CN109021107B

CN109021107B - Monoclonal antibody specifically binding to human PD-L1, and medicine and kit containing monoclonal antibody

Info

Publication number: CN109021107B
Application number: CN201811033908.7A
Authority: CN
Inventors: 苏云鹏; 庄伟亮; 裴丽丽; 张建军; 梁绍勤
Original assignee: Jiangsu Novomab Biopharmaceutical Inc
Current assignee: Jiangsu Novomab Biopharmaceutical Inc
Priority date: 2018-09-05
Filing date: 2018-09-05
Publication date: 2020-08-28
Anticipated expiration: 2038-09-05
Also published as: CN109021107A

Abstract

The invention relates to a monoclonal antibody specifically binding to human PD-L1, which comprises any one of the following groups of CDR regions: 1) 1, 2 and 3 heavy chain variable region CDR1, 2 and 3 sequences shown in SEQ ID NOs, 4, 6 and 7 light chain variable region CDR1, 2 and 3 sequences shown in SEQ ID NOs; 2) 1, 2 and 3 heavy chain variable region CDR1, 2 and 3 sequences shown in SEQ ID NOs, 5, 6 and 7 light chain variable region CDR1, 2 and 3 sequences shown in SEQ ID NOs; 3) the heavy chain variable region CDR1, 2 and 3 sequences shown in SEQ ID NO 1, 2 and 3, respectively, and the light chain variable region CDR1, 2 and 3 sequences shown in SEQ ID NO 4, 6 and 8. The provided PD-L1 antibody has high affinity and specificity with human PD-L1, and simultaneously remarkably inhibits the binding of PD-L1 and receptor PD 1.

Description

Monoclonal antibody specifically binding to human PD-L1, and medicine and kit containing monoclonal antibody

Technical Field

The invention relates to a monoclonal antibody, in particular to a human programmed death factor ligand 1 monoclonal antibody and a pharmaceutical composition containing the same, belonging to the technical field of genetic engineering antibodies.

Background

Human programmed death factor ligand 1(PD-L1), also known as B7-H1, is a member of the B7 family and is widely distributed in peripheral tissues and hematopoietic cells. The PD-L1 full-length cDNA 870 bp encodes a I-type transmembrane protein containing 290 amino acids. PD-L1 contains an IgV-like, IgC-like domain, a hydrophilic transmembrane domain and an endodomain (30 aa). Programmed death factor 1(PD 1) is the main receptor of PD-L1, is mainly distributed in T cells, B cells, NK cells and other immune-related cells, and plays an important role in the immune response process of autoimmune diseases, tumors, infections, organ transplantation, allergy, immune privileges and the like.

PD-L1 inhibits activation of T cells or induces apoptosis of mature T cells by interacting with its receptor programmed death factor 1(PD 1), so that the immune response is suppressed. During the development of tumor, cancer cells can induce apoptosis of T cells by up-regulating PD-L1 expression, and avoid the elimination of T cells by immune system, thereby leading to the progression of disease. Therefore, the PD1/PD-L1 pathway has become an important target for the research of antitumor drugs. At present, antibody drugs inhibiting the PD1/PD-L1 pathway have been clinically successful greatly, wherein Nivolumab of BaishiGuibao, MK-3475 of Daosha and MPDL3280A of Roche are listed in sequence on the market. Antibody drugs directed against the PD1/PD-L1 pathway would be the most promising area in the tumor therapy market.

In addition, studies have shown that the PD1/PD-L1 pathway is associated with several infectious diseases. For example, in HIV patients, PD1 was found to be highly expressed on CD8+ T cells that specifically recognize HIV, and the virus inhibited the activity of CD8+ T cells by activating PD-L1/PD 1 signals, thereby greatly impairing autoimmune function. Studies have also shown that the PD1/PD-L1 pathway is associated with immune diseases, such as intestinal mucositis, and that inhibition of PD-L1 is effective in preventing colitis-related wasting disease (Kanai et al (2003), J Immunol171: 4156-63).

In conclusion, the antibody medicament targeting the PD1/PD-L1 pathway has great application value and wide market prospect in the fields of treating cancers, infectious diseases or autoimmune diseases and the like.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a monoclonal antibody or an antigen binding part thereof which specifically binds to human PD-L1, wherein the provided PD-L1 antibody has high affinity and specificity with human PD-L1, simultaneously remarkably inhibits the binding of PD-L1 and receptor PD1, and does not bind with other members of the family, and can activate T cells by inhibiting PD1 signals, stimulate the T cells to secrete IL2 and remarkably inhibit tumor growth, a pharmaceutical composition or a kit containing the antibody or the antigen binding part thereof, and application in preparing medicaments for treating cancers, infectious diseases or autoimmune diseases.

The technical scheme for solving the technical problems is as follows: a monoclonal antibody or antigen-binding portion thereof that specifically binds human PD-L1, comprising any one of the following sets of CDR regions:

a first set of CDR regions: the amino acid sequence of CDR1 of the heavy chain variable region shown in SEQ ID NO.1, the amino acid sequence of CDR2 of the heavy chain variable region shown in SEQ ID NO.2, the amino acid sequence of CDR3 of the heavy chain variable region shown in SEQ ID NO. 3, the amino acid sequence of CDR1 of the light chain variable region shown in SEQ ID NO. 4, the amino acid sequence of CDR2 of the light chain variable region shown in SEQ ID NO. 6, and the amino acid sequence of CDR3 of the light chain variable region shown in SEQ ID NO. 7;

a second set of CDR regions: the amino acid sequence of CDR1 of the heavy chain variable region shown in SEQ ID NO.1, the amino acid sequence of CDR2 of the heavy chain variable region shown in SEQ ID NO.2, the amino acid sequence of CDR3 of the heavy chain variable region shown in SEQ ID NO. 3, the amino acid sequence of CDR1 of the light chain variable region shown in SEQ ID NO. 5, the amino acid sequence of CDR2 of the light chain variable region shown in SEQ ID NO. 6, and the amino acid sequence of CDR3 of the light chain variable region shown in SEQ ID NO. 7;

third set of CDR regions: the amino acid sequence of CDR1 in the heavy chain variable region shown in SEQ ID NO.1, the amino acid sequence of CDR2 in the heavy chain variable region shown in SEQ ID NO.2, the amino acid sequence of CDR3 in the heavy chain variable region shown in SEQ ID NO. 3, the amino acid sequence of CDR1 in the light chain variable region shown in SEQ ID NO. 4, the amino acid sequence of CDR2 in the light chain variable region shown in SEQ ID NO. 6, and the amino acid sequence of CDR3 in the light chain variable region shown in SEQ ID NO. 8.

The invention has the beneficial effects that: the CDR region provided by the invention enables the PD-L1 antibody to have high affinity and specificity with human PD-L1, simultaneously remarkably inhibits the binding of PD-L1 and receptor PD1, does not bind with other members of the same family, and can remarkably inhibit the growth of tumors.

On the basis of the technical scheme, the invention can be further improved as follows.

Further, the monoclonal antibody or an antigen-binding portion thereof comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.9 and a heavy chain variable region having an amino acid sequence of SEQ ID NO.10, SEQ ID NO:11 or SEQ ID NO:12, or a light chain variable region of any one of seq id nos.

The beneficial effect of adopting the above further scheme is that the variable region provided by the invention enables the PD-L1 antibody to have high affinity and specificity with human PD-L1, and simultaneously significantly inhibits the binding of PD-L1 and receptor PD1, and does not bind with other members of the same family, and the antibody can activate T cells by inhibiting PD1 signals, stimulate the T cells to secrete IL2, and significantly inhibit tumor growth.

Further, the monoclonal antibody or antigen-binding portion thereof comprises a heavy chain variable region having the amino acid sequence of SEQ ID No.9, the amino acid sequence of SEQ ID NO:11, light chain variable region.

The beneficial effect of adopting the above further scheme is that the variable region provided by the invention enables the PD-L1 antibody to have high affinity and specificity with human PD-L1, and simultaneously remarkably inhibits the binding of PD-L1 and receptor PD1, and does not bind with other members of the family, and the antibody can activate T cells by inhibiting PD1 signals, stimulate the T cells to secrete IL2, and remarkably inhibit tumor growth.

Further, the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody or the antigen binding portion thereof are identical to the amino acid sequences SEQ ID NO:9 and SEQ ID NO:11 have an overall sequence identity of at least 90%.

The advantage of using the above further protocol is to provide alternative antibodies with high affinity and specificity to human PD-L1, while significantly inhibiting the binding of PD-L1 to the receptor PD1 and not to other members of the same family, which activate T cells by inhibiting PD1 signaling, stimulate the secretion of IL2 by T cells, and significantly inhibit tumor growth.

Furthermore, the heavy chain amino acid sequence of the monoclonal antibody or the antigen binding part thereof is shown as SEQ ID NO.13, and the light chain amino acid sequence is shown as SEQ ID NO. 14.

The beneficial effect of adopting the further scheme is that the heavy chain and light chain amino acid sequences provided by the invention enable the PD-L1 antibody to have high affinity and specificity with human PD-L1, simultaneously significantly inhibit the binding of PD-L1 to receptor PD1, and not bind with other members of the same family, and the antibody can activate T cells by inhibiting PD1 signals, stimulate the T cells to secrete IL2, and significantly inhibit tumor growth.

Further, the monoclonal antibody is a full length antibody of the IgG1, IgG2, or IgG4 isotype.

The beneficial effect of adopting the further proposal is that the immunoglobulin is easier to diffuse into the extravascular space, thereby having important functions in the aspects of combining complement, enhancing the capability of immune cells to phagocytose pathogenic microorganisms and neutralize bacterial toxin and effectively resisting infection. On the other hand, immunoglobulins have a longer half-life and better stability in vivo.

Further, the monoclonal antibody is an IgG1 mutant, and the glycosylation site N on the FC of the monoclonal antibody is mutated into A.

The beneficial effect of using the above further protocol is a reduction in antibody dependent cell mediated cytotoxicity.

Further, the monoclonal antibody is an antibody fragment or a single-chain antibody.

The advantage of using the above further protocol is to provide more alternative forms of antibodies.

A medicament for treating or alleviating the symptoms of cancer, an infectious disease, or an autoimmune disease, comprising the monoclonal antibody that specifically binds to human PD-L1 or an antigen-binding portion thereof described above.

The beneficial effect of adopting the further scheme is that the antibody or the antigen binding part thereof is applied to the treatment or alleviation of the symptoms of cancer, infectious diseases or autoimmune diseases, and the application range is expanded.

A kit comprising the monoclonal antibody or antigen-binding portion thereof described above.

The beneficial effect of adopting the further scheme is that the antibody or the antigen binding part thereof is applied to the kit, and the related diseases are effectively detected and diagnosed.

Drawings

FIG. 1 is a graph showing the results of measuring the binding activity of the PD-L1 antibody to PD-L1-overexpressing cells in example 4;

FIG. 2 is a graph showing the results of the detection of the binding activity of the PD1 and the PD-L1 overexpression cells by the PD-L1 antibody in example 5;

FIG. 3 is a graph showing the results of the assay for the binding activity of the PD1 and the PD-L1 recombinant protein inhibited by the PD-L1 antibody of example 6;

FIG. 4 is a graph showing the results of measuring the binding activity of HUL02 antibody to recombinant PD-L1 protein in PD-L1 in example 7;

FIG. 5 is a graph showing the results of affinity assay of HUL02 antibody PD-L1 in example 8;

FIG. 6 is a diagram showing the results of detection of inter-species cross-reaction and specificity of antibodies in example 9;

FIG. 7 is a graph showing the results of detecting IL-2 secretion from T lymphocytes stimulated by the PD-L1 antibody of example 10;

FIG. 8 is a graph showing the results of the therapeutic effect of the anti-PD-L1 antibody of example 11 on tumors in vivo.

Detailed Description

The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.

Example 1 hybridoma selection

Murine monoclonal antibodies were prepared by hybridoma cell technology. For experimental protocols see the literature (Ed Harlow, David Lane. Antibody: A laboratory Manual, 1988).

The BALB/c mice were first immunized by intraperitoneal injection of recombinant PD-L1-His protein (Hibiscus, Yi) mixed with complete Freund's adjuvant (Sigma), after which mice were boosted by intraperitoneal injection after mixing with recombinant protein and incomplete Freund's adjuvant (Sigma) at 14d and 35d, respectively, and BALB/c mice were finally immunized by intravenous injection with recombinant PD-L1-His (PBS dilution) at 56d, and spleens were harvested for 4 days before fusion.

Murine splenocytes were mixed with SP2/0 cells (ATCC No. crl-1581) in a quantitative ratio of 4: 1 proportion fusion, culturing in a HAT (GBICO) culture medium in a 96-well plate (Corning), then carrying out hybridoma cell screening, and carrying out identification screening on the obtained cell clone;

the identification and screening process comprises three steps:

immobilizing recombinant PD-L1-His (Yi Qiao Shen) on a 96-hole enzyme-linked immunosorbent plate (Costar), adding a clone expression supernatant, incubating for 1h, washing with PBST for 3 times, identifying a cell clone supernatant with PD-L1 binding activity by using goat anti-mouse IgG-HRP (Jackson Immuno), washing with PBST for 3 times, and developing with TMB (Tetramethylbenzidine), thereby obtaining a positive clone capable of being directly bound with PD-L1;

secondly, transferring the positive clone in the step (i) into a 24-well plate (Corning) for culture to obtain more expression products, inoculating a PD-L1 stable overexpression CHO cell strain on a 96-well plate (Corning) for overnight culture, blocking with 1% BSA (1% BSA in the specification is BSA with the mass concentration of 1% in all), adding the positive clone expression supernatant for co-incubation for 1h, washing with PBS for 2 times, and detecting by using EU-labeled goat anti-mouse IgG (Perkin Elmer), thereby identifying the positive clone capable of binding with the cell surface PD-L1;

③ inoculating PD-L1 stable over-expression cell strain on 96-well plate (Corning) for overnight culture, blocking with 1% BSA the next day, adding antibody supernatant, adding biotin recombinant PD1 protein, washing with PBS for 2 times, adding EU labeled streptavidin (Perkin Elmer), and reading to identify positive clone capable of inhibiting binding.

The screened hybridoma cells of the positive clone are cracked, RNA is extracted and then reverse transcription is carried out to obtain cDNA, the cDNA is taken as a template, the light chain variable region nucleic acid sequence and the heavy chain variable region nucleic acid sequence of a mouse IgG antibody are respectively amplified by adopting a PCR method, the heavy chain variable region and the light chain variable region are sequenced, the sequence of the coded heavy chain variable region is shown as SEQ ID NO.16, the sequence of the coded light chain variable region is shown as SEQ ID NO.17, and the sequence of the coded heavy chain variable region is shown as EVQLQESGPSLVQPSQTLSLTCSVTGDSFTSGYWNWIRKFPDNKLQYMGYVSYTGSTYYIPSLKSRISITRDTSKNQYFLQLNSVTSEDTATYFCAGYRDWLHGYFDYWGQGTTLTVSS, SEQ ID NO.17 and is DIVMTQSQKFMSTSIGDRVSVTCKASQNVGTNVAWYQLKPGQSPKALIYSASYRFSGVPDRFTGSGSGTEFTLTITNVQSEDLTDYFCQQYNGYPLTFGAGTKLEVK.

Example 2 humanization

In this example, the murine monoclonal antibody specifically binding to PD-L1 obtained in example 1 was humanized by the following steps:

analyzing the sequence of the murine antibody, aligning with the human germline (germ line) gene of IMGT to finally determine IGKV 1-17X 01 and IGKJ 2X 01 as the humanized framework sequences of the light chain, and IGHV 4-31X 03 and IGHJ 6X 01 as the humanized framework sequences of the heavy chain, juxtaposing the CDRs of the heavy chain and the light chain to the framework sequences by CDR-grafting, and simultaneously, performing back mutation design on key sites on the heavy chain framework region including Q1E, G27D, I29F, W47Y, V3671 23 and R94G to construct a humanized antibody called HUL01 whose amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.9, the light chain variable region is shown as SEQ ID NO.10, SEQ ID NO.9 is EVQLQESGPGLVKPSQTLSLTCTVSGDSFSSGYWNWIRQHPGKGLEYIGYVSYTGSTYYIPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCAGYRDWLHGYFDYWGQGTTVTVSS, SEQ ID NO.9 including SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3, CDRs SEQ ID NO.1 is YRDWLHGYFDY, and SGID NO. YRDWLHGYFDY is YRDWLHGYFDY, the DIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKRLIYSASYRFSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYNGYPLTFGQGTKLEIK, SEQ ID NO.10 of SEQ ID NO.10 includes the light chain CDRs shown in SEQ ID NO. 4, SEQ ID NO. 6 and SEQ ID NO. 7, the KASQNVGTNVA, SEQ ID NO. 4 is SASYRFS 6, and the QQYNGYPLT SEQ ID NO. 7.

Example 3 affinity maturation

In this example, HUL01 antibody in example 2 was subjected to affinity maturation screening, and scfv fragment of HUL01 antibody was genetically synthesized, DNA sequence is shown as SEQ ID No.20, SEQ ID No.20 is GAGGTGCAACTGCAGGAGAGCGGCCCTGGCCTGGTGAAACCCTCCCAGACACTGTCCCTGACCTGCACCGTGTCCGGCGATTCCTTCAGCAGCGGCTACTGGAACTGGATCAGGCAGCATCCCGGAAAGGGACTGGAGTACATCGGCTACGTGTCCTACACCGGCAGCACCTACTACATCCCCTCCCTCAAGTCCCGGGTGACCATCTCCAGGGACACCTCCAAGAACCAGTTCTCCCTGAAGCTGTCCTCCGTGACAGCTGCCGACACCGCCGTGTACTACTGTGCCGGCTACAGGGACTGGCTGCACGGCTACTTCGACTACTGGGGCCAAGGAACCACCGTGACAGTGTCCTCCGGCGGCGGCGGCTCCGGCGGCGGCGGTTCTGGCGGCGGAGGCAGCGACATTCAGATGACCCAGAGCCCTTCCTCCCTCTCCGCTTCCGTGGGAGACAGGGTCACCATCACCTGTAAGGCCTCCCAGAACGTGGGCACCAACGTGGCCTGGTATCAACAGAAGCCCGGCAAGGCTCCCAAGCGGCTGATTTACAGCGCCTCCTACCGGTTCTCCGGAGTGCCTTCCAGGTTCTCCGGCTCCGGTTCTGGAACCGAGTTCACCCTGACCATCTCCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAACGGCTACCCCCTGACCTTCGGCCAGGGAACCAAGCTGGAGATCAAG, amino acid is shown as SEQ ID No.15, SEQ ID No.15 is EVQLQESGPGLVKPSQTLSLTCTVSGDSFSSGYWNWIRQHPGKGLEYIGYVSYTGSTYYIPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCAGYRDWLHGYFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKRLIYSASYRFSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYNGYPLTFGQGTKLEIK, scfv was ligated to pcmob 3 phage plasmid, DNA sequence of CDR of murine antibody was analyzed, mutation hot spot in CDR of variable region was determined, primer sequence was designed, primer position where mutation hot spot site is located was designed as NNS, so that it encodes arbitrary amino acid, antibody library of HUL01 antibody scfv was used as template, PCR was amplified, antibody library of scfv was constructed into pcmob 3 phage plasmid through sfiI cleavage site, antibody library was constructed, and then high affinity antibody screening was performed by phage display, specifically steps are as follows:

electrically transforming phage plasmids containing an antibody library of scfv into TG1, adding helper phase into a bacterial liquid after recovery at 37 ℃ and 250rpm for 1h, adding Ampicillin at 37 ℃ and 250rpm, centrifuging for 1h and 2500rpmx for 5min to remove supernatant, blowing and suspending bacterial sludge with 2 xYT-AK at 37 ℃ and 250rpm for overnight culture; ② envelope antigen: diluting PD-L1 protein with coating buffer solution, mixing uniformly, adding into an immune tube, and coating overnight at 4 ℃; collecting recombinant phage: centrifuging the overnight culture solution at 2500rpm for 5min, collecting supernatant 10ml, adding 2ml PEG/NaCl, mixing, standing on ice for 1h, centrifuging at 10000g × 20 min, removing supernatant, and dissolving phage library with PBS; sealing: washing the immune tube twice with PBS, adding a sealing solution, and keeping the temperature at room temperature for 1 h; in addition, mixing the sealing liquid with the same volume with the phage library, and sealing for 10-15min at room temperature; incubating the phage library: washing the immune tube with PBS for 2 times, adding the phage library which is sealed, and culturing in a 37-degree incubator for 2-3 h; sixthly, elution: 100TG1 bacterial liquid (inoculated on the previous day) is taken to be added into 10ml2x YT, and cultured at 37 ℃ and 250rpm until the A600 value is 0.4-0.5. Washing the immune tube with PBST for 8 times, then washing with PBS for 2 times, adding 5ml of log-phase growth bacterial liquid, 37 degrees, 250rpm,1 h; seventhly, diluting the bacterial liquid 10-1 and 10-2, and respectively coating 100ul of the diluted bacterial liquid on flat plates. Eighthly, next-round screening: adding 200 ul helper phase into 5ML of eluted bacterial liquid, adding 5ul Ampicillin at 37 ℃,250rpm,1h and 2500rpm for x5min, centrifuging to remove supernatant, blowing suspended bacterial sludge with 10ML2x YT-AK, culturing overnight at 37 ℃ and 250rpm, and repeating the steps of (i) - (c).

After three rounds of screening, monoclonal antibodies are selected, and the activity of the phase is detected by a phage ELISA method, which specifically comprises the following steps:

① coating PD-L1 protein, 0.5ug/ml, 4 degree overnight, ② PBST washing twice, adding 50ul phage supernatant and 50ul 1% BSA, 37 deg.C, 1h, ③ PBST washing three times, adding 100ul1:10000 diluted anti-M13-Ab-HRP (Yi Qiao Shen), 37 deg.C, 1h, ④ PBST washing three times, adding 100ul preheated TMB, room temperature 10min, ⑤ adding 50ul 1M H₂The reaction was stopped with SO4 and absorbance was measured at OD 450.

Selecting a positive monoclonal antibody, and detecting the cell binding activity of the monoclonal antibody through a phage and PD-L1 overexpression cell binding experiment, wherein the cell binding activity is as follows:

① pancreatin PD-L1 overexpression cells, 200gX5min, 1% BSA heavy suspension of cells, room temperature, 1h, ② blocking the phase, 50ul of phase supernatant and 50ul of 1% BSA, room temperature, 1h, ③ cell plating, 100ul/well, 2500rpmx5min, ④ discarding supernatant, 100ul/well heavy suspension of cells, room temperature, 1h, ⑤ cell PBS washing twice, 100ul1:10000 diluted anti-M13-Ab-HRP, room temperature, 1h, ⑥ cell PBST washing twice, 100ul preheated TMB, room temperature, 10min, ⑦ adding 50ul of 1M H₂SO₄The reaction was stopped and the absorbance was measured by OD 450.

Five cell binding clones were selected, clone1, clone2, clone3, clone4 and clone5, and sequenced to identify 3 different monoclonal antibodies, wherein the sequences of clone1, clone2 and clone3 are the same as HUL01, the antibody from clone4 is named HUL02 and the antibody from clone5 is named HUL 03. Wherein, the heavy chain variable region amino acid sequence of HUL02 is shown as SEQ ID NO.9, the light chain variable region is shown as SEQ ID NO.11, the SEQ ID NO.11 is DIQMTQSPSSLSASVGDRVTITCKASQNVMDNVAWYQQKPGKAPKRLIYSASYRFSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYNGYPLTFGQGTKLEIK, SEQ ID NO.11 and comprises light chain CDRs shown as SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7, the heavy chain variable region amino acid sequence of SEQ ID NO. 5 is KASQNVMDNVA, HUL03 is shown as SEQ ID NO.9, the light chain variable region is shown as SEQ ID NO.12, the SEQ ID NO.12 is DIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKRLIYSASYRFSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYNSYPLTFGQGTKLEIK, SEQ ID NO.12 and comprises light chain CDRs shown as SEQ ID NO. 4, SEQ ID NO. 6 and SEQ ID NO. 8, and SEQ ID NO. 8 is QQYNSYPLT.

Construction of full length antibodies for HUL01, HUL02 and HUL 03: the heavy chain variable region DNA fragment of the antibody is connected with the constant region (wherein the glycosylation site N on FC is mutated into A) of heavy chain IgG1 by a PCR method to obtain a full-length heavy chain, and the light chain variable region DNA fragment is respectively connected with the constant region of a light chain K chain to obtain a full-length light chain. Full-length heavy and light chain DNA fragments, both containing signal peptides, were cloned into PCDNA3.1 (Invitrogen) plasmid, respectively. The HUL01, HUL02, and HUL03 expression plasmids were transiently transferred to 293C18 cell line (ATCC No. crl-10852), and the supernatant was purified using Protein a (GE) to obtain purified antibodies.

The heavy chain amino acid sequence and the light chain amino acid sequence of HUL01 are shown as SEQ ID NO.13 and SEQ ID NO.18, the heavy chain full-length DNA sequence and the light chain DNA sequence of which SEQ ID NO.13 is EVQLQESGPGLVKPSQTLSLTCTVSGDSFSSGYWNWIRQHPGKGLEYIGYVSYTGSTYYIPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCAGYRDWLHGYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, SEQ and the ID NO.18 is DIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKRLIYSASYRFSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYNGYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC, HUL01 are shown as SEQ ID NO.21 and SEQ ID NO.22, and the heavy chain amino acid sequence and the light chain DNA sequence of which SEQ ID NO.21 is GAGGTGCAACTGCAGGAGAGCGGCCCTGGCCTGGTGAAACCCTCCCAGACACTGTCCCTGACCTGCACCGTGTCCGGCGATTCCTTCAGCAGCGGCTACTGGAACTGGATCAGGCAGCATCCCGGAAAGGGACTGGAGTACATCGGCTACGTGTCCTACACCGGCAGCACCTACTACATCCCCTCCCTCAAGTCCCGGGTGACCATCTCCAGGGACACCTCCAAGAACCAGTTCTCCCTGAAGCTGTCCTCCGTGACAGCTGCCGACACCGCCGTGTACTACTGTGCCGGCTACAGGGACTGGCTGCACGGCTACTTCGACTACTGGGGCCAAGGAACCACCGTGACAGTGTCCTCCGCCAGCACCAAGGGACCATCCGTGTTCCCACTGGCTCCAAGCTCTAAATCCACTAGCGGAGGCACCGCAGCCCTGGGATGTCTGGTGAAGGATTACTTCCCAGAGCCCGTCACAGTGTCATGGAACTCCGGGGCTCTGACCTCTGGTGTCCACACATTTCCAGCAGTGCTGCAGAGTTCAGGCCTGTACTCCCTGTCCAGCGTGGTCACAGTGCCCTCTAGTTCACTGGGAACTCAGACCTATATCTGCAACGTGAATCACAAGCCATCCAATACTAAAGTCGACAAGAAAGTGGAGCCCAAGAGCTGTGATAAAACACATACTTGCCCCCCTTGTCCTGCACCAGAACTGCTGGGAGGACCATCCGTGTTCCTGTTTCCACCCAAGCCTAAAGACACTCTGATGATTTCTCGAACACCCGAGGTCACTTGCGTGGTCGTGGACGTGTCCCACGAGGATCCTGAAGTCAAGTTTAACTGGTACGTGGATGGAGTCGAAGTGCATAATGCTAAGACAAAACCTAGAGAGGAACAGTACGCCAGTACATATAGAGTCGTGTCAGTCCTGACTGTGCTGCATCAGGACTGGCTGAACGGGAAGGAGTATAAGTGCAAAGTGTCCAATAAGGCTCTGCCCGCACCTATCGAGAAAACTATTAGCAAGGCTAAAGGCCAGCCTAGGGAACCACAGGTGTACACCCTGCCTCCATCTCGGGAGGAAATGACTAAGAACCAGGTCAGTCTGACCTGTCTGGTGAAAGGCTTCTATCCTTCCGACATCGCAGTGGAGTGGGAAAGCAATGGACAGCCAGAGAACAATTACAAGACCACACCCCCTGTGCTGGACAGCGATGGGTCTTTCTTTCTGTATAGTAAGCTGACCGTGGATAAATCACGGTGGCAGCAGGGTAATGTCTTTTCTTGTAGTGTGATGCACGAAGCCCTGCACAACCATTACACTCAGAAATCCCTGTCACTGTCCCCTGGAAAGTGA, SEQ and the ID NO.22 is GACATTCAGATGACCCAGAGCCCTTCCTCCCTCTCCGCTTCCGTGGGAGACAGGGTCACCATCACCTGTAAGGCCTCCCAGAACGTGGGCACCAACGTGGCCTGGTATCAACAGAAGCCCGGCAAGGCTCCCAAGCGGCTGATTTACAGCGCCTCCTACCGGTTCTCCGGAGTGCCTTCCAGGTTCTCCGGCTCCGGTTCTGGAACCGAGTTCACCCTGACCATCTCCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAACGGCTACCCCCTGACCTTCGGCCAGGGAACCAAGCTGGAGATCAAGAGGACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAA; the heavy chain amino acid sequence and the light chain amino acid sequence of HUL02 are shown as SEQ ID NO.13 and SEQ ID NO.14, the heavy chain full-length DNA sequence and the light chain DNA sequence of SEQ ID NO.14, which are DIQMTQSPSSLSASVGDRVTITCKASQNVMDNVAWYQQKPGKAPKRLIYSASYRFSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYNGYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC, HUL02, are shown as SEQ ID NO.21 and SEQ ID NO.23, and the SEQ ID NO.23 is GACATTCAGATGACCCAGAGCCCTTCCTCCCTCTCCGCTTCCGTGGGAGACAGGGTCACCATCACCTGTAAGGCCTCCCAGAACGTGATGGACAACGTGGCCTGGTATCAACAGAAGCCCGGCAAGGCTCCCAAGCGGCTGATTTACAGCGCCTCCTACCGGTTCTCCGGAGTGCCTTCCAGGTTCTCCGGCTCCGGTTCTGGAACCGAGTTCACCCTGACCATCTCCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAACGGCTACCCCCTGACCTTCGGCCAGGGAACCAAGCTGGAGATCAAGAGGACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAA; the heavy chain amino acid sequence and the light chain amino acid sequence of HUL03 are shown as SEQ ID NO.13 and SEQ ID NO.19, the heavy chain full-length DNA sequence and the light chain DNA sequence of SEQ ID NO.19, which are DIQMTQSPSSLSASVGDRVTITCKASQNVMDNVAWYQQKPGKAPKRLIYSASYRFSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYNSYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC, HUL03, are shown as SEQ ID NO.21 and SEQ ID NO.24, and the SEQ ID NO.24 is GACATTCAGATGACCCAGAGCCCTTCCTCCCTCTCCGCTTCCGTGGGAGACAGGGTCACCATCACCTGTAAGGCCTCCCAGAACGTGGGCACCAACGTGGCCTGGTATCAACAGAAGCCCGGCAAGGCTCCCAAGCGGCTGATTTACAGCGCCTCCTACCGGTTCTCCGGAGTGCCTTCCAGGTTCTCCGGCTCCGGTTCTGGAACCGAGTTCACCCTGACCATCTCCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAACAGCTACCCCCTGACCTTCGGCCAGGGAACCAAGCTGGAGATCAAGAGGACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAA.

Example 4 determination of binding Activity with PD-L1 overexpressing cells

Plating PD-L1 overexpression cells on a black 96-well plate, culturing overnight, blocking by 1% BSA, incubating at 37 ℃ for 1 hour; antibody 5ug/ml start, 3 fold dilution, 8 concentration, 100ul/well, 25 ℃ incubation for 1 hour; PBS was washed once, then anti-human-IgG-EU (Perkin Elmer), 50ng/ml, 100ul per well, 25 ℃ reaction for 0.5 hours; PBS washing twice, finally, adding fluorescence enhancement liquid, excitation light 337 nm/emission light 620nm reading. The results showed that the selected monoclonal antibodies HUL01, HUL02 and HUL03 bound to the overexpressing cells EC50 at 130ng/ml,69.51ng/ml and 128.2ng/ml, respectively, as shown in FIG. 1.

Example 5 inhibition of binding Activity of PD1 with PD-L1 overexpressing cells

Plating the PD-L1 overexpression cells on a black 96-well plate, and culturing overnight; blocking with 1% BSA, 25 ℃ and incubating for 1 hour; starting an antibody with the concentration of 10ug/ml, diluting by 3 times, adding 50ul of biotin recombinant PD1 protein, incubating at 25 ℃ for 1 hour, and washing with PBS once; then, add EU-labeled streptavidin (Perkin Elmer) 100ul per well, react at 25 ℃ for 0.5 hour, wash twice with PBS; finally, a fluorescence enhancing fluid was added and the excitation 337 nm/emission 620nm reading. The results show that the screened monoclonal antibodies HUL01, HUL02 and HUL03 and the IC50 inhibiting the binding of PD1 and PD-L1 over-expressing cells are 228.3ng/ml,114.9ng/ml and 206.2ng/ml respectively, as shown in FIG. 2.

Example 6 inhibition of binding Activity of PD1 with PD-L1 recombinant protein

A 96-well plate (Costar) is coated with recombinant PD-L1-Fc protein, the concentration is 1ug/ml, and the temperature is 4 ℃ overnight; blocking with 1% BSA, 37 ℃ and incubating for 1 hour; PBST washing twice, starting antibody final concentration of 1ug/ml, diluting 3 times, biotinylating recombinant PD1 protein final concentration of 0.5ug/ml, adding simultaneously, and incubating at 37 ℃ for 1 hour; PBST washing twice, adding streptavidin-HRP (1: 10000 dilution), 37 degrees, and incubating for 1 hour; PBST is washed for three times, 100ul of preheated TMB is added, and the room temperature is 10 min; the reaction was stopped by adding 50ul 1M H2SO4 and absorbance was measured by OD 450. The results showed that the screened monoclonal antibodies HUL01, HUL02 and HUL03 and the IC50 inhibiting the binding of PD1 and PD-L1 recombinant proteins were 117.1ng/ml,71.61 ng/ml and 118ng/ml, respectively, as shown in FIG. 3.

Example 7 determination of binding Activity with recombinant PD-L1 protein

Coating the recombinant PD-L1-HIS protein on a 96-well plate (Costar) at 4 ℃ overnight; blocking with 1% BSA, 37 ℃ and incubating for 1 hour; PBST washing twice, starting with the final antibody concentration of 1ug/ml, diluting 3 times, and incubating at 37 ℃ for 1 hour; PBST washing twice, adding donkey anti-human IgG-HRP (Jackson immuno), and incubating for 1 hour at 37 ℃; PBST is washed for three times, 100ul of preheated TMB is added, and the room temperature is 10 min; the reaction was stopped by adding 50ul 1M H2SO4 and absorbance was measured by OD 450. The results show that the selected monoclonal antibody HUL02 has 20.61ng/ml direct binding EC50 to PD-L1 protein as shown in FIG. 4.

Example 8 affinity assay for monoclonal antibodies

The affinity constant KD of the monoclonal antibody to PD-L1 was determined using BIAcore T200 by first immobilizing goat anti-human IgG on a CM5 chip, adding 10. mu.g/ml of monoclonal antibody at 10. mu.l/min for 100 seconds, then adding 300nM PD-L1-HIS at 30. mu.l/min flow rate for 300 seconds, and observing the dissociation for 1200 seconds. Biacore T200 analysis software analyzed the antibody for affinity constants KD, KD/ka. The results show (FIG. 5) that the affinity constant KD of HUL02 is 1.7E-10M.

Example 9 Cross-species reaction assay and specificity

Recombinant human B7-1, human B7-2, human PDL2, murine PD-L1, monkey PD-L1 and human PD-L1 proteins were coated on a 96-well plate (Costar) respectively, overnight at 4 ℃; blocking with 1% BSA and incubating at 37 ℃ for 1 hour; PBST washing twice, antibody final concentration of 1ug/ml, 37 degrees were incubated for 1 hours; PBST washing twice, adding donkey anti-human IgG antibody-HRP (Jackson immuno), 37 degrees, and incubating for 1 hour; PBST is washed for three times, 100ul of preheated TMB is added, and the room temperature is 10 min; the reaction was stopped by adding 50ul 1M H2SO4 and absorbance was measured by OD 450. The results show (FIG. 6) that HUL02 does not bind to other proteins of the same family, human B7-1, human B7-2 and human PDL2, and binds to monkey PD-L1.

Example 10 stimulation of IL-2 secretion by T lymphocytes by antibodies

The method comprises the steps of diluting blood by 1:1 with PBS buffer solution, transferring 3ml of lymphocyte separation liquid into a centrifugal tube, adding 4ml of diluted blood, ensuring that the diluted blood is placed on the upper layer of the lymphocyte separation liquid and cannot be uniformly mixed when taking care of adding, centrifuging for 30-40min at 400g and RT, finally sucking out the separated PBMC, centrifuging for 10min at 100g, separating CD4+ T cells by using a CD4+ cell separation kit (BD company), separating DC cells by using DC cell separation magnetic beads (BD company), plating cells, and detecting the number of the CD4+ T cells in each hole by 1 × 10⁵Number of DCs 1 × 10⁴Each volume totaled 100ul., and different concentrations of antibody were added, and IL-2 concentration was measured using a Human IL-2 HTRF kit (Cisbio, cat #64IL2PEB) 5 days after incubation with the marketed PD1 antibody drug Keytruda as a control antibody. The results show (FIG. 7) that HUL02 stimulated T lymphocyte IL-2 secretion with an EC50 of 0.062ug/ml, which is superior to the control antibody.

Example 11 treatment of in vivo tumor models Using anti-PD-L1 antibody

The in vivo effect of the PD-L1 antibody on tumor growth was examined using a MC38-hPDL1 cell subcutaneous tumor-bearing mouse model. MC38.hPDL1 cells were harvested at log phase growth, the culture was removed and washed twice with PBS and inoculated into B6-Pdl1tm (hPDL1)/Nju mice at 5X 105/100 ul/mouse (no matrigel added). When the average tumor volume reached about 100mm3, mice with tumor volumes meeting the grouping criteria were randomly divided into 3 groups of Vehicle control (PBS), positive drug (Tecentriq in PBS) and drug to be tested (HUL 02 in PBS), and then administration was started at a period of q2d x8 in an i.p. dose of 10 mg/kg. Mouse body weight and tumor volume were measured on the day of dosing, followed by 2 measurements per week. When the tumor reached the tumor endpoint (1500 mm') or showed a weight loss of more than 15%, the experiment was terminated and all mice were euthanized. The results show (fig. 6) that HUL02 significantly inhibited tumor growth in the MC38-hPDL1 murine tumor model, with superior efficacy to positive drugs.

The antibody of the present invention can be produced by genetic engineering techniques, since the DNA sequence encoding the humanized antibody of the present invention can be obtained by conventional means well known to those skilled in the art, such as artificial synthesis of the amino acid sequence according to the disclosure of the present invention or amplification by PCR, and thus can be used by recombinant DNA methods, and the sequence can be ligated into an appropriate expression vector by various methods well known in the art.

Once the antibody molecule of the invention is prepared, it can be purified by any method known in the art for purifying immunoglobulin molecules, for example, by chromatography (e.g., ion exchange chromatography, affinity chromatography, particularly by affinity chromatography of protein a for a specific antigen and other column chromatography), centrifugation, use of solubility differences, or by any other standard technique for purifying proteins. In many embodiments, the antibody is secreted from the cell into the culture medium, and the antibody is obtained by collecting the culture medium and purifying it.

In the present invention, unless otherwise defined, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms, and laboratory procedures used herein are all terms and routine steps that are widely used in the relevant arts. Meanwhile, in order to better understand the present invention, the definitions and explanations of the related terms are provided below.

In the present invention, the term "antibody" includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof. An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains that are linked to each other by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains, CH1, CH2, and CH 3. Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The VH and VL regions can be further divided into hypervariable regions, termed Complementarity Determining Regions (CDRs), interspersed with more conserved regions termed Framework Regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4. The variable regions of the heavy and light chains contain binding domains that can interact with antigens. The constant region of the antigen may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). The extent of the framework regions and complementarity determining regions has been precisely defined (see "sequences of Proteins of Immunological Interest," E. Kabat et al, U.S. Department of health and Human Services, 1991). All antibody amino acid sequences discussed herein are ordered with reference to the Kabat system. The term "antibody" is not limited by any particular method of producing an antibody, and includes, for example, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies, among others.

In the present invention, the term "antigen binding portion" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been demonstrated that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" include: (1) fab fragments, i.e., monovalent fragments consisting of the VL, VH, CL and CH1 domains; (2) a F (ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (3) an Fd fragment consisting of the VH and CH1 domains; (4) (ii) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (5) dAb fragments consisting of VH domains (Ward et al (1989) Nature 341: 544-546); and (6) an isolated Complementarity Determining Region (CDR).

In the present invention, the term "isotype" refers to the class of antibodies (e.g., IgM or IgG 1) encoded by the heavy chain constant region gene.

The term "humanized antibody" refers to an antibody in which CDR sequences derived from the germline of another mammalian species, such as a mouse, are grafted onto human framework sequences. Other modifications may also be made within the human framework sequences.

The term "chimeric antibody" is an antibody whose heavy and light chain genes have been constructed, particularly using genetically engineered antibody variable and constant region genes belonging to different species. For example, a variable region fragment of a murine monoclonal antibody gene can be ligated to a human antibody constant region fragment such as γ 1 and γ 3. Of course, other mammalian species may be used as the genetic source for the chimeric antibody.

"KD" in the present invention refers to the dissociation constant, which is obtained from the ratio of Kd and Ka (i.e., Kd/Ka), and is expressed as molar concentration (M). KD values for antibodies can be determined using methods established in the art. A preferred method of determining KD is to use surface plasmon resonance, preferably using a biosensor system, such as the Biacore system.

The invention of at least 90% overall sequence identity means sequence identity of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, the monoclonal antibody or antigen binding portion thereof heavy chain variable region and light chain variable region of the amino acid sequences and the amino acid sequences of SEQ ID NO:9 and SEQ ID NO:11, which has at least 90% overall sequence identity to the monoclonal antibody or antigen binding portion thereof, refers to the amino acid sequence of the heavy chain variable region and the light chain variable region combined together with the amino acid sequences SEQ ID No.9 and SEQ ID NO:11 has at least 90% sequence identity to the sum of the sequences.

The PD-L1 antibodies of the invention also include immunoglobulin constant regions.

The cancer of the invention includes but is not limited to lung cancer, colon cancer, melanoma, renal cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematological malignancy, gastric cancer, cervical cancer, prostate cancer, ovarian cancer and pancreatic cancer; such infectious diseases include, but are not limited to, bacterial, fungal, protozoan and viral infectious diseases; the autoimmune disease includes, but is not limited to, chronic inflammatory diseases (such as lichen planus or T cell mediated chronic inflammatory skin mucositis).

The heavy chain and the light chain of the PD-L1 antibody and the PD-L1 antibody can also be used for scientific research related to PD-L1, such as scientific research in multiple fields of developmental biology, cell biology, metabolism, structural biology, functional genomics and the like, or medical and pharmaceutical application research of tumors, autoimmune diseases and the like.

The PD-L1 antibody of the invention may be a single chain antibody, diabody, chimeric antibody, humanized antibody, and derivatives, functional equivalents and homologues of the foregoing, including antibody fragments and any polypeptide comprising an antigen binding domain.

The pharmaceutical composition of the invention also comprises a pharmaceutically acceptable carrier and/or diluent.

The invention may also be a reagent or chip comprising the aforementioned PD-L1 antibody.

The invention also discloses a method for inhibiting the PD1 signal path by adopting the PD-L1 antibody, and the antibody is used for treating related diseases or carrying out related diagnosis and detection by using a kit containing the antibody.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

<110> Jiangsu Nuomibo biomedical science and technology Co., Ltd

<120> monoclonal antibody specifically binding to human PD-L1, and medicine and kit comprising same

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Tyr Val Ser Tyr Thr Gly Ser Thr Tyr Tyr Ile Pro Ser Leu Lys Ser

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<212>PRT

<213> Artificial Sequence (Artificial Sequence)

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Tyr Arg Asp Trp Leu His Gly Tyr Phe Asp Tyr

1 5 10

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<211>11

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<213> Artificial Sequence (Artificial Sequence)

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Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala

1 5 10

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<213> Artificial Sequence (Artificial Sequence)

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Lys Ala Ser Gln Asn Val Met Asp Asn Val Ala

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<213> Artificial Sequence (Artificial Sequence)

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Ser Ala Ser Tyr Arg Phe Ser

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<213> Artificial Sequence (Artificial Sequence)

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Gln Gln Tyr Asn Gly Tyr Pro Leu Thr

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Gln Gln Tyr Asn Ser Tyr Pro Leu Thr

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Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

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20 25 30

Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Tyr Ile

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Gly Tyr Val Ser Tyr Thr Gly Ser Thr Tyr Tyr Ile Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

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Gly Tyr Arg Asp Trp Leu His Gly Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

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Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn

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Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

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Tyr Ser Ala Ser Tyr Arg Phe Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Gly Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

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<211>107

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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

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Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Met Asp Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Phe Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Gly Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

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<211>107

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<213> Artificial Sequence (Artificial Sequence)

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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

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Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Phe Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>13

<211>449

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>13

Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Phe Ser Ser Gly

20 25 30

Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Tyr Ile

35 40 45

Gly Tyr Val Ser Tyr Thr Gly Ser Thr Tyr Tyr Ile Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Gly Tyr Arg Asp Trp Leu His Gly Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu ProLys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala ValGlu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210>14

<211>214

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>14

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Met Asp Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Phe Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Gly Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>15

<211>241

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>15

Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Phe Ser Ser Gly

20 25 30

Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Tyr Ile

35 40 45

Gly Tyr Val Ser Tyr Thr Gly Ser Thr Tyr Tyr Ile Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Gly Tyr Arg Asp Trp Leu His Gly Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

130 135 140

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser

145 150 155 160

Gln Asn Val Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys

165 170 175

Ala Pro Lys Arg Leu Ile Tyr Ser Ala Ser Tyr Arg Phe Ser Gly Val

180 185 190

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr

195 200 205

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

210 215 220

Tyr Asn Gly Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

225 230 235 240

Lys

<210>16

<211>119

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>16

Glu Val Gln Leu Gln Glu Ser Gly Pro Ser Leu Val Gln Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Asp Ser Phe Thr Ser Gly

20 2530

Tyr Trp Asn Trp Ile Arg Lys Phe Pro Asp Asn Lys Leu Gln Tyr Met

35 40 45

Gly Tyr Val Ser Tyr Thr Gly Ser Thr Tyr Tyr Ile Pro Ser Leu Lys

50 55 60

Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Tyr Phe Leu

65 70 75 80

Gln Leu Asn Ser Val Thr Ser Glu Asp Thr Ala Thr Tyr Phe Cys Ala

85 90 95

Gly Tyr Arg Asp Trp Leu His Gly Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser

115

<210>17

<211>107

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>17

Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Ile Gly

1 5 10 15

Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn

20 25 30

Val Ala Trp Tyr Gln Leu Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile

3540 45

Tyr Ser Ala Ser Tyr Arg Phe Ser Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Thr Asn Val Gln Ser

65 70 75 80

Glu Asp Leu Thr Asp Tyr Phe Cys Gln Gln Tyr Asn Gly Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Val Lys

100 105

<210>18

<211>214

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>18

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Phe Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Gly Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>19

<211>214

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>19

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Met Asp Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Phe Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>20

<211>723

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>20

gaggtgcaac tgcaggagag cggccctggc ctggtgaaac cctcccagac actgtccctg 60

acctgcaccg tgtccggcga ttccttcagc agcggctact ggaactggat caggcagcat 120

cccggaaagg gactggagta catcggctac gtgtcctaca ccggcagcac ctactacatc 180

ccctccctca agtcccgggt gaccatctcc agggacacct ccaagaacca gttctccctg 240

aagctgtcct ccgtgacagc tgccgacacc gccgtgtact actgtgccgg ctacagggac 300

tggctgcacg gctacttcga ctactggggc caaggaacca ccgtgacagt gtcctccggc 360

ggcggcggct ccggcggcgg cggttctggc ggcggaggca gcgacattca gatgacccag 420

agcccttcct ccctctccgc ttccgtggga gacagggtca ccatcacctg taaggcctcc 480

cagaacgtgg gcaccaacgt ggcctggtat caacagaagc ccggcaaggc tcccaagcgg 540

ctgatttaca gcgcctccta ccggttctcc ggagtgcctt ccaggttctc cggctccggt 600

tctggaaccg agttcaccct gaccatctcc tccctgcagc ccgaggactt cgccacctac 660

tactgccagc agtacaacgg ctaccccctg accttcggcc agggaaccaa gctggagatc 720

aag 723

<210>21

<211>1350

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>21

gaggtgcaac tgcaggagag cggccctggc ctggtgaaac cctcccagac actgtccctg 60

acctgcaccg tgtccggcga ttccttcagc agcggctact ggaactggat caggcagcat 120

cccggaaagg gactggagta catcggctac gtgtcctaca ccggcagcac ctactacatc 180

ccctccctca agtcccgggt gaccatctcc agggacacct ccaagaacca gttctccctg 240

aagctgtcct ccgtgacagc tgccgacacc gccgtgtact actgtgccgg ctacagggac 300

tggctgcacg gctacttcga ctactggggc caaggaacca ccgtgacagt gtcctccgcc 360

agcaccaagg gaccatccgt gttcccactg gctccaagct ctaaatccac tagcggaggc 420

accgcagccc tgggatgtct ggtgaaggat tacttcccag agcccgtcac agtgtcatgg 480

aactccgggg ctctgacctc tggtgtccac acatttccag cagtgctgca gagttcaggc 540

ctgtactccc tgtccagcgt ggtcacagtg ccctctagtt cactgggaac tcagacctat 600

atctgcaacg tgaatcacaa gccatccaat actaaagtcg acaagaaagt ggagcccaag 660

agctgtgata aaacacatac ttgcccccct tgtcctgcac cagaactgct gggaggacca 720

tccgtgttcc tgtttccacc caagcctaaa gacactctga tgatttctcg aacacccgag 780

gtcacttgcg tggtcgtgga cgtgtcccac gaggatcctg aagtcaagtt taactggtac 840

gtggatggag tcgaagtgca taatgctaag acaaaaccta gagaggaaca gtacgccagt 900

acatatagag tcgtgtcagt cctgactgtg ctgcatcagg actggctgaa cgggaaggag 960

tataagtgca aagtgtccaa taaggctctg cccgcaccta tcgagaaaac tattagcaag 1020

gctaaaggcc agcctaggga accacaggtg tacaccctgc ctccatctcg ggaggaaatg 1080

actaagaacc aggtcagtct gacctgtctg gtgaaaggct tctatccttc cgacatcgca 1140

gtggagtggg aaagcaatgg acagccagag aacaattaca agaccacacc ccctgtgctg 1200

gacagcgatg ggtctttctt tctgtatagt aagctgaccg tggataaatc acggtggcag 1260

cagggtaatg tcttttcttg tagtgtgatg cacgaagccc tgcacaacca ttacactcag 1320

aaatccctgt cactgtcccc tggaaagtga 1350

<210>22

<211>645

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>22

gacattcaga tgacccagag cccttcctcc ctctccgctt ccgtgggaga cagggtcacc 60

atcacctgta aggcctccca gaacgtgggc accaacgtgg cctggtatca acagaagccc 120

ggcaaggctc ccaagcggct gatttacagc gcctcctacc ggttctccgg agtgccttcc 180

aggttctccg gctccggttc tggaaccgag ttcaccctga ccatctcctc cctgcagccc 240

gaggacttcg ccacctacta ctgccagcag tacaacggct accccctgac cttcggccag 300

ggaaccaagc tggagatcaa gaggactgtg gctgcaccat ctgtcttcat cttcccgcca 360

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420

cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480

gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540

ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600

ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttaa 645

<210>23

<211>645

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>23

gacattcaga tgacccagag cccttcctcc ctctccgctt ccgtgggaga cagggtcacc 60

atcacctgta aggcctccca gaacgtgatg gacaacgtgg cctggtatca acagaagccc 120

ggcaaggctc ccaagcggct gatttacagc gcctcctacc ggttctccgg agtgccttcc 180

aggttctccg gctccggttc tggaaccgag ttcaccctga ccatctcctc cctgcagccc 240

gaggacttcg ccacctacta ctgccagcag tacaacggct accccctgac cttcggccag 300

ggaaccaagc tggagatcaa gaggactgtg gctgcaccat ctgtcttcat cttcccgcca 360

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420

cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480

gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540

ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600

ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttaa 645

<210>24

<211>645

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>24

gacattcaga tgacccagag cccttcctcc ctctccgctt ccgtgggaga cagggtcacc 60

atcacctgta aggcctccca gaacgtgggc accaacgtgg cctggtatca acagaagccc 120

ggcaaggctc ccaagcggct gatttacagc gcctcctacc ggttctccgg agtgccttcc 180

aggttctccg gctccggttc tggaaccgag ttcaccctga ccatctcctc cctgcagccc 240

gaggacttcg ccacctacta ctgccagcag tacaacagct accccctgac cttcggccag 300

ggaaccaagc tggagatcaa gaggactgtg gctgcaccat ctgtcttcat cttcccgcca 360

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420

cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480

gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540

ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600

ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttaa 645

Claims

1. A monoclonal antibody or an antigen binding portion thereof that specifically binds human PD-L1, said antigen binding portion being a Fab fragment or a F (ab') 2 fragment, comprising any one of the following sets of CDR regions:

2. The monoclonal antibody or antigen-binding portion thereof of claim 1, which specifically binds to human PD-L1, comprising a heavy chain variable region having the amino acid sequence of SEQ ID No.9 and a heavy chain variable region having the amino acid sequence of SEQ ID No.10, SEQ ID NO:11 or SEQ ID NO:12, or a light chain variable region of any one of seq id nos.

3. The monoclonal antibody or antigen-binding portion thereof of claim 2, which specifically binds to human PD-L1, wherein the monoclonal antibody or antigen-binding portion thereof comprises a heavy chain variable region having the amino acid sequence of SEQ ID No.9, and wherein the amino acid sequence of SEQ ID NO:11, light chain variable region.

4. The monoclonal antibody or antigen-binding portion thereof of claim 3, wherein the heavy chain variable region and the light chain variable region amino acid sequences of the monoclonal antibody or antigen-binding portion thereof are identical to the amino acid sequences of SEQ ID NO 9 and SEQ ID NO:11 have an overall sequence identity of at least 90%.

5. The monoclonal antibody or antigen-binding portion thereof that specifically binds to human PD-L1 of claim 3, wherein the monoclonal antibody has the heavy chain amino acid sequence set forth in SEQ ID NO.13 and the light chain amino acid sequence set forth in SEQ ID NO. 14.

6. The monoclonal antibody or antigen-binding portion thereof that specifically binds to human PD-L1 of any one of claims 1-4, wherein the monoclonal antibody is a full-length antibody of the IgG1, IgG2, or IgG4 isotype.

7. The monoclonal antibody or antigen-binding portion thereof of claim 6, which specifically binds to human PD-L1, wherein the monoclonal antibody is an IgG1 mutant having a N mutation of the glycosylation site on FC to A.

8. The monoclonal antibody or the antigen-binding portion thereof that specifically binds to human PD-L1 according to any one of claims 1-7, wherein the monoclonal antibody is a single chain antibody.

9. A medicament for treating or ameliorating cancer or an infectious disease, wherein the medicament comprises the monoclonal antibody or an antigen-binding portion thereof that specifically binds to human PD-L1 according to any one of claims 1 to 8.

10. A kit comprising the monoclonal antibody or antigen-binding portion thereof of any one of claims 1-8.