CN113087796B - anti-PD-L1 antibody and application thereof - Google Patents

Abstract

Translated fromChinese

本发明提供了一种抗PD‑L1抗体或其抗原结合片段及其制备方法和应用。本发明还提供了编码抗PD‑L1抗体或其抗原结合片段的核酸、包含核酸的载体、包含载体的细胞，以及PD‑L1的检测方法。本发明所述的抗PD‑L1抗体或其抗原结合片段具有特异性强、亲和力高优势，可以用于与PD‑L1表达相关的疾病的治疗以及PD‑L1表达水平的检测。

The present invention provides an anti-PD-L1 antibody or an antigen-binding fragment thereof, and a preparation method and application thereof. The present invention also provides a nucleic acid encoding an anti-PD-L1 antibody or an antigen-binding fragment thereof, a vector comprising the nucleic acid, a cell comprising the vector, and a method for detecting PD-L1. The anti-PD-L1 antibody or its antigen-binding fragment of the present invention has the advantages of strong specificity and high affinity, and can be used for the treatment of diseases related to PD-L1 expression and the detection of PD-L1 expression level.

Description

anti-PD-L1 antibody and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to an anti-PD-L1 antibody or an antigen binding fragment thereof and application thereof in detecting PD-L1 or preparing products for treating diseases related to PD-L1 expression.

Background

Programmed cell death-ligand 1 (PD-L1), also known as cluster of differentiation 274 (CD 274) or B7 homolog (B7 homolog 1, B7-H1), is a single transmembrane protein of 40kDa in size. PD-L1 is mainly expressed in T cells, B cells, dendritic cells, macrophages and the like. The study shows that PD-L1 has higher expression in tumor tissues. PD-L1 is one of the major ligands of the programmed cell death receptor-1 (PD-1). PD-1 is mainly expressed in T cells, and PD-L1 expressed on the surface of tumor cells can inhibit the anti-tumor immune response of local T cells after being combined with PD-1, so that the tumor cells can escape from the immune surveillance of NK cells or T cells. Meanwhile, the combination of PD-1/PD-L1 can reduce the hyperplasia of lymph node CD8+ T cells and inhibit the secretion of cytokines and cytotoxic activity. Preclinical studies show that blocking the interaction between PD-1/PD-L1 increases the number of effector T cells, enhances the cytotoxic activity of tumor-specific T cells, promotes the production of pro-inflammatory cytokines, and reduces the number of tregs at tumor sites. In recent years, targeted immunotherapy which blocks PD-1/PD-L1 has become one of the important directions for treating tumors. Therefore, the development of antibody drugs with strong specificity and high affinity provides possibility for the treatment of various cancers, and has huge application potential and market value.

Disclosure of Invention

The invention provides an anti-PD-L1 antibody or an antigen binding fragment thereof with strong specificity and high affinity, which can be used for treating diseases related to PD-L1 expression and detecting the expression level of PD-L1. Specifically, the method comprises the following steps:

in a first aspect of the invention, an anti-PD-L1 antibody or antigen-binding fragment thereof is provided, said anti-PD-L1 antibody or antigen-binding fragment thereof comprising VH-CDRs 1-3 of the heavy chain variable region and/or VL-CDRs 1-VL-CDRs 3 of the light chain variable region.

The VH-CDR 1-VH-CDR 3 comprises SEQ ID NO: 1-3 or a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 1-3, and a VL-CDR 1-3 comprising the amino acid sequence of SEQ ID NO: 4-6 or a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 4-6 have an amino acid sequence with at least 80% identity.

In one embodiment of the invention, the VH-CDR 1-VH-CDR 3 are as set forth in SEQ ID NO: 1-3, wherein the VL-CDR 1-VL-CDR 3 is shown as SEQ ID NO: 4-6.

Preferably, the amino acid sequence of the heavy chain variable region comprises SEQ ID NO: 7 or a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 7 has an amino acid sequence with at least 80% identity.

Preferably, the amino acid sequence of the light chain variable region comprises SEQ ID NO: 8 or a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 8, an amino acid sequence having at least 80% identity.

Preferably, the anti-PD-L1 antibody or antigen-binding fragment thereof further comprises_ab、F_ab’、F_ab’-SH、Fv、scFv、(F_ab’)₂A single domain antibody, a diabody (dAb) or a fragment of a linear antibody.

Preferably, the antibody may be a monoclonal antibody or a polyclonal antibody.

Preferably, the antibody may be a monospecific antibody or a multispecific antibody (e.g., bispecific antibody).

Preferably, the antibody can be a fully human antibody, a humanized antibody, an animal derived antibody. Wherein, the animal can be mouse, rabbit, cow, monkey, etc.

The anti-PD-L1 antibody or antigen-binding fragment thereof of the invention specifically binds to PD-L1 protein. The PD-L1 protein is human or TNFR2 protein of other primates.

Specifically, the other primates mentioned above include various classes of monkeys including, but not limited to, cynomolgus monkeys, macaques, baboons, mandrill, and the like; specifically, the macaque includes rhesus macaque, cynomolgus macaque, macaque and the like.

In a specific embodiment of the invention, the anti-PD-L1 antibody or antigen-binding fragment thereof specifically binds to human PD-L1 protein and blocks the interaction of PD-1 with PD-L1.

In a second aspect of the invention, there is provided a chimeric antigen receptor whose extracellular domain comprises the anti-PD-L1 antibody or an antigen-binding fragment thereof described above.

Preferably, the chimeric antigen receptor further comprises any transmembrane, intracellular, hinge and/or intracellular signaling region disclosed in the prior art.

In a third aspect of the invention, there is provided a nucleic acid encoding the anti-PD-L1 antibody or antigen-binding fragment thereof described above or the chimeric antigen receptor described above.

In a fourth aspect of the present invention, there is provided a nucleic acid encoding the heavy chain variable region described above. Preferably, the nucleic acid sequence encoding the heavy chain variable region is as set forth in SEQ ID NO: shown at 9.

In a fifth aspect of the present invention, there is provided a nucleic acid encoding the light chain variable region described above. Preferably, the nucleic acid sequence encoding the light chain variable region is as shown in SEQ ID NO: shown at 10.

The nucleic acid of the present invention may be DNA or RNA, and may be single-stranded or double-stranded.

In a sixth aspect of the invention, there is provided a vector comprising a nucleic acid as described above.

The vector can be expressed in vivo or in vitro, and is preferably an expression vector. Preferably, the expression vector is a prokaryotic expression vector, a lentiviral expression vector, a plasmid, a cosmid, a phage, a virus, or the like. More specifically, the prokaryotic expression vector is an Escherichia coli series. In a seventh aspect of the invention, there is provided a host cell comprising a nucleic acid as described above and/or a vector as described above.

Preferably, the host cell may be eukaryotic or prokaryotic. More preferably, the host cell is a yeast cell, 293 cell, CHO cell, E.coli, etc.

In an eighth aspect of the invention, there is provided a recombinant lentivirus comprising a nucleic acid as described above.

In a ninth aspect of the invention, there is provided an immune cell expressing the chimeric antigen receptor described above. Preferably, the immune cell refers to a cell that plays a role in an immune response, including but not limited to lymphocytes (including T cells, B cells), natural killer cells, bone marrow cells (e.g., monocytes/macrophages, granulocytes, mast cells, eosinophils or basophils, etc.) or dendritic cells.

In a tenth aspect of the present invention, there is provided a method for producing the above immune cell, wherein the method is obtained by transfecting a nucleic acid sequence encoding the above chimeric antigen receptor into an immune cell and expressing the nucleic acid sequence.

In an eleventh aspect of the invention, there is provided a CAR-T cell comprising an anti-PD-L1 antibody or antigen-binding fragment thereof as described above.

In a twelfth aspect of the invention, there is provided an immunoconjugate or antibody drug conjugate comprising the anti-PD-L1 antibody or antigen-binding fragment thereof described above of the invention conjugated to a therapeutic or diagnostic agent.

In a thirteenth aspect of the invention, there is provided a medicament or a test kit comprising the above anti-PD-L1 antibody or antigen-binding fragment thereof, the above chimeric antigen receptor, the above nucleic acid, the above vector, the above host cell, the above recombinant lentivirus, the above immune cell, the above CAR-T cell, or the above immunoconjugate.

Preferably, the drug specifically targets tumor cells expressing PD-L1.

Preferably, the medicament also comprises pharmaceutically acceptable auxiliary materials. Further preferably, the pharmaceutically acceptable excipients include, but are not limited to, diluents, binders, humectants, surfactants, lubricants or disintegrants, and the like.

In a fourteenth aspect of the present invention, a method for preparing the above anti-PD-L1 antibody or an antigen-binding fragment thereof is provided, wherein the method for preparing comprises a protein immunization method and/or a DNA immunization method.

Preferably, the protein immunization method comprises immunizing by taking PD-L1 protein as an immunogen to obtain an anti-PD-L1 antibody or an antigen-binding fragment thereof; collecting splenocytes from the immunized non-human animal, fusing the collected splenocytes with SP2/0 cells to obtain hybridoma cells, culturing the hybridoma cells, and isolating and purifying to obtain the anti-PD-L1 antibody or antigen-binding fragment thereof. The DNA immunization method comprises the step of immunizing by taking DNA plasmid for coding PD-L1 protein as immunogen to obtain the anti-PD-L1 antibody or antigen binding fragment thereof.

In a fifteenth aspect of the present invention, there is provided a method for preparing the above anti-PD-L1 antibody or antigen-binding fragment thereof, the method comprising:

A) obtaining a nucleic acid sequence encoding an anti-PD-L1 antibody or antigen-binding fragment thereof;

B) transforming the nucleic acid sequence obtained in the step A) into a host cell, and then inducing the expression and purification of the nucleic acid sequence.

In a sixteenth aspect of the present invention, there is provided an application of the above anti-PD-L1 antibody or antigen binding fragment thereof, the above chimeric antigen receptor, the above nucleic acid, the above vector, the above host cell, the above recombinant lentivirus, the above immune cell, the above CAR-T cell or the above immunoconjugate, or the above drug or the above detection kit, in the preparation of a product for diagnosing, preventing and/or treating diseases associated with PD-L1 expression.

Preferably, the product is a targeted drug.

Preferably, the product can be a kit, a chip or an antibody drug conjugate, and the like.

In a seventeenth aspect of the present invention, there is provided a method for detecting PD-L1, the method comprising contacting a sample to be detected with the anti-PD-L1 antibody or antigen-binding fragment thereof described above, and then detecting a complex formed by PD-L1 and the anti-PD-L1 antibody or antigen-binding fragment thereof.

Preferably, the detection method detects the presence or content of PD-L1. Wherein the presence or absence is indicated, and the content may be an expression amount, a protein concentration, or the like.

Preferably, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a detectable label.

Preferably, the anti-PD-L1 antibody or antigen-binding fragment thereof is linked to a detectable moiety, which may be biotin, streptavidin, a luminescent agent, an enzyme, a fluorophore, a dye, a radiolabel, a chromophore, a metal ion, a gold particle, a silver particle, a magnetic particle, a polypeptide, or an oligonucleotide.

Preferably, the detection method may be for diagnostic or non-diagnostic purposes. Wherein, the diagnosis purpose is to obtain the result of whether the individual is ill or not after detecting the existence or the content of PD-L1. The non-diagnostic purpose is that on the one hand the detection method is not necessarily an ex vivo tissue or cell of an organism, on the other hand the concentration or expression level of PD-L1 is not directly directed to a disease.

In an eighteenth aspect of the present invention, there is provided a method for diagnosing a tumor, which comprises sampling, contacting the sample with the anti-PD-L1 antibody or an antigen-binding fragment thereof according to the present invention, and then detecting a complex formed between PD-L1 and the anti-PD-L1 antibody or an antigen-binding fragment thereof.

In a nineteenth aspect of the present invention, there is provided a method for treating or preventing a tumor, the method comprising administering to a subject an effective amount of the anti-PD-L1 antibody or an antigen-binding fragment thereof, a chimeric antigen receptor, an immune cell, a drug, or the like, according to the present invention.

The "antibody" of the present invention may be of any class (e.g., IgA, IgD, IgE, IgG and IgM) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 or IgA 2).

"antigen-binding fragments" as described herein include, but are not limited to: a Fab fragment having VL, CL, VH and CH1 domains; a Fab' fragment, which is a Fab fragment having one or more cysteine residues at the C-terminus of the CH1 domain; an Fd fragment having VH and CH1 domains; an Fd' fragment having VH and CH1 domains and one or more cysteine residues C-terminal to the CH1 domain; fv fragments having the VL and VH domains of a single arm of an antibody; a dAb fragment consisting of a VH domain or a VL domain; an isolated CDR region; a F (ab ')2 fragment which is a bivalent fragment comprising two Fab' fragments connected by a disulfide bridge at the hinge region; single chain antibody molecules (e.g., single chain Fv; scFv); a "diabody" having two antigen-binding sites, which comprises a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain; a "linear antibody" comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) that together with a complementary light chain polypeptide form a pair of antigen binding regions; and modified forms of any of the foregoing which retain antigen binding activity.

The term "CDR" as used herein refers to complementarity determining regions within the variable sequences of an antibody. For each variable region, there are three CDRs, called CDR1, CDR2, and CDR3, in each variable region of the heavy and light chains. The exact boundaries of these CDRs are defined differently for different systems. The systems described by Kabat et al (Kabat et al, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) provide not only a clear residue numbering system suitable for antibody variable regions, but also residue boundaries defining the three CDRs, which may be referred to as Kabat CDRs, each of which may comprise amino acid residues from a "complementarity determining region" as defined by Kabat: (Chothia & Lesk, J.Mol.biol., 196:901-, it has boundaries that overlap with the Kabat CDRs. Still other CDR boundary definitions may not strictly follow one of the above systems, but will still overlap with the Kabat CDRs, and the methods used herein may utilize CDRs defined according to any of these systems, although preferred embodiments use Kabat or Chothia defined CDRs. The term "antibody variable region" refers to the portions of the light and heavy chains of an antibody molecule that include the amino acid sequences of the complementarity determining regions (CDRs, i.e., CDR1, CDR2, and CDR3) and the Framework Region (FR). VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain.

The term "and/or" as used herein includes a list of items in the alternative as well as any number of combinations of items.

As used herein to describe a sequence of a protein or nucleic acid, the "comprising" of the invention may consist of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still possess the activity described herein.

"diagnosis" as used herein refers to the determination of whether a patient has suffered from a disease or condition in the past, at the time of diagnosis, or in the future, or the determination of the progression or likely progression of a disease in the future, or the assessment of a patient's response to a therapy.

"treating" as referred to herein means slowing, interrupting, arresting, controlling, stopping, reducing, or reversing the progression or severity of one sign, symptom, disorder, condition, or disease, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders, and refers to therapeutic intervention that ameliorates the signs, symptoms, etc. of a disease or pathological state after the disease has begun to develop.

"prevention" as referred to herein means suppression of symptoms or delay of all actions of a particular symptom stress by administration of a product as described herein.

An "effective amount" as referred to herein refers to an amount or dose of a product of the invention which provides the desired treatment or prevention after administration to a patient or organ in single or multiple doses.

The individual or subject can be a human or non-human animal, and the non-human animal can be a non-human mammal such as a mouse, a cow, a sheep, a rabbit, a pig, a monkey and the like.

The "diseases related to the expression of PD-L1" can be persistent infectious diseases, asthma, transplant rejection, inflammatory diseases and tumors. The persistent infectious disease may be a bacterial infection or a viral infection, including but not limited to Helicobacter (Helicobacter), Mycobacterium (Mycobacterium), Porphyromonas (Porphyromonas), or Chlamydia (Chlamydia); the viral infection includes but is not limited to cytomegalovirus, epstein-barr virus, hepatitis b virus, hepatitis c virus, herpes virus, human immunodeficiency virus, human lymphotropic T-lymphotropic virus, lymphocytic choriomeningitis virus, respiratory syncytial virus, or rhinovirus. The tumor includes but is not limited to solid tumor, leukemia, bladder cancer, brain cancer, breast cancer, colon cancer, stomach cancer, glioma, head cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, neck cancer, ovarian cancer, melanoma, pancreatic cancer, kidney cancer, salivary gland cancer, stomach cancer, thymic epithelial cancer or thyroid cancer. The inflammatory disorder includes, but is not limited to, acute disseminated encephalomyelitis, addison's disease, ankylosing spondylitis, antiphospholipid antibody syndrome, autoimmune hemolytic anemia, autoimmune hepatitis, arthritis, behcet's disease, bullous pemphigoid, coeliac disease, chagas ' disease, crohn's disease, dermatomyositis, type 1 diabetes, goodpasture's syndrome, graft-versus-host disease, graves ' disease, guillain-barre syndrome, hashimoto's disease, hyper IgE syndrome, idiopathic thrombocytopenic purpura, lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus, pernicious anemia, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, sjogren's syndrome, temporal arteritis, vasculitis, or wegener's granulomatosis. Such transplant rejection includes, but is not limited to, organ rejection, bone marrow transplant rejection, and/or non-myeloablative bone marrow transplant rejection. In one embodiment of the present invention, the "disease associated with PD-L1 expression" may be multiple myeloma, myelodysplastic syndrome, acute myelogenous leukemia, melanoma or parkinson's disease.

The "sample to be detected" or "specimen" according to the present invention may be a biological specimen or any non-biological specimen. The biological sample includes, but is not limited to, cells, tissues, blood or bone marrow, etc.

The "antibody" of the present invention may further comprise a light chain constant region and/or a heavy chain constant region. The light chain constant region may be one having the amino acid sequence of the constant region of either a Kappa chain or a Lambda chain, and the Lambda chain (Lambda chain) is higher in sheep, cats, dogs, and horses, and the Kappa chain (Kappa chain) is higher in mice, rats, humans, and pigs, as compared to the presence ratio. The heavy chain constant region may have the amino acid sequence of the constant region of rat IgG2 a. Heavy chains are classified into γ, μ, α, δ, and ε chains according to the differences in the constant regions, and IgG, IgM, IgA, IgD, and IgE5 types (isotypes) of immunoglobulin are formed according to the differences.

The term "identity" as used herein means that, in the context of using an amino acid sequence or a nucleotide sequence, a person skilled in the art can adjust the sequence according to the actual working requirement without changing the structure or activity of the original sequence, such that the used sequence has (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 100% identity. For example, the term "a sequence identical to SEQ ID NO: 1-3 "is an amino acid sequence having at least 80% identity to the CDR regions, i.e. the amino acid sequence of SEQ ID NO: 1-3, for example, by substitution, deletion, and/or insertion of one or more amino acids. Wherein said at least 80% includes but is not limited to 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%.

Drawings

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

FIG. 1: the binding condition of the anti-PD-L1 antibody and PD-L1 is detected by flow cytometry, wherein 4T 1-is not stained into a breast cancer cell line which is not stained by the antibody, 4T1-hPD-L1 is not stained into a breast cancer cell line which is overexpressed by human PD-L1 which is not stained by the antibody, 4T1-5B4 is a breast cancer cell line which is stained by 5B4 antibody, and 4T1-hPD-L1-5B4 is an breast cancer cell line which is overexpressed by human PD-L1 which is stained by 5B4 antibody.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

EXAMPLE 1 preparation of antibodies

A mouse is immunized by using human PD-L1 protein as an antigen, and a hybridoma is separated to obtain a mouse monoclonal antibody.

The method comprises the following specific steps: the PD-L1 extracellular domain nucleic acid sequence was derived from the RefSeq database (NM-014143.3) and was synthesized by Kingchi corporation. The C-terminal of the PD-L1 extracellular region is added with a 6Xhis label and constructed in eukaryotic expression plasmids. The purified PD-L1 extracellular region protein was mixed with the immunological adjuvant QuickAntibody-Mouse according to the following ratio of 1: 1, injecting 50uL of the mixture into the left and right hind legs of a BALB/C mouse respectively, and injecting the mixture once every two weeks for 5 times of immunization. Killing the mice by dislocation of the neck, taking the spleen, and squeezing by using forceps to release the spleen cell suspension; preparing SP2/0 myeloma cells (purchased from Chinese academy of sciences), mixing the two cells, adding preheated PEG, centrifuging at 1000rpm for 10 minutes, discarding the supernatant, adding HAT culture medium, adding appropriate amount of abdominal macrophages (obtained from abdominal cavity of healthy mice), culturing in 37 deg.C and 5% CO2 incubator, observing growth of hybridoma cells, and sucking appropriate amount of cell supernatant for antibody detection. During detection, the supernatant stock solution is used for being incubated with 4T1 cells over-expressing PD-L1 for 30 minutes, and then an anti-mouse secondary antibody (APC Goat anti-mouse IgG, biolegend,405308) is added for incubation for 30 minutes, and then flow detection is carried out. And (3) separating the single cell clone of the detected positive hybridoma cell group by a dilution method, carrying out supernatant detection after the single cell clone grows, and finally screening the single clone expressing the PD-L1 antibody. The antibody antigen-binding region in the hybridoma was cloned by PCR and sequenced to obtain the PD-L1 antibody sequence (see Table 1).

TABLE 1 amino acid sequence and nucleotide sequence

Example 2 antibody affinity assays

1. The experimental process comprises the following steps: adherent cells 4T1 (breast cancer cell line, purchased from Chinese academy of cells) and 4T1-hPDL1 (stably overexpressing human PD-L1 breast cancer cell line constructed by infecting lentivirus expressing the full length of human PD-L1 (NM-014143.3) in 4T1 cells) were first digested with PBS + EDTA (2mM), centrifuged, and one aliquot of each of the 5B4 monoclonal supernatant selected in example 1 was stained for 30 minutes with 500uL, and the other aliquot was added as a control; after 30 minutes of washing with PBS, an anti-mouse secondary antibody (APC coat anti-mouse IgG, biolegend,405308) was added to the four samples, and after 30 minutes of staining, PBS was washed and subjected to flow-through detection.

2. Results of the experiment

The result of using the mouse breast cancer cell line 4T1 as a detection cell is shown in FIG. 1, and the result shows that the 5B4 monoclonal antibody can specifically recognize and bind to the human PD-L1 protein.

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.

Claims (11)

1. An anti-PD-L1 antibody or antigen-binding fragment thereof, wherein said anti-PD-L1 antibody or antigen-binding fragment thereof comprises VH-CDR 1-VH-CDR 3 of the heavy chain variable region and VL-CDR 1-VL-CDR 3 of the light chain variable region, wherein said VH-CDR 1-VH-CDR 3 is as set forth in SEQ ID NO: 1-3, wherein the VL-CDR 1-VL-CDR 3 is shown as SEQ ID NO: 4-6.

2. The anti-PD-L1 antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 7 is shown in the specification; the amino acid sequence of the light chain variable region is shown as SEQ ID NO: shown in fig. 8.

3. The anti-PD-L1 antibody or antigen-binding fragment thereof according to any one of claims 1-2, wherein said anti-PD-L1 antibody or antigen-binding fragment thereof further comprises a peptide as F_ab、F_ab’、F_ab’-SH、Fv、scFv、(F_ab’)₂A single domain antibody, a diabody (dAb) or a fragment of a linear antibody.

4. A nucleic acid encoding the anti-PD-L1 antibody or antigen-binding fragment thereof according to any one of claims 1-3.

5. A vector comprising the nucleic acid of claim 4.

6. A host cell comprising the nucleic acid of claim 4 or the vector of claim 5.

7. A medicament comprising the anti-PD-L1 antibody or antigen-binding fragment thereof of any one of claims 1-3, the nucleic acid of claim 4, the vector of claim 5, or the host cell of claim 6.

8. A test kit comprising the anti-PD-L1 antibody or antigen-binding fragment thereof of any one of claims 1-3, the nucleic acid of claim 4, the vector of claim 5, or the host cell of claim 6.

9. A method of producing the anti-PD-L1 antibody or antigen-binding fragment thereof of any one of claims 1-3, comprising:

A) obtaining a nucleic acid sequence encoding an anti-PD-L1 antibody or antigen-binding fragment thereof;

B) transforming the nucleic acid sequence obtained in the step A) into a host cell, and then inducing the expression and purification of the nucleic acid sequence.

10. Use of the anti-PD-L1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the nucleic acid according to claim 4, the vector according to claim 5, the host cell according to claim 6, the medicament according to claim 7 or the test kit according to claim 8 for the manufacture of a product for the diagnosis, prevention and/or treatment of a disease associated with PD-L1 expression.

11. A method for detecting PD-L1, comprising contacting a sample to be detected with the anti-PD-L1 antibody or antigen-binding fragment thereof of any one of claims 1-3, and detecting the complex formed between PD-L1 and the anti-PD-L1 antibody or antigen-binding fragment thereof, wherein said method is for non-diagnostic purposes.

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