CN109593135B

CN109593135B - Anti-human PD-L1 monoclonal antibody and application thereof

Info

Publication number: CN109593135B
Application number: CN201811645073.0A
Authority: CN
Inventors: 杨毅; 杨放; 陆程远; 沈月雷; 倪健; 郭雅南; 陈云云; 谢婧书; 董春艳
Original assignee: Biocytogen Jiangsu Gene Biotechnology Co ltd; Beijing Biocytogen Co Ltd
Current assignee: Baccetus (Beijing) Pharmaceutical Technology Co.,Ltd.; BIOCYTOGEN JIANGSU GENE BIOTECHNOLOGY Co.,Ltd.
Priority date: 2018-12-29
Filing date: 2018-12-29
Publication date: 2021-01-15
Anticipated expiration: 2038-12-29
Also published as: CN109593135A

Abstract

The present invention relates to monoclonal antibodies or antigen-binding portions thereof that specifically bind to human-derived PD-L1. On one hand, the heavy chain variable region and the light chain variable region of the antibody, or the amino acid sequences of the CDR regions thereof, encoding DNA, vectors and cells thereof are provided, and on the other hand, the application of the product is provided, including the evaluation of the function of PD-L1, the screening of drugs, the treatment of diseases in combination with other drugs and the like.

Description

Anti-human PD-L1 monoclonal antibody and application thereof

Technical Field

The invention relates to the field of biological medicine. More particularly, it relates to a monoclonal antibody of anti-human PD-L1 and its application.

Background

Cancer is one of the diseases that currently causes the highest mortality in humans. According to the statistics of the world health organization, the number of cases of morbidity and mortality of malignant tumors in the whole world in 2012 reaches 1400 ten thousand and 820 ten thousand, the number of new cancer cases diagnosed in China is 307 ten thousand, and the number of deaths is 220 ten thousand. In recent years, development of antibody drugs aiming at immune checkpoints is considered to be a potential target for overcoming various cancers.

PD-1 and PD-L1 are called programmed cell death-1 and programmed cell death-ligand 1 respectively, are important members of immunoglobulin superfamily costimulatory molecules and mainly participate in the body immune regulation processes of autoimmunity, tumor immunity and the like. It has been found that PD-1 is a 40kDa type I transmembrane protein, which is mainly expressed in inhibitory receptors on activated T cells, and can significantly inhibit the activation and proliferation of T cells after binding with its ligand PD-L1. Two ligands of PD-1 are known at present, namely PD-L1 (also known as B7-H1) and PD-L2 (also known as B7-DC). The human PD-L1 gene is located in chromosome 9p24, is I-type transmembrane protein of 290 amino acids and consists of an extracellular region IgV, an IgC structural domain, a hydrophobic transmembrane structural domain and an intracellular domain of 30 amino acids. PD-L1 is widely expressed on the surface of a variety of immune, epithelial and tumor cells. While PD-L2 is mainly expressed on the surface of immune cells.

The current research finds that the expression of PD-L1 on the surface of tumor cells becomes a driving factor for the tumor escape immune cells to chase and kill to cause the tumor growth, and the tumor cells are combined with a receptor PD-1 on the surface of T cells through high-expression PD-L1 molecules to transmit negative regulation signals and inhibit the activation and proliferation of tumor antigen specific T cells, thereby avoiding the immune monitoring and killing of organisms. In recent years, monoclonal antibody medicines targeting PD-1/PD-L1 protein are utilized to block the combination of PD-1/PD-L1, so that the activation and proliferation of T cells in vivo are promoted, and the aim of killing tumor cells is fulfilled. Tumor immunotherapy has become one of the worldwide research hotspots and new drug development directions. However, when this treatment is implemented into clinical practice, a number of problems are posed. As clearly suggested in the NCCN guideline, PD-L1 can be detected for the patient with advanced NSCLC lung cancer without the initial diagnosis of clear driver gene mutation, and PD-1 monoclonal antibody can be selected for the initial treatment if the expression of PD-L1 is more than or equal to 50 percent. Therefore, the research on the expression of PD-L1 in the tumor has important scientific significance and clinical value on the pathogenesis and treatment prognosis of the tumor.

Disclosure of Invention

The invention provides a monoclonal antibody or an antigen binding portion thereof that specifically binds to PD-L1, wherein the PD-L1 is human PD-L1.

Preferably, the monoclonal antibody or antigen binding portion thereof comprises a heavy chain variable region and a light chain variable region.

More preferably, the heavy chain variable region comprises CDR1, CDR2 and CDR3, said CDR1-3 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology with the amino acid sequences shown in SEQ ID No.3, SEQ ID No.4 and SEQ ID No.5, respectively; and, the light chain variable region comprises CDR1, CDR2 and CDR3, said CDR1-3 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology with the amino acid sequences shown in SEQ ID No.6, SEQ ID No.7 and SEQ ID No.8, respectively.

Or

The heavy chain variable region comprises CDR1, CDR2 and CDR3, the CDR1-3 having at least 90% of the amino acid sequences shown in SEQ ID No.9, SEQ ID No.10 and SEQ ID No.11, respectively. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology; and the light chain variable region comprises CDR1, CDR2 and CDR3, wherein the CDR1-3 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% homology with the amino acid sequences shown in SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, respectively.

More preferably, the CDR1-3 of the heavy chain variable region is shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, respectively, and the CDR1-3 of the light chain variable region is shown as SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, respectively.

Or the CDR1-3 of the heavy chain variable region is respectively shown as SEQ ID NO.9, SEQ ID NO.10 and SEQ ID NO.11, and the CDR1-3 of the light chain variable region is respectively shown as SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO. 14.

Preferably, the monoclonal antibody or antigen binding portion thereof of the present invention, the heavy chain variable region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology with the amino acid sequence shown in SEQ ID No.1, and the light chain variable region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology with the amino acid sequence shown in SEQ ID No. 2.

More preferably, the heavy chain variable region is represented by SEQ ID NO.1, and the light chain variable region is represented by SEQ ID NO. 2.

Preferably, the monoclonal antibody or antigen binding portion thereof of the invention, wherein the antibody or antigen binding portion thereof is a whole antibody, bispecific antibody, scFv, Fab ', F (ab')₂Or Fv.

Preferably, the monoclonal antibody or antigen-binding portion thereof of the present invention is obtained by a protein immunization method or a DNA immunization method; further, the protein immunization method comprises the steps of immunizing by taking the human PD-L1 protein as an immunogen to obtain an antibody or an antigen binding part thereof; the DNA immunization method comprises the step of immunizing by taking a DNA plasmid coded by the human PD-L1 as an immunogen to obtain an antibody or an antigen binding part thereof.

In another aspect of the present invention, there is provided an isolated DNA encoding the monoclonal antibody or antigen binding portion thereof described above.

In another aspect of the present invention, there is provided a vector comprising the DNA of any one of the above.

In another aspect of the present invention, there is provided a cell comprising the vector described above.

In another aspect of the present invention, there is provided a method for producing the above-mentioned monoclonal antibody or an antigen-binding portion thereof, which comprises producing the monoclonal antibody or the antigen-binding portion thereof by a protein immunization method or a DNA immunization method.

Preferably, the protein immunization method comprises immunizing with human PD-L1 protein as immunogen to obtain an antibody or an antigen-binding part thereof; the DNA immunization method comprises the step of immunizing by taking a DNA plasmid coded by the human PD-L1 as an immunogen to obtain an antibody or an antigen binding part thereof.

In another aspect of the present invention, there is provided a use of the monoclonal antibody or an antigen-binding portion thereof, the isolated DNA, the vector or the cell as described above in connection with PD-L1.

Preferably, the use comprises assessing PD-L1 function, detecting expression of PD-L1, screening for drugs, treating diseases. Preferably, the assay is an immunohistochemical assay.

More preferably, the disease is a disease associated with the activation of T cells, such as a tumor, including malignancies, such as cancer, hematologic malignancies, and the like. In a specific embodiment, the malignant tumor is melanoma, lymphoma, bladder cancer, non-small cell lung cancer, head and neck cancer, colon cancer.

More preferably, the use comprises the use in the preparation of a pharmaceutical composition.

In another aspect of the present invention, there is provided a pharmaceutical composition comprising: the above monoclonal antibody or an antigen-binding portion thereof, the above isolated DNA, the above vector or the above cell; and, optionally, a pharmaceutically acceptable carrier.

Preferably, the pharmaceutically acceptable carrier may be one or more, and includes, but is not limited to, diluents, binders, humectants, surfactants, lubricants, disintegrants, and the like.

Further, the gene-carrying vector of the present invention is various vectors known in the art, such as commercially available vectors, including plasmids, cosmids, phages, viruses, and the like.

More preferably, the pharmaceutical composition may be administered comprised in a nanocarrier, a viral vector, a microcapsule, a liposome, or the like.

Preferably, the pharmaceutical composition is used in combination with other drugs for therapy.

In another aspect of the present invention, there is provided a use of the monoclonal antibody or the antigen-binding portion thereof, the isolated DNA, the vector or the cell in the preparation of a kit.

In another aspect of the present invention, there is provided a kit comprising: the above monoclonal antibody or an antigen-binding portion thereof, the above isolated DNA, the above vector or the above cell.

In another aspect of the invention, the invention provides a method of treating a disease, comprising administering to the subject a therapeutically effective amount of the above-described pharmaceutical composition.

Preferably, the method comprises the step of treating the disease by combining the pharmaceutical composition with other medicines.

Further, the diseases of the present invention include diseases associated with the activation of T cells, such as tumors, including malignant tumors, such as cancers, hematological malignancies, and the like. In a specific embodiment, the malignant tumor is melanoma, lymphoma, bladder cancer, non-small cell lung cancer, head and neck cancer, colon cancer.

More preferably, the present invention provides a method of combined treatment of cancer in a subject comprising administering to the subject a therapeutically effective amount of the above pharmaceutical composition, further comprising administering to the subject a therapeutically effective amount of another agent for treating cancer or administering another method for treating cancer.

The term "treating" (or "treatment") as used herein means slowing, interrupting, arresting, controlling, stopping, alleviating, or reversing the progression or severity of one sign, symptom, disorder, condition, or disease, but does not necessarily refer to the complete elimination of all disease-related signs, symptoms, conditions, or disorders.

The invention has the beneficial effects that: the monoclonal antibody or the antigen binding fragment thereof which is obtained by screening and binds to the human PD-L1 can be specifically bound with the human PD-L1, has high affinity and stable performance, can be used for preparing an immunohistochemical detection tool for specifically detecting the PD-L1 protein, and provides important clinical references for medical research and diagnosis of tumors and selection and prognosis of treatment schemes. In addition, the monoclonal antibody or the antigen binding fragment thereof which is obtained by screening and binds to the human PD-L1 can effectively block the binding between the PD-L1 and the receptor PD-1 thereof, has the blocking effect equivalent to that of a positive control antibody, and can be used for detection, treatment, function evaluation, drug screening and the like related to the PD-L1.

The practice of the present invention will employ, without limitation, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology. These techniques are explained in detail in the following documents. For example: molecular Cloning A Laboratory Manual, 2nd Ed., ed.by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (d.n. glovered., 1985); oligonucleotide Synthesis (m.j. gaited., 1984); transformation And transformation (B.D. Hames & S.J. Higgins.1984); B.Perbal, A Practical Guide To Molecular Cloning (1984); (ii) a Immunochemical Methods In Cell And Molecular Biology (Mayer And Walker, eds., Academic Press, London, 1987); handbook Of Experimental Immunology, Volumes V (d.m.weir and c.c.blackwell, eds., 1986).

The foregoing is merely a summary of aspects of the invention and is not, and should not be taken as, limiting the invention in any way.

All patents and publications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication was specifically and individually indicated to be incorporated herein by reference. Those skilled in the art will recognize that certain changes may be made to the invention without departing from the spirit or scope of the invention. The following examples further illustrate the invention in detail and are not to be construed as limiting the scope of the invention or the particular methods described herein.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1: results of blocking effect of monoclonal antibody on binding of human PD-L1 and Biotin-hPD1 receptor

FIG. 2: expression staining chart for detecting PD-L1 protein in lung cancer tissue by immunohistochemical method

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

In each of the following examples, the primary equipment and materials were obtained from several companies as indicated below:

BALB/c mice were purchased from Experimental animals technology, Inc. of Wei Tongli, Beijing;

NOD-Prkdc^scidIL-2rg^null(B-NDG) mouse-derived Beijing Baiosai atlas Gene Biotechnology Co., Ltd, cat # B-CM-001;

humanized PD-L1 recombinant Protein (Human PD-L1/B7-H1Protein, Fc Tag) was purchased from Peking Baipussels Biotech, Inc., cat # PD 1-H5258;

the mouse two-step method kit (mouse polymer method detection system) is purchased from Beijing China fir Jinqiao biotechnology, Inc., and has a product number of PV-6002;

the citrate repair solution pH6.0 is purchased from Beijing China fir Jinqiao biotechnology, Inc., and has a product number of ZLI-9065;

DAB color developing agent (DAB kit) is purchased from Beijing China fir Jinqiao biotechnology, Inc., and the product number is ZLI-9018;

normal sheep serum (working solution) for sealing was purchased from Beijing Zhonghua Jinqiao biotechnology, Inc., under the product number ZLI-9056;

murine CD3e antibody magnetic beads (CD3 epsilon MicroBead Kit, mouse) from Miltenyi Biotec, cat # 130-094-973;

anti-Mouse IgM antibody magnetic beads (Mouse IgM-isotype control antibodies) come from Miltenyi Biotec with the cargo number of 130-120-156;

hematoxylin (Hematoxylin Solution, Harris Modified) was purchased from Sigma under the trade designation HHS 16;

Anti-PD-L1 antibody [28-8] is derived from Abcam with the cargo number ab205921

EXAMPLE 1 preparation and purification of anti-PD-L1 monoclonal antibody

1 animal immunization

1.1 Pre-immune serum Collection

The mice to be immunized are collected by using EP tube orbital blood collection, and 50-100 mu l of preimmune serum is used as a blank control of a subsequent detection test. The collected serum is placed in an incubator at 37 ℃ for 30min, transferred to 4 ℃ overnight, centrifuged at 4000rmp the next morning for 5min, and the supernatant is transferred to a new EP tube for storage.

1.2 immunization

The Balb/c mice are immunized by a classical immunization method, and the immunogen is human PD-L1 recombinant protein (purchased from Popseuos) or human PD-L1 coding DNA plasmid so that the animals can generate anti-human PD-L1 antibodies.

Protein immunization: BALB/c female mice 6-8 weeks old were taken, mixed with His-tagged human PD-L1 protein in an equal volume with Complete Freund's Adjuvant (CFA), and injected with 20. mu.g (100. mu.g/ml) per subcutaneous injection for the first immunization. After 14 days, a second subcutaneous injection of 20. mu.g (100. mu.g/ml) was administered after mixing the PD-L1 protein with an equal volume of Incomplete Freund's Adjuvant (IFA). A third immunization was performed 14 days later, in the same manner as the second immunization. 3 days after the third immunization, serum was collected by a method of collecting blood from mouse orbit, and the antibody titer was measured by ELISA.

DNA immunization: the 6-8 week-old BALB/c female mice were immunized by intramuscular injection with 60. mu.g (1000. mu.g/. mu.l) per mouse, using the expression plasmid encoding human PD-L1 gene. Mice were immunized at least 4 consecutive times, each time with 14 days intervals. After 7 days of the last immunization, serum was collected by a method of collecting blood from mouse orbit, and antibody titer was measured by ELISA.

The enhanced immunization was carried out 14 days after the last immunization, and CHO cells expressing human PD-L1 protein were added in a volume of 200. mu.l and a volume of 1X 10⁷One cell/mouse was injected into the body by tail vein injection, and 4 days later, spleen of the mouse was collected by carbon dioxide euthanasia for subsequent experiments.

2 cell fusion

After the collected mouse spleen was ground, and spleen cells were separated and purified using mouse CD3e antibody magnetic beads and anti-mouse IgM antibody magnetic beads, the spleen cells were separated from mouse myeloma cells SP2/0 by PEG in a 10: 1 to 5: 1, the fused hybridoma cells were seeded in a 96-well cell culture plate, and cultured and expanded for 7 days in a medium containing hypoxanthine-aminopterin-thymidine (HAT).

3 preliminary screening of hybridoma cells

Hybridoma cells were initially screened by flow cytometry (BD FACS Aria II) using Fluorescence-Activated Cell Sorting (FACS). CHO cells (2X 10) expressing human PD-L1 protein were added to 96-well plates⁴Cell/well), 50. mu.l of hybridoma cell supernatant culture fluid was added to the wells of the corresponding 96-well culture plates, respectively. And (3) performing specific reaction on the goat anti-mouse IgG secondary antibody fragmented by FITC-AffiniPuf F (ab)2 and the Fc gamma fragment, and screening positive hybridoma cells by a flow cytometer.

4 hybridoma cell subcloning

The primary screening positive hybridoma cells are transferred to a 96-well cell culture plate containing a semi-solid culture medium for culturing for 10-14 days (1 cell per well), and a high-expression positive hybridoma cell line is efficiently screened and picked by using a ClonePix2 automatic platform of Molecular Devices and a FITC-labeled goat anti-mouse IgG secondary antibody, and transferred to a 24-well cell culture plate for subsequent culture amplification.

Preparation of antibodies from ascites

Will be 1 × 10⁶The positive hybridoma cells are injected into a severe immunodeficiency B-NDG mouse (Beijing Baioecto Tu Biotechnology limited) in an abdominal cavity for producing an ascites antibody, and the mouse lacks functional T, B and NK cells so as not to express the antibody, so the method is very suitable for preparing ascites of exogenous hybridoma cell expression antibody,for subsequent detection.

6 antibody purification

Positive hybridoma antibodies collected from ascites fluid of mice were purified using GE AKTA protein chromatography full-automatic purifier (GE Healthcare), and the purification procedure and method were found in GE website "antibody purification Manual" (http:// www.gelifesciences.com.cn/CNLS/jsp/Service% 20 &% 20 support/handbook. html).

The PD-L1 antibody 09-8B8 ("8B 8") described herein was prepared and purified as described above. The amino acid sequence of an antibody, including a heavy chain variable region, a light chain variable region, or a CDR region thereof, is shown in tables 1-3:

table 1: heavy chain variable region and light chain variable region amino acid sequences of antibody

Table 2: kabat CDR sequences of antibodies

Table 3: chothia CDR sequences of antibodies

Example 2 detection of blocking Effect of the antibody on the binding of human PD-L1 to Biotin-hPD1 receptor

This experiment was conducted to examine the ability of the anti-hPD-L1 antibody of example 1 to block the binding of human PD-L1 to PD-1 receptor. A96-well cell culture plate was prepared, and 25. mu.l of CHO-hPD-L1 cells (2X 10) expressing human PD-L1 protein were added to each well⁴Cells/well), purified ascites fluid was diluted at 50, 5, 0.5, 0.05, 0.005 μ g/ml, serum was diluted at a final concentration of 1: after 100 dilution, 25. mu.l of the solution was added to each well, and the mixture was left to stand at 4 ℃ for 30 min. After the Biotin-hPD-1 protein (200. mu.g/ml) was diluted to 0.4. mu.g/ml, 50. mu.l was added to each well, and the mixture was allowed to stand at 4 ℃ for 15 min. Centrifuging at 1200rpm for 5min,PBS was washed twice. Mu.l of 1:100anti-mouse IgG Fc-FITC and 1:100 Streptavidin-PE secondary antibody, standing at 4 ℃ for 30 min. After centrifugation at 1200rpm for 5min and one PBS wash, 200. mu.l of PBS was added to each well and the flow analysis was performed.

As can be seen from FIG. 1, the PE-labeled Biotin-hPD-1 fluorescence intensity gradually decreases with the increase of the concentration of the anti-hPD-L1 antibody 09-8B8, indicating that the anti-hPD-L1 antibody 09-8B8 can block the binding of human PD-L1 and PD-1 receptor.

Example 3 antibody specific detection

Using a Protein L chip, PD-L1 antibody 09-8B8 was captured as a ligand by a capture method, recombinant unrelated proteins (human BTLA, CTLA4 and CD28) were set as analytes at different concentrations, specifically 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM, 1.5625nM and 0nM, respectively, and binding detection was performed with the captured PD-L1 antibody 09-8B 8. The generated data were analyzed by Biacore T200Evaluation software3.0 to obtain the binding response values of the PD-L1 antibody 09-8B8 with different concentrations of different proteins, and curves were drawn to confirm the cross-reaction. The results indicate that PD-L1 antibody 09-8B8 did not bind to these unrelated proteins.

Example 4 immunohistochemical experiments

1, respectively sampling 3 different types of human tumor tissues to manufacture tissue chips, and slicing the tissue chips by an external commercial company, wherein the thickness of each slice is 4 micrometers;

2, immunohistochemical staining test of the antibodies of the invention using conventional methods:

a) paraffin wax slices are firstly baked for 2 hours at 58 ℃ for dewaxing, and a series of ethanol is rehydrated. Adding appropriate amount of reagent 1 (endogenous peroxidase blocker) in mouse two-step method kit (mouse polymer method detection system), incubating at room temperature for 10min to eliminate endogenous peroxidase activity, washing with distilled water for 2min × 3 times, and soaking in PBS buffer for 5 min.

b) Antigen retrieval: the tissue sections were placed in 400mL of 0.1 × citrate repair solution (pH6.0), heated in a microwave oven for 4min, and then allowed to cool naturally. And (3) sealing: laminin polyclonal antibody (Santa Cruz Co.) was blocked with normal sheep serum (diluted in PBS) using 10% blocking and incubated at room temperature for 30 min.

c) The antibody of the present invention was used as a primary antibody, and Anti-PD-L1 antibody [28-8] was used as a control antibody, and the serum was decanted, and a primary antibody (1: 100 dilution), 4 ℃ refrigerator overnight.

d) Taking a reagent 2 (enzyme-labeled goat anti-mouse IgG polymer) in a mouse two-step method kit (mouse polymer method detection system) as a secondary antibody, and dropwise adding 1: 200 diluted secondary antibodies (Zhongshan Biotechnology Co.) were incubated at 37 ℃ for 30 min. Washing with PBS buffer for 5min, and repeating washing 3 times.

e) And observing DAB staining condition under a microscope. Hematoxylin counterstaining and flushing with running water. Dehydrating with ethanol, and sealing with neutral gum.

Microscopic examination, 09-8B8 antibody staining results: cell membrane staining is shown on part of tumor tissues, negative no PD-L1 staining is shown on part of samples, wherein lung cancer staining is shown in figure 2, obvious cell membrane staining can be seen, staining mode is correct, signal is strong, and obvious non-specific staining is not seen. The effect is equivalent to that of a control antibody (not shown in the figure), which indicates that the antibody of the invention can be effectively combined with the human PD-L1, the staining effect of the antibody is equivalent to that of a positive control antibody, and the antibody has better specificity.

The results show that the monoclonal antibody or the antigen binding fragment thereof can be specifically combined with human PD-L1, has high affinity and stable performance, can be used for preparing an immunohistochemical detection tool for specifically detecting PD-L1 protein, and can be used for medical research and provide important clinical references for diagnosis of tumors and selection and prognosis of treatment schemes. In addition, the monoclonal antibody or the antigen binding fragment thereof which is obtained by screening and binds to the human PD-L1 can effectively block the binding between the PD-L1 and the receptor PD-1 thereof, has the blocking effect equivalent to that of a positive control antibody, and can be used for detection, treatment, function evaluation, drug screening and the like related to the PD-L1.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.

Sequence listing

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Claims

1. A monoclonal antibody or antigen binding portion thereof that specifically binds PD-L1, wherein PD-L1 is human PD-L1 and wherein the monoclonal antibody or antigen binding portion thereof comprises a heavy chain variable region comprising CDR1, CDR2 and CDR3 and a light chain variable region comprising the amino acid sequences set forth in SEQ ID No.3, SEQ ID No.4 and SEQ ID No.5 for CDR1-3 of the heavy chain variable region; and, the light chain variable region comprises CDR1, CDR2 and CDR3, the Kabat CDR sequences of CDR1-3 of the light chain variable region are the amino acid sequences shown as SEQ ID No.6, SEQ ID No.7 and SEQ ID No.8, respectively;

alternatively, the first and second electrodes may be,

the heavy chain variable region comprises CDR1, CDR2 and CDR3, the Chothia CDR sequences of CDR1-3 of the heavy chain variable region are the amino acid sequences shown in SEQ ID NO.9, SEQ ID NO.10 and SEQ ID NO.11, respectively; and, the light chain variable region comprises CDR1, CDR2 and CDR3, the Chothia CDR sequences of CDR1-3 of the light chain variable region are the amino acid sequences shown in SEQ ID No.12, SEQ ID No.13 and SEQ ID No.14, respectively.

2. The monoclonal antibody, or antigen binding portion thereof, of claim 1, wherein the antibody, or antigen binding portion thereof, is a whole antibody, scFv, Fab ', or F (ab')₂。

3. The monoclonal antibody, or antigen-binding portion thereof, of claim 1, wherein the antibody, or antigen-binding portion thereof, is a bispecific antibody or Fv.

4. An isolated DNA encoding the monoclonal antibody or antigen binding portion thereof of any one of claims 1-3.

5. A vector comprising the DNA of claim 4.

6. A cell comprising the vector of claim 5.

7. Use of the monoclonal antibody or antigen-binding portion thereof that specifically binds PD-L1 of any one of claims 1-3, the DNA of claim 4, the vector of claim 5, or the cell of claim 6 in association with PD-L1, which is not a diagnostic or therapeutic method of disease.

8. The use of claim 7, wherein the use comprises assessing PD-L1 function, detecting PD-L1 expression, screening for a drug.

9. The use of claim 8, wherein the assay is an immunohistochemical assay.

10. Use of the monoclonal antibody or antigen-binding portion thereof that specifically binds PD-L1 of any one of claims 1-3, the DNA of claim 4, the vector of claim 5, or the cell of claim 6 in the preparation of a pharmaceutical composition.

11. A pharmaceutical composition comprising the monoclonal antibody or antigen-binding portion thereof that specifically binds to PD-L1 of any one of claims 1-3, the DNA of claim 4, the vector of claim 5, or the cell of claim 6.

12. Use of the monoclonal antibody or antigen-binding portion thereof that specifically binds PD-L1 of any one of claims 1-3, the DNA of claim 4, the vector of claim 5, or the cell of claim 6 in the preparation of a kit.

13. A kit comprising the monoclonal antibody or antigen-binding portion thereof that specifically binds to PD-L1 of any one of claims 1-3, the DNA of claim 4, the vector of claim 5, or the cell of claim 6.