Humanized anti-PDL1 antibody and application thereof

Abstract

The invention relates to a humanized anti-PDL1 antibody and an application thereof, and provides the humanized anti-PDL1 antibody and an antigen binding part thereof, and a preparation method and an application of the humanized anti-PDL1 antibody and the antigen binding part thereof. The humanized anti-PDL1 antibody has high affinity with human recombinant PDL1, can effectively stimulate human T cells to generate IL2 and IFN-gamma, and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a humanized anti-PDL1 antibody and an application thereof. Background Art

[0002] The human immune system maintains a dynamic equilibrium state through immune activation and immunosuppressive receptors. Taking T cells as an example, immunosuppression is mainly achieved through immunosuppressive receptors on their surface, including two drug targets that have been used in clinical practice, CTLA-4 and PD1. Programmed death receptor 1 (PD1) can be induced to express in a variety of immune cells including T cells, B cells, dendritic cells and monocytes. Its ligand PDL1 (programmed death ligand 1, PDL1, also known as CD274, B7 homolog, B7-H1) is widely distributed, such as T / B cells, vascular endothelial cells, various mesenchymal cells, hepatocytes, pancreatic islet cells, glial cells, placental cells, and various tumor cells, indicating that it plays an important role in the body's immune regulation. After PD1 binds to PDL1, it will induce T cell kinetic exhaustion and apoptosis. Studies have shown that it is involved in immune rejection, autoimmune diseases, atherosclerosis, tumors and other diseases.

[0003] In recent years, the role of PD1 / PDL1 in tumors has attracted much attention. It has been reported that PDL1 is highly expressed in a variety of tumor cells, such as cervical cancer, pancreatic cancer, urothelial cancer, gastric cancer, esophageal cancer, non-small cell lung cancer, melanoma, glioma, head and neck cell cancer, leukemia, breast cancer, ovarian cancer, bladder cancer, multiple myeloma, etc. In recent years, a variety of drugs targeting PDL1 have been studied and developed, the most important of which include anti-PDL1 antibodies.

[0004] In antibody development, mouse monoclonal antibodies can cause human anti-mouse antibody (HAMA) reactions during clinical treatment, thus limiting their clinical use. The immunogenicity of mouse monoclonal antibodies can be greatly reduced by antibody humanization. Therefore, there is still a need in the art to develop anti-PDL1 humanized antibodies suitable for treating patients. Summary of the invention

[0005] The purpose of the present invention is to provide a humanized anti-PDL1 antibody and its application, which can effectively bind to cancer cells, has a high affinity with human recombinant PD1, and can effectively activate the expression of IL2 and IFN-γ in human T cells, and has good application prospects.

[0006] To this end, in a first aspect, the present invention provides a humanized anti-PDL1 antibody or an antigen-binding portion thereof, wherein the humanized anti-PDL1 antibody comprises a light chain variable region comprising LCDR1, LCDR2 and LCDR3 amino acid sequences and a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3;

[0007] Among them, the amino acid sequence of the LCDR1 is SEQ ID NO: 1, the amino acid sequence of the LCDR2 is SEQ ID NO: 2, the amino acid sequence of the LCDR3 is SEQ ID NO: 3, the amino acid sequence of the HCDR1 is SEQ ID NO: 4, the amino acid sequence of HCDR2 is SEQ ID NO: 5 or SEQ ID NO: 6, and the amino acid sequence of HCDR3 is SEQ ID NO: 7.

[0008] RASQSLSHYLH (SEQ ID NO: 1).

[0009] YASQSIS (SEQ ID NO: 2).

[0010] QNGHSFPPT (SEQ ID NO: 3).

[0011] EYTLH (SEQ ID NO: 4).

[0012] GIHPNYGDSTYTQKFKG (SEQ ID NO: 5).

[0013] GIHPNYGDTTYTQKFKG (SEQ ID NO: 6).

[0014] GHGFAHYVMDY (SEQ ID NO: 7).

[0015] Furthermore, the light chain variable region includes light chain framework regions FL1, FL2, FL3 and FL4, and the light chain framework regions FL1, FL2, FL3 and FL4 have the amino acid sequences shown in SEQ ID NOs: 8, 9, 10 and 11, respectively.

[0016] DIVMTQSPATLSX 1 SPGERX 2 TLSC (SEQ ID NO: 8);

[0017] Among them, X 1 Selected from L or V; X 2 Selected from A or V;

[0018] WlqX 3 X 4 EX 5PRLLIK (SEQ ID NO: 9);

[0019] Among them, X 3 Choose between P or S; X 4 Selected from G or R; X 5 Select from A or S;

[0020] GIPARFSGSGPGX 6 DFTLTISSX 7 EPEDFX 8 VYYC (SEQ ID NO: 10);

[0021] Among them, X 6 Select from T or S; X 7 Selected from L or V; X 8 Select from A or G;

[0022] FGQGTKLEL (SEQ ID NO: 11).

[0023] Furthermore, the heavy chain variable region includes heavy chain framework regions FL1, FL2, FL3 and FL4, and the heavy chain framework regions FL1, FL2, FL3 and FL4 have the amino acid sequences shown in SEQ ID NOs: 12, 13, 14 and 15, respectively.

[0024] EVQLVQSGAEVKKPGASVKVSCKTSGYSFT(SEQ ID NO:12);

[0025] WX 9 QAPGQGLEWIG (SEQ ID NO: 13);

[0026] Among them, X 9 Selected from R or K;

[0027] RVTLTVDX 10 STSTAYMELSSLRSEDTAVYYCAR(SEQ ID NO:14);

[0028] Among them, X 10 Select from T or K;

[0029] WGQGTLVTVSS (SEQ ID NO: 15).

[0030] Furthermore, the amino acid sequence of the light chain variable region is shown in any one of the following groups: SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20.

[0031] DIVMTQSPATLSVIPGDRVSLSCRASQSLSHYLHWYQKKSRESPRLLIKYASQSISGIPSR FSGRGSGSDFTLTINSVEPEDVGVYYCQNGHSFPPTFGAGTKLEL (SEQ ID NO: 16).

[0032] DIVMTQSPATLSLSPGERATLSCRASQSLSHYLHWYQKKPGEAPRLLIKYASQSISGIPA RFSGSGPGTDFTLTISSLEPEDFAVYYCQNGHSFPPTFGQGTKLEL (SEQ ID NO: 17).

[0033] DIVMTQSPATLSLSPGERATLSCRASQSLSHYLHWYQKKPGEAPRLLIKYASQSISGIPA RFSGSGPGTDFTLTISSVEPEDFAVYYCQNGHSFPPTFGQGTKLEL (SEQ ID NO: 18).

[0034] DIVMTQSPATLSVSPGERVTLSCRASQSLSHYLHWYQKKSGESPRLLIKYASQSISGIPA RFSGSGPGSDFTLTISSVEPEDFGVYYCQNGHSFPPTFGQGTKLEL (SEQ ID NO: 19).

[0035] DIVMTQSPATLSVSPGERVTLSCRASQSLSHYLHWYQKKSRESPRLLIKYASQSISGIPA RFSGSGPGSDFTLTISSVEPEDFGVYYCQNGHSFPPTFGQGTKLEL (SEQ ID NO: 20).

[0036] Furthermore, the amino acid sequence of the heavy chain variable region is shown as any one of the following groups: SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23.

[0037] EVQLQQSGPELVKPGASVKVSCKTSGYSFTEYTLHWVKQSHGKSLEWIGGIHPNYGD STYTQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARGHGFAHYVMDYWGQGTSVT VSS (SEQ ID NO: 21).

[0038] EVQLVQSGAEVKKPGASVKVSCKTSGYSFTEYTLHWVRQAPGQGLEWIGGIHPNYGD TTYTQKFKGRVTLTVDTSTSTAYMELSSLRSEDTAVYYCARGHGFAHYVMDYWGQGTLVT VSS (SEQ ID NO: 22).

[0039] EVQLVQSGAEVKKPGASVKVSCKTSGYSFTEYTLHWVKQAPGQGLEWIGGIHPNYGD TTYTQKFKGRVTLTVDKSTSTAYMELSSLRSEDTAVYYCARGHGFAHYVMDYWGQGTLVT VSS (SEQ ID NO: 23).

[0040] In one embodiment, the amino acid sequence of the light chain variable region is SEQ ID NO: 16, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 21.

[0041] In one embodiment, the amino acid sequence of the light chain variable region is SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 22 or SEQ ID NO: 23.

[0042] Furthermore, the humanized anti-PDL1 antibody also includes a human antibody light chain constant region.

[0043] In a certain embodiment, the human antibody light chain constant region is a light chain constant region of the human IgG1 subtype.

[0044] In a certain embodiment, the amino acid sequence of the light chain constant region of the human IgG1 subtype comprises SEQ ID NO:24.

[0045] Furthermore, the humanized anti-PDL1 antibody also includes a human antibody heavy chain constant region.

[0046] In one embodiment, the human antibody heavy chain constant region is a heavy chain constant region of the human IgG1 subtype.

[0047] In one embodiment, the amino acid sequence of the heavy chain constant region of the human IgG1 subtype comprises SEQ ID NO:25.

[0048] In some embodiments, the humanized anti-PDL1 antibody comprises a light chain constant region and a heavy chain constant region of human IgG1 subtype, the amino acid sequence of the light chain constant region of human IgG1 subtype comprises SEQ ID NO: 24; the amino acid sequence of the heavy chain constant region of human IgG1 subtype comprises SEQ ID NO: 25.

[0049] KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 24).

[0050] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 25).

[0051] The second aspect of the present invention provides a nucleic acid molecule encoding the humanized anti-PDL1 antibody or the antigen-binding portion thereof of the present invention.

[0052] The third aspect of the present invention provides a vector comprising the nucleic acid molecule of the present invention.

[0053] The fourth aspect of the present invention provides a host cell, which contains the nucleic acid molecule of the present invention, or contains the vector of the present invention.

[0054] The fifth aspect of the present invention provides a pharmaceutical composition comprising the humanized anti-PDL1 antibody or its antigen-binding portion described in the present invention and a pharmaceutically acceptable carrier; wherein the humanized anti-PDL1 antibody or its antigen-binding portion described in the present invention is present in a therapeutically effective dose.

[0055] In the sixth aspect of the present invention, there is provided a method for preparing the humanized anti-PDL1 antibody or its antigen binding portion described in the present invention, comprising the following steps: expressing the humanized anti-PDL1 antibody or its antigen binding portion described in the present invention in the host cell described in the fourth aspect of the present invention; and isolating the humanized anti-PDL1 antibody or its antigen binding portion from the host cell.

[0056] The seventh aspect of the present invention provides use of the humanized anti-PDL1 antibody or antigen-binding portion thereof of the present invention in the preparation of a medicament for preventing, alleviating or treating cancer:

[0057] In some embodiments, the cancer is selected from liver cancer, cervical cancer, pancreatic cancer, urothelial cancer, gastric cancer, esophageal cancer, non-small cell lung cancer, small cell lung cancer, bile duct cancer, prostate cancer, hematological tumors, osteosarcoma, melanoma, glioma such as brain glioma, head and neck cell cancer, leukemia, breast cancer, ovarian cancer, bladder cancer, multiple myeloma.

[0058] The eighth aspect of the present invention provides a use, wherein the humanized anti-PDL1 antibody or the antigen-binding portion thereof described in the present invention is administered in combination with a chemotherapeutic agent, radiotherapy and / or other agents used for cancer immunotherapy.

[0059] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0060] The present invention provides a humanized anti-PDL1 antibody or an antigen-binding portion thereof, which significantly reduces the immunogenicity of mouse monoclonal antibodies and is suitable for clinical treatment of human patients. The humanized anti-PDL1 antibody provided by the present invention has excellent binding ability to human PDL1 high-expressing stably transfected CHO cell lines and human ovarian cancer cell lines ES-2. The antibody has a high affinity for human recombinant PDL1 and can effectively stimulate human T cells to produce IL2 and IFN-γ, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the following detailed description of the preferred embodiment.The drawings are only for the purpose of illustrating the preferred embodiments and are not to be construed as limiting the invention.

[0062] In the attached picture:

[0063] Figure 1 : Flow cytometry results of the binding ability of the antibody provided by the present invention to the human PDL1 high-expressing stably transfected CHO cell line;

[0064] Figure 2 : Flow cytometry results of the binding ability of the antibody provided by the present invention to the human ovarian cancer cell line ES-2;

[0065] Figure 3 : Flow cytometry results of competitive binding ability of the antibody provided by the present invention and human recombinant PD1 protein on PDL1-CHO cell line;

[0066] Figure 4 : The detection result of IL2 expression of human T cells under the action of the antibody provided by the present invention;

[0067] Figure 5 : The detection results of the IFN-γ expression level of human T cells under the action of the antibody provided by the present invention. DETAILED DESCRIPTION

[0068] The exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although the exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art.

[0069] Unless otherwise indicated, the examples herein employ conventional techniques of molecular biology, microbiology, cell biology, biochemistry, and immunology within the skill of the art.

[0070] Unless otherwise specified, the terms used in this application have the meanings commonly understood by those skilled in the art.

[0071] As used herein, the term "humanized antibody" refers to an antibody obtained by grafting CDR sequences derived from another mammalian species other than human (eg, mouse) onto human framework sequences. Other framework region modifications may be made within the human framework sequences.

[0072] As used herein, the term "antigen binding portion" refers to an antibody fragment formed by a portion of an antibody comprising one or more CDRs. Examples of antigen binding portions include, but are not limited to, variable regions, Fab, Fab", F(ab") 2 , Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv) 2 , multispecific antibodies, nanobodies. The antigen binding module can bind to the same antigen as the parent antibody. In some embodiments, the antigen binding portion may comprise one or more CDRs grafted onto the framework region from one or more different human antibodies.

[0073] As used herein, the term "vector" refers to a nucleic acid vector into which a polynucleotide can be inserted. When a vector allows the expression of a protein encoded by a polynucleotide inserted therein, the vector is referred to as an expression vector. The vector can be transformed, transduced or transfected into a host cell to express the genetic material elements carried in the host cell. Vectors are well known to those skilled in the art, including but not limited to plasmids, bacteriophages, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40). The vector may contain multiple elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may contain an origin of replication.

[0074] As used herein, the term "host cell" refers to a cell into which exogenous nucleic acid has been introduced, including the offspring of such cells. Host cells include cells of initial transformation and offspring derived therefrom (regardless of the number of generations). Offspring may not be identical to parental cells in nucleic acid content, but may contain mutations. Mutant offspring with the same function or biological activity as screened or selected in the original transformed cells are included herein. Host cells are any type of cell systems that can be used to generate fusion proteins of the present invention. Host cells include mammalian cultured cells such as CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, bacterial cells such as Escherichia coli, insect cells and plant cells, etc., and are also included in transgenic animals, transgenic plants or cells contained in cultivated plants or animal tissues.

[0075] As used herein, the term "cancer" refers to any neoplastic or malignant cell growth, proliferation, or metastasis-mediated solid and non-solid tumors that cause a medical condition.

[0076] As used herein, the term "pharmaceutical composition" refers to a preparation whose form permits the biological activity of the active ingredient (humanized anti-PDL1 antibody or antigen-binding portion thereof provided by the present invention) contained therein to be effective, and does not contain other ingredients that are unacceptably toxic to a subject to which the pharmaceutical composition is administered.

[0077] As used herein, the term "therapeutically effective dose" refers to the amount or concentration of the humanized anti-PDL1 antibody or the antigen-binding portion thereof that can produce a beneficial therapeutic effect on the corresponding disease.

[0078] It refers to an amount effective to achieve the desired therapeutic or prophylactic result including, for example, eliminating, reducing, delaying, minimizing or preventing adverse effects of a disease.

[0079] As used herein, the term "pharmaceutically acceptable carrier" refers to a component other than the active ingredient in a pharmaceutical composition that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0080] As used herein, the term "subject" is a mammal. Mammals include, but are not limited to, primates (e.g., humans and non-human primates such as monkeys) or other mammals (e.g., cattle, sheep, cats, dogs, horses, rabbits, and rodents such as mice and rats). In particular, the individual or subject is a human.

[0081] In some embodiments of the present invention, the amino acid sequence of the light chain variable region of the humanized anti-PDL1 antibody provided includes SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20; the amino acid sequence of the heavy chain variable region of the humanized anti-PDL1 antibody provided includes SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, as shown in Table 1.

[0082] Table 1

[0083]

[0084] In some embodiments of the present invention, chimeric antibody HEC41115 and humanized antibodies HEC41116, HEC41117, HEC41118, HEC41119, HEC41120, HEC41121, HEC41122, and HEC41123 are provided, as shown in Table 2.

[0085] Table 2

[0086] Antibody Name Heavy chain variable region sequence number Light chain variable region sequence number HEC41115 SEQ ID NO: 21 SEQ ID NO: 16 HEC41116 SEQ ID NO: 22 SEQ ID NO: 17 HEC41117 SEQ ID NO: 23 SEQ ID NO: 17 HEC41118 SEQ ID NO: 22 SEQ ID NO: 18 HEC41119 SEQ ID NO: 23 SEQ ID NO: 18 HEC41120 SEQ ID NO: 22 SEQ ID NO: 19 HEC41121 SEQ ID NO: 23 SEQ ID NO: 19 HEC41122 SEQ ID NO: 22 SEQ ID NO: 20 HEC41123 SEQ ID NO: 23 SEQ ID NO: 20

[0087] The CDR regions (complementarity determining regions) of antibody HEC41115 are shown in Table 3.

[0088] Table 3

[0089]

[0090] The CDR regions of antibodies HEC41116, HEC41117, HEC41118, HEC41119, HEC41120, HEC41121, HEC41122, and HEC41123 are shown in Table 4.

[0091] Table 4

[0092]

[0093]

[0094] Example 1 Preparation of humanized antibodies

[0095] The human recombinant protein PDL1 (UniProtKB-Q9NZQ7) and complete Freund's adjuvant were mixed into an emulsion and dispersed and injected subcutaneously into Balb / c mice. After three immunizations, the binding titer of the mouse serum to the human recombinant protein PDL1 was determined by ELISA. When the titer reached the standard, the mouse spleen was obtained, and the mouse spleen was fully ground to obtain a single cell suspension, which was fused with myeloma cells by PEG to form hybridoma cells. After culturing the hybridoma cells for 14 days, the supernatant was taken and tested by ELISA to screen ELISA-positive cells that met the standards. After the ELISA-positive cells were further cultured, the supernatant was taken, and the FACs method was used to detect the binding of the antibody in the supernatant to the stable CHO cell line (CHOK1-GS knockout cell line) with high expression of PDL1. The cells that met the standards were subcloned, and the mRNA of the cells was obtained after harvesting the cells, and reverse transcribed into cDNA. The antibody sequence in the hybridoma cell line was obtained and sequenced using commonly used antibody fishing sequence primers. At the same time, the subcloned cells were cultured at 1×10 6 The number of cells was inoculated into the peritoneal cavity of mice, and the ascites in the peritoneal cavity was collected 8 hours later. The mouse monoclonal antibody with a purity of more than 90% was obtained through purification with Protein A medium, and then screened, tested and evaluated. The mouse monoclonal antibody obtained by screening was named HEC401, which has the potential to be a drug for clinical prevention of hematological tumors, solid tumors, viral infections, bacterial infections and improving immunity.

[0096] The HEC401 variable region sequence (SEQ ID NO: 16, SEQ ID NO: 21) and its humanized variable region mutant sequence (SEQ ID NO: 17-20, SEQ ID NO: 22-23) were assembled with the human antibody constant region sequence (the light chain constant region sequence was SEQ ID NO: 24, and the heavy chain constant region sequence was SEQ ID NO: 25) by whole gene synthesis, constructed into the pcDNA3.1 vector, transiently transfected into HEK293 cells, and purified and prepared by Protein A medium to obtain chimeric antibody HEC41115 with a purity of more than 95% and humanized antibodies HEC41116, HEC41117, HEC41118, HEC41119, HEC41120, HEC41121, HEC41122, and HEC41123, and their expression levels in HEK293 cells were detected, as shown in Table 5.

[0097] Table 5

[0098]

[0099]

[0100] According to the expression level detection results, the humanized antibodies provided by the present invention can be effectively expressed in mammalian cells.

[0101] Example 2 Binding of humanized antibodies to PDL1 high-expressing stably transfected CHO cell lines

[0102] In this example, flow cytometry was used to detect the binding ability of the antibodies prepared in Example 1 to the human PDL1 high-expressing stable CHO cell line (CHOK1-GS knockout cell line). The antibodies HEC41115, HEC41116, HEC41117, HEC41118, HEC41119, HEC41120, HEC41121, HEC41122, and HEC41123 prepared in Example 1 (50 μg / ml antibody was taken and diluted 5 times each time to obtain eight concentrations), and each antibody was incubated with the human PDL1 high-expressing stable CHO cell line (cell dosage: 5×10 5cells; incubation conditions: incubated in PBS solution at 37°C for 1 h), followed by binding with FITC-labeled goat anti-human IgG Fc secondary antibody (Jackson), using Isotype IgG antibody as a negative control, and using the following anti-PDL1 antibodies as controls: Avelumab (Merck / Pfizer), BMS-936559 (BMS), Durvalumab (AstraZeneca), Atezolizumab (Roche). The test results are shown in Figure 1 shown.

[0103] The results showed that the humanized antibody provided by the present invention can effectively bind to the human PDL1 membrane-expressed protein, and its binding ability has no significant change compared with that before humanization.

[0104] Example 3 Binding of humanized antibodies to human ovarian cancer cell line ES-2 cells

[0105] In this example, flow cytometry was used to detect the binding ability of the antibodies prepared in Example 1 to the human ovarian cancer cell line ES-2. The antibodies HEC41115, HEC41116, HEC41117, HEC41118, HEC41119, HEC41120, HEC41121, HEC41122, and HEC41123 prepared in Example 1 (2 μg / ml antibody, diluted 2-fold each time to obtain eight concentrations) were incubated with the human ovarian cancer cell line ES-2 (cell dosage: 2.5×10 5 cells; incubation conditions: incubated in PBS solution at 37°C for 1 h), followed by binding with FITC-labeled goat anti-human IgG Fc secondary antibody (Jackson), using Isotype IgG antibody as a negative control, and using the following anti-PDL1 antibodies as controls: Avelumab (Merck / Pfizer), BMS-936559 (BMS), Durvalumab (AstraZeneca), Atezolizumab (Roche). The test results are shown in Figure 2 shown.

[0106] The results showed that the humanized antibody provided by the present invention can effectively bind to the PDL1 protein endogenously expressed in the human tumor cell line ES2, and its binding ability has no significant change compared with that before humanization.

[0107] Example 4 Humanized antibodies compete with human recombinant PD1 protein for binding to PDL1

[0108] In this example, flow cytometry was used to detect the competitive binding ability of the antibodies prepared in Example 1 and the human recombinant PD1 protein on the PDL1-CHO cell line. The antibodies HEC41115, HEC41116, HEC41117, HEC41118, HEC41119, HEC41120, HEC41121, HEC41122, and HEC41123 prepared in Example 1 (30 μg / ml antibody was taken and diluted 3 times each time to obtain eight concentrations) were mixed with 1 μg / ml human recombinant PD1 protein and 5×10 5 cells PDL1-CHO cell line was co-incubated (incubation conditions: 37°C in PBS solution for 1 h), followed by binding with FITC-labeled goat anti-human IgG Fc secondary antibody (Jackson), using Isotype IgG antibody as a negative control, and the following anti-PDL1 antibodies as controls: Avelumab (Merck / Pfizer), BMS-936559 (BMS), Durvalumab (AstraZeneca), Atezolizumab (Roche). The test results are shown in Figure 3 shown.

[0109] The results showed that the humanized antibody provided by the present invention can effectively block the binding of PD1 protein to PDL1 protein, and its binding ability has no significant change compared with that before humanization.

[0110] Example 5: Detection of affinity between humanized antibodies and human PDL1 protein using BLI method

[0111] This example uses bio-layer interferometry (BLI) to determine the affinity of the antibody provided by the present invention to the human recombinant PDL1 protein. Prepare the following antibody solutions, experimental group: antibodies HEC41115, HEC41116, HEC41117, HEC41118, HEC41119, HEC41120, HEC41121, HEC41122, HEC41123 prepared in Example 1 (take 15 μg / ml antibody, dilute with SD-Buffer at 2 times each dilution to obtain six concentrations; SD-Buffer system: 0.1% BSA, 0.02% Tween 20, 20mM PBS solution); negative control group: Isotype IgG antibody; control group: avelumab (Merck / Pfizer), BMS-936559 (BMS), Durvalumab (AstraZeneca), Atezolizumab (Roche). For each antibody to be tested, follow these steps:

[0112] The probe was placed in SD-buffer solution for 180 seconds; the probe was placed in PDL1 protein solution for loading for 600 seconds; the probe was placed in SD-buffer solution for 180 seconds; the probe was placed in antibody solutions of different concentrations for 360 seconds; the probe was placed in SD-buffer solution for 180 seconds; after completion, the probe was placed in regeneration solution to complete the regeneration and preservation of the probe. The data was sorted and analyzed using the instrument's own data processing software to obtain KD\Kon\Kdis(Koff).

[0113] The affinity of each antibody to the human recombinant protein PDL1 was measured using the above-mentioned BLI detection technology as shown in Table 6.

[0114] Table 6

[0115] Sample ID KD(M) kon(1 / Ms) kdis(1 / s) HEC41115 2.57E-10 2.07E+05 5.32E-05 HEC41117 1.16E-11 2.83E+05 3.28E-06 HEC41118 5.78E-10 1.12E+05 6.49E-05 HEC41119 8.97E-12 2.78E+05 2.50E-06 HEC41120 1.31E-11 3.13E+05 4.08E-06 HEC41121 1.34E-11 2.57E+05 3.45E-06 HEC41122 1.83E-11 2.79E+05 5.11E-06 HEC41123 7.25E-10 2.06E+05 1.49E-04 Avelumab 7.05E-11 1.50E+05 1.06E-05 BMS936559 4.02E-10 6.16E+05 2.47E-04 Durvalumab 2.92E-10 3.75E+05 1-10E-04 Atezolizumab 3.00E-10 1.28E+05 3.82E-05 Isotype lgG —— —— ——

[0116] The results showed that the affinity of the humanized antibody provided by the present invention to the human recombinant protein PDL1 did not change significantly compared with that before humanization.

[0117] Example 6 Mixed lymphocyte reaction assay to detect the IL2 / IFN-γ activation effect of antibodies on the system

[0118] This example detects the activation effect of the antibodies provided by the present invention on IL2 and IFN-γ, including the following steps: using a kit (EasySep Human CD4+T Cell Enrichment Kit) to separate human monocytes, and obtaining mDC (mature dendritic cells) after treatment; then incubating the mDC with the antibody, and then adding allogeneically separated human T cells, and detecting the expression levels of IL2 and IFN-γ in the supernatant three days later. Isotype IgG antibody was used as a negative control, and the following anti-PDL1 antibodies were used as controls: Avelumab (Merck / Pfizer), BMS-936559 (BMS), Durvalumab (AstraZeneca), Atezolizumab (Roche). The detection results of IL2 expression are shown in the figure. Figure 4 The results of IFN-γ expression were shown in Figure 5 shown.

[0119] The results showed that the humanized antibodies provided by the present invention can effectively stimulate T cells to produce IL2 and IFN-γ, and no significant changes occurred compared with before humanization.

[0120] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A humanized anti-PDL1 antibody or an antigen-binding portion thereof, characterized in that: The humanized anti-PDL1 antibody comprises a light chain variable region comprising LCDR1, LCDR2 and LCDR3 and a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3; Among them, the amino acid sequence of the LCDR1 is SEQ ID NO: 1, the amino acid sequence of the LCDR2 is SEQ ID NO: 2, the amino acid sequence of the LCDR3 is SEQ ID NO: 3, the amino acid sequence of the HCDR1 is SEQ ID NO: 4, the amino acid sequence of HCDR2 is SEQ ID NO: 5 or SEQ ID NO: 6, and the amino acid sequence of HCDR3 is SEQ ID NO:

7.

2. The humanized anti-PDL1 antibody or antigen-binding portion thereof according to claim 1, wherein The light chain variable region includes light chain framework regions FL1, FL2, FL3 and FL4, and the light chain framework regions FL1, FL2, FL3 and FL4 have the amino acid sequences shown in SEQ ID NOs: 8, 9, 10 and 11, respectively; DIVMTQSPATLSX 1 SPGERX 2 TLSC(SEQ ID NO:8); Among them, X 1 Select from L or V; X 2 Selected from A or V; WYQKKX 3 X 4 EX 5 PRLLIK(SEQ ID NO:9); Among them, X 3 Choose between P or S; X 4 Selected from G or R; X 5 Select from A or S; GIPARFSGSGPGX 6 DFTLTISSX 7 EPEDFX 8 VYYC(SEQ ID NO:10); Among them, X 6 Select from T or S; X 7 Select from L or V; X 8 Select from A or G; FGQGTKLEL (SEQ ID NO: 11).

3. The humanized anti-PDL1 antibody or antigen-binding portion thereof according to claim 1, wherein The heavy chain variable region includes heavy chain framework regions FL1, FL2, FL3 and FL4, and the heavy chain framework regions FL1, FL2, FL3 and FL4 have the amino acid sequences shown in SEQ ID NOs: 12, 13, 14 and 15, respectively; EVQLVQSGAEVKKPGASVKVSCKTSGYSFT(SEQ ID NO:12); WVX 9 QAPGQGLEWIG(SEQ ID NO:13); Among them, X 9 Selected from R or K; RVTLTVDX 10 STSTAYMELSSLRSEDTAVYYCAR(SEQ ID NO:14); Among them, X 10 Select from T or K; WGQGTLVTVSS (SEQ ID NO: 15).

4. The humanized anti-PDL1 antibody or antigen-binding portion thereof according to claim 1, wherein The amino acid sequence of the light chain variable region is shown in any one of the following groups: SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO:

20.

5. The humanized anti-PDL1 antibody or antigen-binding portion thereof according to claim 1, wherein: The amino acid sequence of the heavy chain variable region is shown in any one of the following groups: SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO:

23.

6. The humanized anti-PDL1 antibody or antigen-binding portion thereof according to claim 1, wherein: The humanized anti-PDL1 antibody further comprises a human antibody light chain constant region; Preferably, the human antibody light chain constant region is a light chain constant region of human IgG1 subtype; Preferably, the amino acid sequence of the light chain constant region of the human IgG1 subtype includes SEQ ID NO:

24.

7. The humanized anti-PDL1 antibody or antigen-binding portion thereof according to claim 1, wherein: The humanized anti-PDL1 antibody further comprises a human antibody heavy chain constant region; Preferably, the human antibody heavy chain constant region is a heavy chain constant region of human IgG1 subtype; Preferably, the amino acid sequence of the heavy chain constant region of human IgG1 subtype includes SEQ ID NO:

25.

8. A biological material, which is any one of the following (i), (ii), (iii): (i) a nucleic acid molecule encoding the humanized anti-PDL1 antibody or antigen-binding portion thereof according to any one of claims 1 to 7; (ii) a vector comprising the nucleic acid molecule described in (i); (iii) A host cell comprising the nucleic acid molecule described in (i), or the vector described in (ii).

9. A pharmaceutical composition comprising the humanized anti-PDL1 antibody or its antigen-binding portion according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier; wherein the humanized anti-PDL1 antibody or its antigen-binding portion is present in a therapeutically effective dose.

10. A method for preparing the humanized anti-PDL1 antibody or the antigen-binding portion thereof according to any one of claims 1 to 7, comprising the following steps: expressing the humanized anti-PDL1 antibody or the antigen binding portion thereof in a host cell of one of the biological materials of claim 8; and isolating the humanized anti-PDL1 antibody or the antigen binding portion thereof from the host cell.

11. Use of the humanized anti-PDL1 antibody or the antigen-binding portion thereof according to any one of claims 1 to 7 in the preparation of a medicament for preventing, alleviating or treating cancer; Preferably, the cancer is selected from liver cancer, cervical cancer, pancreatic cancer, urothelial cancer, gastric cancer, esophageal cancer, non-small cell lung cancer, small cell lung cancer, bile duct cancer, prostate cancer, hematological tumors, osteosarcoma, melanoma, glioma such as brain glioma, head and neck cell carcinoma, leukemia, breast cancer, ovarian cancer, bladder cancer, multiple myeloma.

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