEC402, HEC403, HEC404, and HEC405, and the names of the monoclonal antibodies secreted by them (hereinafter referred to as the test antibodies) were consistent with the numbers of the corresponding hybridoma cell lines. The amino acid sequences of the CDR region, variable region, and heavy / light chain of the above-mentioned test antibodies are shown in Table 1.

[0165] Table 1 CDR region, variable region and heavy and light chains of the tested antibodies

[0166]

[0167] Example 2 Binding of Antibodies to Human PDL1 Highly Expressing Stably Transfected CHO Cell Lines

[0168] Flow cytometry was used to detect the binding ability of antibodies to human PDL1 high-expressing stable CHO cell lines. The test antibody (HEC401, HEC402, HEC403, HEC404, HEC405) samples were diluted to 8 concentration gradients with PBS buffer, and the antibodies were incubated with cells. Then, FITC-labeled goat anti-mouse IgG Fc secondary antibodies were used for binding. Isotype IgG antibodies were used as negative controls, and PDL1 antibodies promoted clinically by Merck, BMS and Roche were used as positive control samples. The binding ability of each test antibody to human PDL1 was evaluated at the cellular level. The results are shown in Figure 1 . Figure 1The results showed that the tested antibodies had strong binding ability with human PDL1.

[0169] Example 3 Binding of Antibodies to Monkey-derived PDL1 Highly Expressing Stably Transfected CHO Cell Lines

[0170] Flow cytometry was used to detect the binding ability of antibodies to monkey PDL1 high-expressing stable CHO cell lines. The test antibody (HEC401, HEC402, HEC403, HEC404, HEC405) samples were diluted to 8 concentration gradients with PBS buffer, and the antibodies were incubated with cells. Then, FITC-labeled goat anti-mouse IgG Fc secondary antibodies were used for binding. Isotype IgG antibodies were used as negative controls, and PDL1 antibodies promoted clinically by Merck, BMS, AstraZeneca and Roche were used as control samples. The binding ability of each test antibody to monkey PDL1 was evaluated at the cellular level. The results are shown in Figure 2 . Figure 2 The results showed that, except for HEC403, the tested antibodies had strong binding ability with monkey PDL1.

[0171] Example 4 Binding of Antibodies to T Cells Isolated and Activated in vitro from Human Blood

[0172] T cells were separated from human PBMC cell populations using a kit, and CD3 and CD28 antibodies were coupled to magnetic beads to stimulate T cells in vitro for 48-72 hours. Flow cytometry was used to detect the binding ability of antibodies to stimulated T cells. The test antibody (HEC401, HEC402, HEC403, HEC404, HEC405) samples were diluted to 8 concentration gradients with PBS buffer, and the antibodies were incubated with cells. Then, FITC-labeled goat anti-mouse IgG Fc secondary antibodies were used for binding, and Isotype IgG antibodies were used as negative controls. PDL1 antibodies promoted clinically by Merck, BMS, AstraZeneca and Roche were used as control samples to evaluate the binding ability of each test antibody to T cells at the cellular level. The results are shown in Figure 3 . Figure 3 The results showed that HEC401, HEC402, HEC403, HEC404, and HEC405 can all effectively bind to human T cells.

[0173] Example 5 Binding of Antibodies to Human Ovarian Cancer Cell Line ES-2 Cells

[0174] After routine cultured ES-2 cells were digested with trypsin, they were collected and flow cytometry was used to detect the binding ability of the antibody to the human ovarian cancer cell line ES-2. After the antibody was incubated with the cells, it was then bound with a FITC-labeled goat anti-mouse IgG Fc secondary antibody, an Isotype IgG antibody was used as a negative control, and a PDL1 antibody promoted clinically by Merck, BMS, AstraZeneca and Roche was used as a control sample. The results are shown in Figure 4 . Figure 4 The results showed that PDL1 can effectively bind to ES-2 within a certain concentration range, providing a basis for the possible inhibitory effect of the tested antibody on tumors.

[0175] Example 6 Antibodies compete with human recombinant PD1 protein for binding to PDL1

[0176] Flow cytometry was used to detect the competitive binding ability of antibodies and human recombinant PD1 proteins to human PDL1 high-expressing stably transfected CHO cell lines. The test antibody (HEC401, HEC402, HEC403, HEC404, HEC405) samples and human recombinant PD1 proteins were diluted to 8 concentration gradients with PBS buffer, and the antibodies and mammalian cells expressing human PD1 proteins were incubated with cells. Then, FITC-labeled goat anti-human IgG Fc secondary antibodies were used for binding, and Isotype IgG antibodies were used as negative controls. PDL1 antibodies promoted in clinical practice by Merck, BMS and Roche were used as control samples. The results are shown in Figure 5 . Figure 5 The normalized competition ratio reflects the competition situation. The lower the reading, the greater the effect of the antibody in inhibiting the binding of human PD1 protein to PDL1. Figure 5 As shown, as the concentration of each tested antibody increases, the competition rate gradually decreases until it approaches zero (i.e., the binding of human PD1 protein to PDL1 is completely inhibited). It can be seen that within a certain concentration range, each tested antibody can inhibit the binding of human PD1 protein to PDL1 in a dose-dependent manner at the cellular level.

[0177] Example 7 BLI method to detect the affinity of antibodies and human PDL1 protein

[0178] Bio-Layer Interferometry (BLI) was used to measure affinity. The human PDL1 protein was fixed on the sensor for 300 seconds, and then the sensor was immersed in PBST solution to balance the baseline for 180 seconds. The probe was then immersed in antibody solutions with different concentration gradient dilutions for a binding duration of 600 seconds. The probe was then placed in PBST solution for a dissociation duration of 1200 seconds, and finally placed in regeneration solution to complete the regeneration and preservation of the probe. BLI kinetics / affinity software analysis was used to obtain the dissociation constant (kdis) and binding constant (kon) of the test antibody and the human recombinant protein PDL1. The affinity constant (KD) was calculated based on the ratio of the dissociation constant to the binding constant. BMS-93669 was used as a reference. The results are shown in Table 2. The test antibodies all have high affinity.

[0179] Table 2 Affinity of antibodies to human recombinant PDL1 protein

[0180] serial number KD(M) <![CDATA[ kon (1 / Ms )]]> <![CDATA[ kdis (1 / s)]]> HEC401 584E-11 679E+05 396E-05 HEC402 132E-10 597E+05 790E-05 HEC403 7.37E-11 114E+06 840E-05 HEC404 1.29E-10 2.57E+05 3.32E-05 HEC405 2.46E-10 2.45E+05 6.01E-05 BMS-936559 4.72E-11 1.41E+06 6.67E-05

[0181] Example 8 Mixed lymphocyte reaction experiment

[0182] The antibody was co-incubated with mature DC cells and CD4+T cells cultured in vitro, and the relative expression of IL2 in the detection system was used to reflect the activation effect of different antibodies on T cells. After the human monocytes were separated by the kit, they were treated with the kit reagents to become mDC (mature dendritic cells), which were then incubated with antibodies and then added with allogeneic human T cells. The expression of IL2 in the supernatant was detected three days later. Isotype IgG antibody was used as a negative control, and the PDL1 antibody promoted clinically by Merck, BMS and Roche was used as a control sample. The results are as follows Figure 6 shown. Figure 6 The results showed that the IL2 produced by T cells stimulated by the tested antibodies was higher than that of Avelumab and comparable to that of BMS-936559 and Durvalumab. Therefore, the tested antibodies can effectively activate T cells.

[0183] Example 9 Mixed lymphocyte reaction experiment

[0184] The antibody is co-incubated with mature DC cells and CD4+T cells cultured in vitro, and the relative expression of IFN-γ in the detection system is used to reflect the activation effect of different antibodies on T cells. After the human monocytes are separated by the kit, they are treated with the kit reagents to become mDC (mature dendritic cells), which are then incubated with antibodies, and then allogeneically separated human T cells are added. The expression of IFN-γ in the supernatant is detected after five days. Isotype IgG antibody is used as a negative control, and the PDL1 antibody promoted clinically by Merck, BMS and Roche is used as a control sample. The results are as follows Figure 7 shown. Figure 7 The results showed that within a certain concentration range, the tested antibodies could effectively stimulate T cells to produce IFN-γ.

[0185] Example 10: In vitro stimulation of human PBMC by SEA to detect the enhancing effect of the test antibody on the production of IL2 in the system

[0186] The test antibody of the present invention is used to evaluate the degree of T cell activation that a specific subject has or may have in the presence of the test antibody. After PBMC from human blood is plated, a certain concentration of SEA (Staphylococcus superantigen) and antibody are added, and IL2 in the supernatant is detected after three days of incubation. Isotype IgG antibody is used as a negative control, and PDL1 antibody promoted clinically by Merck, BMS and Roche is used as a control sample. The results are as follows Figure 8 shown. Figure 8 The results showed that in the presence of the test antibody, the production of IL2 was increased, which was equivalent to or slightly lower than the enhancement effect of the positive control group. Therefore, the test antibody effectively enhanced the activation of T cells.

[0187] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0188] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. An isolated antibody or antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof contains a CDR sequence selected from at least one of the following or an amino acid sequence having at least 95% identity thereto: Heavy chain variable region CDR sequence: SEQ ID NO: 1-13; Light chain variable region CDR sequence: SEQ IN NO: 14-27.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof comprises: The heavy chain variable region CDR1, CDR2, CDR3 sequences are shown in SEQ ID NOs: 1, 2 and 3, respectively, or amino acid sequences that are at least 95% identical to SEQ ID NOs: 1, 2 and 3; or The heavy chain variable region CDR1, CDR2, CDR3 sequences shown in SEQ ID NOs: 4, 5 and 6, respectively, or amino acid sequences having at least 95% identity to SEQ ID NOs: 4, 5 and 6; or The heavy chain variable region CDR1, CDR2, CDR3 sequences are shown in SEQ ID NOs: 4, 5 and 7, respectively, or amino acid sequences that are at least 95% identical to SEQ ID NOs: 4, 5 and 7; or The heavy chain variable region CDR1, CDR2, CDR3 sequences shown in SEQ ID NOs: 8, 9 and 10, respectively, or amino acid sequences having at least 95% identity to SEQ ID NOs: 8, 9 and 10; or The heavy chain variable region CDR1, CDR2, CDR3 sequences are shown in SEQ ID NOs: 11, 12 and 13, respectively, or amino acid sequences that are at least 95% identical to SEQ ID NOs: 11, 12 and 13.

3. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof comprises: The light chain variable region CDR1, CDR2, CDR3 sequences are shown in SEQ ID NOs: 14, 15 and 16, respectively, or amino acid sequences that are at least 95% identical to SEQ ID NOs: 14, 15 and 16; or The light chain variable region CDR1, CDR2, CDR3 sequences are shown in SEQ ID NOs: 17, 18 and 19, respectively, or amino acid sequences that are at least 95% identical to SEQ ID NOs: 17, 18 and 19; or The light chain variable region CDR1, CDR2, CDR3 sequences are shown in SEQ ID NOs: 20, 18 and 21, respectively, or amino acid sequences that are at least 95% identical to SEQ ID NOs: 20, 18 and 21; or The light chain variable region CDR1, CDR2, CDR3 sequences are shown in SEQ ID NOs: 22, 23 and 24, respectively, or amino acid sequences that are at least 95% identical to SEQ ID NOs: 22, 23 and 24; or The light chain variable region CDR1, CDR2, CDR3 sequences are shown in SEQ ID NOs: 25, 26 and 27, respectively, or amino acid sequences that are at least 95% identical to SEQ ID NOs: 25, 26 and 27.

4. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof specifically binds to PDL1 or PDL1-Fc fusion protein.

5. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: It contains at least one of a heavy chain framework region sequence and a light chain framework region sequence, at least a portion of at least one of the heavy chain framework region sequence and the light chain framework region sequence is from a rodent, a human, a chicken, a camel, an ostrich, a sheep, a cow, a non-human primate or a shark.

6. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: It contains a heavy chain variable region comprising an amino acid sequence as shown in any one of SEQ ID NOs: 28-32 or an amino acid sequence having at least 95% identity with any one of SEQ ID NOs: 28-32.

7. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: It contains a light chain variable region comprising an amino acid sequence as shown in any one of SEQ ID NOs: 33-37 or an amino acid sequence having at least 95% identity with any one of SEQ ID NOs: 33-37.

8. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody contains at least one of a heavy chain constant region and a light chain constant region, at least a portion of at least one of the heavy chain constant region and the light chain constant region is from a rodent, a human, a chicken, a camel, an ostrich, a sheep, a cow, a non-human primate or a shark.

9. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The heavy chain constant region of the antibody is of IgG1 subtype, IgG2 subtype, IgG3 subtype or IgG4 subtype.

10. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The light chain constant region of the antibody is of κ subtype or λ subtype.

11. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The heavy chain constant region and the light chain constant region of the antibody are both derived from a murine antibody or a mutant thereof. Optionally, the heavy chain constant region and the light chain constant region of the antibody are both derived from murine IgG2.

12. The antibody or antigen-binding fragment thereof according to claim 11, characterized in that: The antibody has a heavy chain having an amino acid sequence shown in any one of SEQ ID NOs: 38 to 42 and a light chain having an amino acid sequence shown in any one of SEQ ID NOs: 43 to 47.

13. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody is a monoclonal antibody.

14. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof is at least one of a whole antibody, a single-chain antibody, a multimeric antibody, a CDR-grafted antibody, a multivalent antibody, a fusion protein, a Fab fragment, a Fv fragment, a single domain antibody or a minimum recognition unit.

15. The antibody or antigen-binding fragment thereof according to claim 14, characterized in that: The single-chain antibody comprises a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 28 to 32 and a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 33 to 37, wherein the C-terminus of the heavy chain variable region is connected to the N-terminus of the light chain variable region via a connecting peptide linker, or the C-terminus of the light chain variable region is connected to the N-terminus of the heavy chain variable region via a connecting peptide linker.

16. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15.

17. The nucleic acid molecule according to claim 16, characterized in that The nucleic acid molecule is DNA.

18. An expression vector, characterized in that: Carrying the nucleic acid molecule according to any one of claims 16 to 17.

19. A recombinant cell, characterized in that The recombinant cell carries the nucleic acid molecule according to any one of claims 16 to 17, or expresses the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15.

20. The recombinant cell according to claim 19, characterized in that The recombinant cell is obtained by introducing the expression vector according to claim 18 into a host cell.

21. The recombinant cell according to claim 19, characterized in that The expression vector is introduced into the host cell by electrotransduction.

22. The recombinant cell according to claim 19, characterized in that The recombinant cell is a eukaryotic cell.

23. The recombinant cell according to claim 19, characterized in that The recombinant cell is a mammalian cell.

24. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15 using the recombinant cell according to any one of claims 19 to 23, characterized in that: The following steps are involved: (1) expressing the antibody or antigen-binding fragment thereof in the recombinant cell; and (2) isolating the antibody or antigen-binding fragment thereof from the recombinant cell or cell culture.

25. A bispecific molecule, immunoconjugate, chimeric antigen receptor, engineered T cell receptor or oncolytic virus, characterized in that: A composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15.

26. A pharmaceutical composition, characterized in that A method comprising: comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, the nucleic acid molecule according to any one of claims 16 to 17, the expression vector according to claim 18 or the recombinant cell according to any one of claims 19 to 23.

27. Use of the antibody according to any one of claims 1 to 15, the nucleic acid molecule according to any one of claims 16 to 17, the expression vector according to claim 18 or the recombinant cell according to any one of claims 19 to 23, or the pharmaceutical composition according to claim 26 in the preparation of a drug for treating or preventing tumors, infections and enhancing immunity.

28. The use according to claim 27, characterized in that The tumor is a blood tumor or a solid tumor selected from lymphoma, leukemia, multiple myeloma, melanoma, colon adenocarcinoma, pancreatic cancer, colon cancer, gastrointestinal cancer, prostate cancer, bladder cancer, kidney cancer, ovarian cancer, cervical cancer, breast cancer, lung cancer, renal cell carcinoma, nasopharyngeal carcinoma or any combination thereof.

29. The use according to claim 27, characterized in that The infection is selected from a viral infection, a bacterial infection, a fungal infection or a parasitic infection.

30. A kit, characterized in that It comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, the nucleic acid molecule according to any one of claims 16 to 17, the expression vector according to claim 18, the recombinant cell according to any one of claims 19 to 23, or the pharmaceutical composition according to claim 26.

31. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, the nucleic acid molecule according to any one of claims 16 to 17, the expression vector according to claim 18, the recombinant cell according to any one of claims 19 to 23, or the pharmaceutical composition according to claim 26 in the preparation of a kit for diagnosing or treating PDL1-mediated related diseases.

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