CN108997500B - An anti-human PD-L1 antibody and its application - Google Patents

Abstract

The invention relates to the technical field of immunity, in particular, to an anti-human PD-L1 antibody and an application thereof. The antibody comprises an isolated binding protein of an antigen binding domain, wherein the antigen binding domain comprises at least one complementarity determining region selected from the following amino acid sequence, or: has at least 80% of the complementarity determining region with the following amino acid sequence The sequence identity of the protein and the PD-L1 protein have Kd≤8.0× ^10-9 mol/L affinity; the amino acid sequences of the complementarity determining regions CDR-VL1, CDR-VL2, CDR-VL3 are respectively as SEQ ID NO:1-3 The amino acid sequences of the complementarity determining regions CDR-VH1, CDR-VH2, and CDR-VH3 are respectively shown in SEQ ID NO: 4-6. The binding protein has strong activity and high affinity with human PD-L1 protein.

Description

An anti-human PD-L1 antibody and its application

technical field

The present invention relates to the field of immunization technology, in particular, to an anti-human PD-L1 antibody and an application thereof.

Background technique

The incidence of lung cancer, especially female lung cancer, is on the rise worldwide. Lung cancer is the malignant tumor with the highest incidence in my country, and its mortality rate ranks first among malignant tumors. At present, international developed countries have achieved results in the prevention and treatment of lung cancer, and the mortality rate of lung cancer is decreasing year by year. In addition to tobacco control and early detection, targeted therapy has also benefited lung cancer patients. Especially in recent years, the immunotherapy of lung cancer has benefited. A substantial breakthrough has been achieved, and its effective treatment significantly prolongs the survival time compared with traditional chemotherapy. Lung cancer immunotherapy is mainly aimed at an expression molecule PD-1 (programmed death receptor-1) expressed in exhausted T lymphocytes, and the use of this molecule-specific antibody or its ligand (PD-L1) antibody to block PD-1-PD-L1 mediated negative regulation of T cells, thereby reversing the exhaustion of T cells and restoring their tumor-killing activity. In addition to lung cancer, the largest indication, PD-1-PD-L1 blockade therapy Widely used in melanoma, kidney cancer, bladder cancer, Hodgkin lymphoma, head and neck cancer, cervical cancer, gastric cancer, bowel cancer and other MSI-H (highly microsatellite instability) or DNA mismatch repair defects Spectrum of anti-tumor activity. Currently, the main PD-1 target therapeutic antibodies for lung cancer are Keytruda and Opdivo, both of which have an effective rate of about 20% in the treatment of lung cancer patients. The 5-year survival rate of advanced lung cancer patients treated with PD-1 antibody increased by 5- 6 times, for this reason, it is of great clinical significance to screen effective candidate patients for treatment, and blind medication can also be avoided. At present, prediction methods with clinical guidance value include tumor mutation load (TMB), MSI detection and PD-L1 expression analysis. PD-L1 detection is the most classic detection method. There are two internationally used detection reagents, namely Ventana PD-L1 Rabbit monoclonal antibody (clone SP142) and PD-L1, IHC 22C3 pharmDx mouse monoclonal antibody atezolizumab (clone 22C3), used for the accompanying detection of immunohistochemistry, has been approved as a standard antibody for the detection of PD-L expression levels in clinical tumor tissues , Keytruda can be used for first-line non-small cell lung cancer treatment when the tumor cell expression rate exceeds 50%. In addition, PD-L1 expression is associated with a variety of tumor prognosis, and its detection can predict survival.

In view of the potential prediction with clinical guidance value, it is limited to several methods such as tumor mutation load (TMB), MSI detection and tissue PD-L1 expression analysis. The former two detections involve molecular biology methods and nucleic acid whole-genome sequencing, which are expensive , and relying on surgery or puncture to obtain tumor tissue, the detection application in patients with non-surgical or metastatic lesions is difficult to obtain tissue specimens is limited, in addition, TMB and MSI detection have not been determined as clinical standard detection methods. In recent years, immunohistochemical PD-L1 expression analysis of the above-mentioned approved antibody preparations (currently the only diagnostic partner approved by the FDA is PD-L1IHC22C3pharmDx - used to screen patients treated with pembrolizumab) is relatively simple and more common in clinical applications. , the approved antibodies of these two reagents are limited to the accompanying detection of immunohistochemistry (IHC), targeting tumor tissue, and also relying on the acquisition of tissue samples; in addition, PD-L1 negativity is still unreliable, and the test results may be due to different antibodies and tissue samples. The same is true, tumor tissue heterogeneity, low expression, and induced genes may also lead to sampling errors or false negatives, especially in immunohistochemical staining analysis, whose clinical PD-L1 tissue expression cutoff value read has subjective factors, Therefore, at present, PD-L1-IHC positivity is still an incomplete response biomarker and cannot be used as a "deterministic" indicator for screening patients treated with PD-1-PD-L1 inhibitors. Systematic multicomponent predictive markers.

In view of this, the present invention is proposed.

SUMMARY OF THE INVENTION

The present invention relates to a novel isolated binding protein comprising an antigen binding domain, and studies on the preparation and application of the binding protein. It can be efficiently used to supplement PD-L1 analysis methods that do not depend on tissue samples of tumor patients, and establish an enzyme-linked immunosorbent assay (ELISA) technology suitable for the determination of sPD-L1 levels in blood samples of lung cancer and other malignant tumors. A small amount of blood samples from patients are tested, and blood collection is non-invasive or minimally invasive, which is convenient for repeated testing and has high detection sensitivity.

The antigen binding domain comprises at least one complementarity determining region selected from the following amino acid sequence, or: having at least 80% sequence identity with the complementarity determining region of the following amino acid sequence, and having Kd≤8.0 with PD-L1 protein ×10 ^-9 mol/L affinity;

The amino acid sequences of the complementarity determining regions CDR-VL1, CDR-VL2, and CDR-VL3 are respectively shown in SEQ ID NOs: 1-3;

The amino acid sequences of the complementarity determining regions CDR-VH1, CDR-VH2, and CDR-VH3 are shown in SEQ ID NOs: 4-6, respectively.

An important advantage is that the binding protein is highly active and has a high affinity for human PD-L1 protein.

Description of drawings

In order to illustrate the specific embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that need to be used in the description of the specific embodiments or the prior art. Obviously, the accompanying drawings in the following description The drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without creative efforts.

Fig. 1 is the result diagram of the specificity of PD-L1 antibody identified by ELISA in Experimental Example 1 of the present invention;

FIG. 2 is the result of identifying the specificity of PD-L1 antibody by Western blot in Experimental Example 1 of the present invention; M: Marker; 1: PD-L1-Fc; 2: PD-1-his; 3: PD-L1-his ;

3 is the detection of PD-L1 antibody and PD-L1 protein affinity in Experimental Example 2 of the present invention;

Figure 4 is the flow cytometry in Experimental Example 3 of the present invention to detect the transfection rate of pcDNA3.1-PD-L1 plasmid; FITC-A: GFP; APC-A: PD-L1;

FIG. 5 is a graph showing the results of the flow cytometry detection of the binding of PD-L1 antibody to PD-L1 protein on the cell membrane in Experimental Example 3 of the present invention; FITC-A: GFP; APC-A: PD-L1;

Figure 6 is a graph showing the results of the flow cytometry detection of the binding of PD-L1 antibody to PD-L1 in lung adenocarcinoma H2009 cells in Experimental Example 3 of the present invention; PE-A: PD-L1;

Fig. 7 is the standard curve diagram of the establishment of sandwich ELISA with PD-L1 antibody in Experimental Example 4 of the present invention;

8 is a graph showing the results of detecting sPD-L1 in the serum of lung cancer patients and normal people by the PD-L1 antibody in Experimental Example 4 of the present invention;

FIG. 9 is the result of flow cytometry detection of PD-L1 expression in transfected 293 cells in Experimental Example 5 of the present invention; APC-A: PD-L1;

FIG. 10 is a graph showing the blocking result of the binding of PD-L1 to PD-1 detected by flow cytometry in Experimental Example 5 of the present invention;

FIG. 11 is a graph showing the blocking result of ELISA detecting the binding of PD-L1 antibody to PD-1 and PD-L1 in Experimental Example 5 of the present invention.

Detailed ways

In order that the present invention may be more easily understood, selected terms are defined below.

The term "isolated binding protein" is a protein which, by virtue of its origin or source of derivation, is not bound to a naturally associated component with which it is associated in its native state; substantially free of Other proteins; expressed by cells from different species; or not found in nature. Thus, a protein that is chemically synthesized or synthesized in a cellular system other than its natural origin will be "isolated" from the components with which it is naturally associated. Proteins can also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.

The term "isolated binding protein comprising an antigen binding domain" refers broadly to all proteins/protein fragments comprising CDR regions. The term "antibody" includes polyclonal and monoclonal antibodies and antigenic compound-binding fragments of these antibodies, including Fab, F(ab')2, Fd, Fv, scFv, bispecific antibodies, and antibody minimal recognition units, as well as these antibodies and single-chain derivatives of fragments. The type of antibody can be selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD. In addition, the term "antibody" includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, chimeric, bifunctional, and humanized antibodies, as well as related synthetic isoforms (isoforms). The term "antibody" is used interchangeably with "immunoglobulin".

The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of an antibody. The variable domain of the heavy chain may be referred to as "VH". The variable domain of the light chain can be referred to as "VL". These domains are usually the most variable part of the antibody and contain the antigen binding site. The light or heavy chain variable region (VL or VH) consists of a framework region interrupted by three hypervariable regions called "complementarity determining regions" or "CDRs". The range of framework regions and CDRs has been precisely defined, for example, in Kabat (see "Sequences of Proteins of Immunological Interest", E. Kabat et al., U.S. Department of Health and Human Services ), (1983)) and Chothia. The framework regions of antibodies, ie, the framework regions that make up the combination of the essential light and heavy chains, serve to position and align the CDRs, which are primarily responsible for binding to the antigen.

As used herein, "framework" or "FR" regions mean regions of an antibody variable domain excluding those regions defined as CDRs. Each antibody variable domain framework can be further subdivided into adjacent regions (FR1, FR2, FR3, and FR4) separated by CDRs.

In general, the variable regions VL/VH of the heavy and light chains can be obtained by linking the following numbered CDRs and FRs in the following combinations: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

As used herein, the term "purified" or "isolated" in connection with a polypeptide or nucleic acid means that the polypeptide or nucleic acid is not in its native medium or native form. Thus, the term "isolated" includes a polypeptide or nucleic acid removed from its original environment, eg, if it occurs in nature, from its natural environment. For example, an isolated polypeptide is typically free of at least some proteins or other cellular components with which it is ordinarily associated or which is ordinarily mixed or in solution. Isolated polypeptides include naturally produced said polypeptides contained in cell lysates, said polypeptides in purified or partially purified form, recombinant polypeptides, said polypeptides expressed or secreted by cells, and in heterologous host cells or cultures of the polypeptide. In connection with a nucleic acid, the term isolated or purified indicates, for example, that the nucleic acid is not in its native genomic context (eg, in a vector, as an expression cassette, linked to a promoter, or artificially introduced into a heterologous host cell).

Since PD-L1 is mainly expressed in tumor cells or other tumor stromal cells in the tumor microenvironment in vivo, PD-L1 expression can be shed from the surface of tumors and other cells by interstitial protease digestion or released into the blood circulation as different mRNA splicing proteins. The level of PD-L1 potentially reflects the expression status of PD-L1 in the tumor environment, and its expression level is directly related to tumor size, necrosis, and patient prognosis. Soluble PD-L1 (sPD-L1) also plays a role in suppressing T cell activity. sPD-L1 can better represent the level of PD-L1 from different sources, including tumor cells in the tumor microenvironment and other tumor stromal cells. In addition, sPD-L1 can be expressed in the serum of patients with various tumors and autoimmune diseases other than tumors and infectious diseases. detected.

The present invention provides an isolated binding protein comprising an antigen binding domain, the antigen binding domain comprising at least one complementarity determining region selected from the following amino acid sequence, or: having at least one complementarity determining region with the following amino acid sequence 80% sequence identity and affinity with PD-L1 protein with Kd≤8.0×10 ^-9 mol/L;

The amino acid sequences of the complementarity determining regions CDR-VL1, CDR-VL2, and CDR-VL3 are respectively shown in SEQ ID NOs: 1-3;

The amino acid sequences of the complementarity determining regions CDR-VH1, CDR-VH2, and CDR-VH3 are shown in SEQ ID NOs: 4-6, respectively. It is known in the art that the binding specificity and affinity of antibodies are mainly determined by the CDR sequences. According to mature and well-known existing technologies, the amino acid sequences of non-CDR regions can be easily changed to obtain variants with similar biological activities. body. Accordingly, the present invention also includes "functional derivatives" of the binding proteins. "Functional derivatives" refers to amino acid substitution variants (which may be antibodies or fragments with the same or similar activity formed by substitution, deletion or addition of one or several amino acids), a functional derivative that retains detectable binding The protein activity is preferably the activity of an antibody capable of binding to cTnI. "Functional derivatives" can include "variants" and "fragments", which have the exact same CDR sequences as the binding proteins of the present invention, and thus have similar biological activities.

In some embodiments, the antigen binding domain has at least 85%, or 90%, or 91%, or 92%, or 93%, or 94%, or 95% of the complementarity determining regions of the following amino acid sequences, Or 96%, or 97%, or 98%, or 99% sequence identity, and have Kd≤8.0× ^10-9 mol/L with PD-L1 protein, Kd value can also be selected as 7.8× ^10-9 mol/ L, 7.5×10 ^-9 mol/L, 7×10 ^-9 mol/L, 6.96×10 ^-9 mol/L, 6.7×10 ^-9 mol/L, 6×10 ^-9 mol/L, 5.5×10 ^{- 9} mol/L, 4×10 ^-9 mol/L, 2×10 ^-9 mol/L, 1.0×10 ^-9 mol/L, 9.0×10 ^-10 mol/L, 8.0×10 ^-10 mol/L , 7.0×10 ^-10 mol/L, 6.0×10 ^-10 mol/L, 5.0×10 ^-10 mol/L, 4.0×10 ^-10 mol/L affinity, etc.

Wherein, the affinity is determined according to the method in the specification of the present invention.

In some embodiments, the binding protein includes at least 3 CDRs (eg, 3 heavy chain CDRs or 3 light chain CDRs); alternatively, the binding protein includes at least 6 CDRs.

In some embodiments, the binding protein comprises light chain framework regions FR-L1, FR-L2, FR-L3 and FR-L4 whose sequences are shown in SEQ ID NOs: 7-10 in sequence, and/or, sequenced in sequence Heavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4 as set forth in SEQ ID NOs: 11-14.

In some embodiments, the binding protein further comprises antibody constant region sequences.

In some embodiments, the binding protein comprises an antibody constant region Fc.

In some embodiments, the constant region Fc includes a light chain constant region and a heavy chain constant region.

In some embodiments, the light chain constant region sequence is set forth in SEQ ID NO:15.

In some embodiments, the heavy chain constant region sequence is set forth in SEQ ID NO:16.

In some embodiments, the sequence of the constant region Fc is selected from the sequence of any one of the constant regions of IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD.

In some embodiments, the sequence of the constant region Fc is selected from the sequence of an IgG2 constant region.

In some embodiments, the sequence of the constant region Fc is selected from the sequence of an IgG2b constant region.

In some embodiments, the constant region sequence is selected from the sequence of the IgG2b/kappa constant region.

In some embodiments, the species source of the constant region is bovine, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck , goose, turkey, cockfight or man.

In some embodiments, the constant region is derived from a mouse.

In some embodiments, the constant region Fc is derived from human.

In some embodiments, the composition of the light chain amino acids of the binding protein comprises:

FR-L1-CDR-VL1-FR-L2-CDR-VL2-FR-L3-CDR-VL3-FR-L4-Constant region;

or;

Leader sequence-FR-L1-CDR-VL1-FR-L2-CDR-VL2-FR-L3-CDR-VL3-FR-L4-Constant region-Stop codon (see SEQ ID NO:20 for Leader sequence sequence).

In some embodiments, the heavy chain amino acid composition of the binding protein comprises:

FR-H1-CDR-VH1-FR-H2-CDR-VH2-FR-H3-CDR-VH3-FR-H4-Constant region;

or;

Leader sequence-FR-H1-CDR-VH1-FR-H2-CDR-VH2-FR-H3-CDR-VH3-FR-H4-Constant region-Stop codon (see SEQ ID NO:21 for Leader sequence sequence).

Constant region is the constant region Fc.

In some embodiments, the binding protein is an intact antibody comprising variable and constant Fc regions.

In some embodiments, the binding proteins are "functional fragments" of antibodies, such as Nanobodies, F(ab')2, Fab', Fab, Fv, Fd, scFv, scFv-Fc chimeric fragments/diabodies and one of the smallest recognition units of antibodies.

The "functional fragment" in the present invention particularly refers to an antibody fragment with the same specificity for PD-L1 as the parent antibody. In addition to the functional fragments described above, any fragment that has an increased half-life is also included. These functional fragments generally have the same binding specificity as the antibody from which they were derived. Those skilled in the art infer from the contents described in the description of the present invention that the antibody fragments of the present invention can obtain the above-mentioned functions by methods such as enzymatic digestion (including pepsin or papain) and/or by chemical reduction methods to split disulfide bonds Fragment. Antibody fragments can also be obtained by peptide synthesis by recombinant genetics techniques also known to those skilled in the art or by, for example, automated peptide synthesizers such as those sold by Applied BioSystems and the like.

In some embodiments, the binding protein is a scFv-Fc chimeric fragment comprising the above-mentioned constant region Fc, the above-mentioned complementarity determining region, the above-mentioned light chain framework region, and the above-mentioned heavy chain framework region .

In some embodiments, the constant region Fc is a human Ig-Fc amino acid.

In some embodiments, the human Ig-Fc amino acid sequence is set forth in SEQ ID NO:17.

scFv (single chain antibody fragment, scFv) is a single chain antibody fragment, which is formed by connecting the variable region of the heavy chain and the variable region of the light chain of an antibody through an artificial flexible linker (Linker) of 15-20 amino acids.

The scFv-Fc chimeric fragment, the single-chain antibody chimeric form, is composed of scFv and constant region Fc.

In some embodiments, the scFv-Fc chimeric fragment further comprises an artificial flexible linker peptide.

In some embodiments, the number of amino acids of the artificial flexible linking peptide is 10-29, the sequence is (GGGGS)n, and n is 2, 3, 4, 5, 6, or the sequence is GTSSGAGKSSEGKG. The artificial flexible linking peptide also includes other amino acid sequences with linking effect.

In some embodiments, the composition of the scFv-Fc chimeric fragment, namely the single chain antibody chimeric form, comprises: Variable region of heavy chain+Linker+Variable region of light chain+Human Fctag;

or;

Signal peptide+Variable region of heavy chain+Linker+Variable region of light chain+Human Fc tag+Stop codon.

The Variable region of heavy chain is the heavy chain variable region (FR-H1-CDR-VH1-FR-H2-CDR-VH2-FR-H3-CDR-VH3-FR-H4).

The Variable region of light chain is the light chain variable region (FR-L1-CDR-VL1-FR-L2-CDR-VL2-FR-L3-CDR-VL3-FR-L4).

The Signal peptide is the signal peptide amino acid, and the sequence is shown in SEQ ID NO:22.

Another aspect of the present invention is to provide an isolated nucleic acid molecule, wherein the nucleic acid molecule is DNA or RNA, which encodes the binding protein as described above.

The nucleic acid sequences therein are operably linked to at least one regulatory sequence. "Operably linked" means that the coding sequence is linked to regulatory sequences in a manner that allows for the expression of the coding sequence. Regulatory sequences are selected to direct expression of the protein of interest in suitable host cells, and include promoters, enhancers and other expression control elements.

In some embodiments, the DNA sequences encoding light chain amino acids and heavy chain amino acids are set forth in SEQ ID NOs: 23-24, respectively.

As used herein, nucleic acids include conservatively substituted variants thereof (eg, substitutions of degenerate codons) and complementary sequences. The terms "nucleic acid" and "polynucleotide" are synonymous and include genes, cDNA molecules, mRNA molecules, and fragments thereof such as oligonucleotides.

Another aspect of the present invention is to provide a vector comprising the above-mentioned nucleic acid molecule.

The vector may contain a selectable marker, and an origin of replication that matches the cell type specified by the cloning vector, while the expression vector contains regulatory elements necessary to affect expression in the specified target cells. The vectors can be cloning vectors and expression vectors, including plasmid vectors, phage vectors, viral vectors, etc. When expressing or preparing antibodies or fragments, prokaryotic expression vectors and eukaryotic expression vectors are often involved. Prokaryotic expression vectors are commonly used PET series, In the pGEX series, eukaryotic expression vectors commonly used are pcDNA3.1, pcDNA3.4, pEGFP-N1, pEGFP-N1, pSV2, etc. The viral vectors can be lentivirus, retrovirus, adenovirus or adeno-associated virus.

In some embodiments, the vector is the pcDNA3.1 expression system.

Another aspect of the present invention is to provide a host cell, which comprises the above-mentioned nucleic acid molecule or the above-mentioned vector.

The host cells mainly involve eukaryotic cells, and eukaryotic cells include mammalian cells, yeast, and insect cells. Especially for the preparation of whole antibody or full-length antibody, mammalian cells are commonly used, which can be CHO, 293, and NSO cells.

In some embodiments, the host cell is a mammalian cell293.

In some embodiments, the method for introducing the vector into the host cell includes lipofection methods and electroporation methods, such as Lipofectamine ^™ , RNAiMAX, HiPerFect, DharmaFECT, X-tremeGENEsiLentFect ^™ , TransIntro™ EL Transfection Reagent and the like. Viral vectors are introduced into mammalian cells by their natural infection mode, such as retrovirus or lentivirus by preparing whole virus particles and directly adding them to cultured cells to infect mammalian cells.

Another aspect of the present invention is to provide a method for producing the above-mentioned binding protein, comprising the steps of: culturing the above-mentioned host cell in a culture medium, and recovering the so-produced binding from the culture medium or from the cultured host cell protein.

Another aspect of the present invention is to provide the use of the above-mentioned binding protein in the preparation of a medicament for diagnosing diseases; the diseases include cancer and/or immune-related diseases.

In some embodiments, the cancer includes lung cancer, melanoma, NSCLC, classical Hodgkin lymphoma, HNSCC, renal cell carcinoma, urothelial carcinoma, head and neck cancer, gastric cancer, hematological malignancies, prostate cancer, One or more of cervical cancer, brain cancer, hepatocellular carcinoma, and colorectal cancer.

In some embodiments, the immune-related disease includes viral infection, bacterial infection, fungal infection, parasite, and one of rheumatoid arthritis, ulcerative colitis, pemphigus, dermatomyositis, Alzheimer's disease or more.

In some embodiments, the cancer comprises a microsatellite instability high (MSI-H) or mismatch repair gene (MMR) deficient cancer.

Another aspect of the present invention is to provide an antibody combination product, which includes the above-mentioned binding protein and a second antibody; the second antibody specifically recognizes PD-L1, and its recognition epitope is different from the binding protein.

In some embodiments, the combination product is packaged in a box.

In some embodiments, the binding protein and the second antibody are contained in different containers (eg, EP tubes).

In some embodiments, the amino acid sequence of the light chain variable region VL of the second antibody is shown in SEQ ID NO:18; the amino acid sequence of the heavy chain variable region VH is shown in SEQ ID NO:19.

In some embodiments, the antibody combination further comprises one or more of PBS, BSA, ddH2O, glycerol, _sodium azide, and gentamicin.

The present invention also relates to a method for detecting PD-L1 protein in a test sample, comprising:

a) contacting the PD-L1 protein in the test sample with the above-described binding protein under conditions sufficient for an antibody/antigen binding reaction to occur to form an immune complex; and

b) detecting the presence of the immune complex, the presence of the complex being indicative of the presence of the PD-L1 protein in the test sample.

In some embodiments, the immune complex further includes a second antibody that binds to the PD-L1 protein or the binding protein.

In some embodiments, the second antibody binds to the binding protein.

In some embodiments, the binding protein comprises a detectable label.

In some embodiments, the test item is blood, cells or tissue.

In some embodiments, the binding protein acts as a non-blocking antigen capture antibody, which can be paired with PD-L1 antigen capture or anti-PD-L1 antibodies with different epitopes, such as PD-1-PD-L1 anti-PD -L1 capture antibody, detected using sandwich ELISA.

The binding protein or secondary antibody can be labeled with an indicator that displays signal strength to allow the complex to be easily detected.

In some embodiments, the indicator showing signal intensity includes fluorescent substances, quantum dots, digoxigenin-labeled probes, biotin, radioisotopes, radiocontrast agents, paramagnetic ion fluorescent microspheres, electron-dense substances, chemical Any of luminescent labels, ultrasound contrast agents, photosensitizers, colloidal gold or enzymes.

In some embodiments, the fluorescent substance includes Alexa 350, Alexa 405, Alexa 430, Alexa488, Alexa 555, Alexa 647, AMCA, Aminoacridine, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY- R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxy-4', 5'-dichloro-2', 7'-dimethoxyfluorescein, 5-carboxy-2', 4', 5', 7 '-Tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxyrhodamine, 6-carboxytetramethylrhodamine, Cascade Blue, Cy2, Cy3, Cy5, Cy7, 6-FAM, Dan Sulfonyl chloride, Fluorescein, HEX, 6-JOE, NBD (7-nitrobenzo-2-oxa-1,3-oxadiazole), Oregon Green 488, Oregon Green 500, Oregon Green514, Pacific Blue, o-phenylene Dicarboxylic acid, terephthalic acid, isophthalic acid, cresyl fast violet, cresyl blue violet, brilliant cresyl blue, p-aminobenzoic acid, erythrosine, phthalocyanine, azomethine, cyanine, xanthine , succinyl fluorescein, rare earth metal cryptate, tribispyridyldiamine europium, europium cryptate or chelate, diamine, dicyanidin, La Jolla blue dye, allophycocyanin, allococyanin B, Phycocyanin C, Phycocyanin R, Thiamine, Phycoerythrin, Phycoerythrin R, REG, Rhodamine Green, Rhodamine Isothiocyanate, Rhodamine Red, ROX, TAMRA, TET, TRIT (four methyl rhodamine isothiol), tetramethyl rhodamine and any of Texas Red.

In some embodiments, the radioisotope includes 110In, 111In, 177Lu, 18F, 52Fe, 62Cu, 64Cu, 67Cu, 67Ga, 68Ga, 86Y, 90Y, 89Zr, 94mTc, 94Tc, 99mTc, 120I, 123I, 124I, 125I , 131I, 154-158Gd, 32P, 11C, 13N, 15O, 186Re, 188Re, 51Mn, 52mMn, 55Co, 72As, 75Br, 76Br, 82mRb and 83Sr.

In some embodiments, the enzyme includes any one of horseradish peroxidase, alkaline phosphatase, and glucose oxidase.

In some embodiments, the fluorescent microspheres are: polystyrene fluorescent microspheres, and the interior is wrapped with rare earth fluorescent ion europium.

Another aspect of the present invention is to provide a kit comprising one or more of the above-mentioned binding protein, the nucleic acid molecule, the carrier and the antibody combination product .

The kit can be used for the assessment of tumors and various other immune-related diseases.

In some embodiments, the antibody containing the binding protein can be added with a detectable label to prepare a labeled detection tracer reagent; or, as a second antigen, combined with the antigen or the first antibody that captures the antigen, and works together to detect the antigen, Improve the sensitivity and accuracy of detection; or, as the first antibody binds to the antigen or captures the antigen, and works together with the second antibody to detect the antigen to improve the sensitivity and accuracy of the detection.

In this embodiment, flow cytometry, Western blot, ELISA or immunohistochemistry can be selected for detection.

In some embodiments, the anti-human PD-L1 monoclonal antibody is specific to a monoclonal antibody secreted by a mouse-derived anti-human PD-L1 hybridoma that encodes an antibody specific for its specific antibody nucleic acid sequence. Binding to the extracellular domain of human PD-L1, the antibody protein completely blocks the binding of PD-1-PD-L1, and its basic characteristics such as binding affinity to PD-L1 are clear.

In some embodiments, the present invention relates to the combination of the antibody with other anti-human PD-L1 antibodies of different epitopes to establish a sensitive sandwich ELISA method for lung cancer, melanoma, kidney cancer, bladder cancer, Hodge Blood soluble PD-L1 (sPD) in patients with MSI-H (high microsatellite instability) or DNA mismatch repair deficiency (MSI/dMMR) tumor types such as lymphoma, head and neck cancer, cervical cancer, gastric cancer, and bowel cancer -L1) concentration assessment, determine the concentration level of sPD-L1 in patients, and its sensitivity reaches the single-digit effective detection concentration (5pg/mL). Antibodies can also be used to detect PD-L1 in tumor tissue, guide the prognosis evaluation of clinical PD-1-PD-L1 pathway blockers, and potentially expand for the analysis of inflammation in other immune-related diseases.

The full sequence of the antibody encoding nucleic acid of the present invention is convenient for PD-L1 antibody engineering, such as immunoglobulin class, subclass conversion and various subunit functional small molecule antibody engineering. The antibody sequence is an effective expression strategy in the mammalian cell system and is suitable for genetic Trait stable antibody production.

The embodiments of the present invention will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be obtained from the market.

Example 1 Construction of PD-L1 single-chain antibody

The PD-L1scFv-Fc chimeric fragment of the present invention, namely the single-chain antibody chimeric form, is designed as follows: heavy chain variable region + connecting flexible peptide + light chain variable region + human Fc fusion protein tag + stop codon.

It encodes 486 amino acids, including 259 murine antibody amino acids, and the heavy chain variable region (FR-H1-CDR-VH1-FR-H2-CDR-VH2-FR-H3-CDR-VH3-FR-H4) 138 including 19 signal peptide amino acids whose sequence is shown in SEQ ID NO: 22, 15 artificial flexible linker peptide (Linker) amino acids, followed by light chain variable region (FR-L1-CDR-VL1- FR-L2-CDR-VL2-FR-L3-CDR-VL3-FR-L4) 106 amino acids, the light chain variable region amino acids do not include the leader sequence amino acids. Subsequently, the adaptor sequence is the 227 human Ig-Fc amino acid sequence (Human Fc tag) shown in SEQ ID NO: 17. The tag sequence can be used for purification and detection of expressed proteins. Its amino acid design structure is as follows:

Signal peptide+Variable region of heavy chain+Linker+Variable region of light chain+Human Fc tag+Stop codon

Example 2 Expression and purification of PD-L1 antibody in host cells

The full-length DNA containing the light chain (sequence shown in SEQ ID NO: 23) and the full-length DNA containing the heavy chain (sequence shown in SEQ ID NO: 24) were cloned into pcDNA3.1 or pcDNA3.4, respectively, and transformed into competent large intestine. Bacillus, amplify the plasmid, after enzyme digestion and sequencing to confirm the correctness of the sequence, use a spectrophotometer to quantify, the following plasmid dosage is the DNA mass (μg) after mixing the light chain vector and the heavy chain expression vector according to 2:1, using Expressed by transient transfection in mammalian cells ExpiCHO-S ^™ (ThemoFisher).

Day 1: Freshly thawed ExpiCHO-S ^™ cells were thawed and expanded to a cell density of approximately ^4-6 x 106 viable cells/mL.

Day 2: Split ^ExpiCHO -S ^™ cultures, adjust cell density to 3-4 x 106 viable cells/mL, and grow cells overnight.

Day 3: Viable cell density and percent viability were determined. The cell density should reach approximately 7-10 x ¹⁰⁶ viable cells/mL. The viability should be 95-99% before proceeding with transfection.

Dilute the cells to a final density of 6 x 10 ⁶ viable cells/mL using freshly pre-warmed ExpiCHO ^TM expression medium at 37°C, and mix the cells by gently shaking the culture flask.

Day 4: Transfection. Prepare ExpiFectamine ^™ CHO/plasmid by mixing well with cold reagents (4°C), ExpiFectamine ^™ CHO and cold plasmid solution. Take the concentration of 0.5-1.0 μg DNA/mL of the total plasmid of the light chain variable region as the transfection culture volume (take a 1-liter culture flask system as an example, the culture volume is 280 mL, the volume of plasmid DNA is 8 mL, and the total amount of plasmid needs 4.0- 8.0 μg).

Specifically, follow the steps described below: (i) Gently invert the ExpiFectamine ^™ CHO reagent bottle upside down 4-5 times and mix thoroughly. Dilute plasmid DNA with cold OptiPRO ^™ medium (ThemoFisher), shake or invert the tube, mix with DNA at room temperature, make total plasmid DNA 0.5-1.0 μg/mL culture volume, and mix well. (ii) Dilute 640 μL of ExpiFectamine ^™ CHO reagent (ThemoFisher) with OptiPRO ^™ 7.4 mL medium, shake or invert the tube, and mix. (iii) Add the diluted ExpiFectamine ^™ CHO reagent to the diluted DNA, shake or invert the tube, and mix. (iV) Incubate the ExpiFectamine ^™ CHO/plasmid DNA complex for 1-5 minutes at room temperature (immediately add the ExpiFectamine ^™ CHO/DNA complex to the cells, mix for no more than 5 minutes, then slowly transfer the solution to the flask in step 4) , gently shake the culture flask during the addition process. Place the cells on an orbital shaker and culture in a 37°C incubator with 8% _CO in humidified air at a shaking speed of 120±5rpm (25-mm orbital). .

Day 5: The next day after transfection (Day 1, 18-22 hours after transfection), add ExpiFectamine ^TM CHO Enhancer 1.2 mL and ExpiCHO ^TM Adjuvant 48 mL according to the chosen protocol: Transfer the flask to 32 Cultivate with shaking in a humidified air incubator containing 5% CO ₂ .

Day 12-14: Collect culture supernatant. The optimal collection time for antibody protein is usually 8-10 days after transfection.

Antibody purification: centrifuge at 4000g for 20min, remove cell particles or debris, filter with 0.22μm or 0.45μm membrane filter, measure the pH of the supernatant, use 0.2M Na ₂ HPO ₄ or 0.2M NaH ₂ PO ₄ (1M NaOH or 1M HCl) to adjust the pH to 7.0-7.4, fully equilibrate a protein A purification column (Sigma) with buffer (PBS, pH 7-7.4), and load the cell culture supernatant at 4°C with an AKTA system or a peristaltic pump into a fully equilibrated On the column, equilibrate the column with 3CV-5CV of binding buffer; elute the antibody/protein from the column using the AKTA system from the elution buffer. The eluate was collected at 1-3 mL/min according to UV280 absorbance, desalted through a desalting column, quantified by spectrophotometer, and stored in aliquots at -80 degrees or freeze-dried.

Example 3 Preparation method of anti-human PD-L1 monoclonal antibody

1. Immunization of animals

The immunogen is soluble antigen PD-L1 tag (Fc) protein (Yiqiao Company). Purebred BALB/c mice aged 4-6 weeks were selected, and the initial immunization started 2 months before fusion. In order to increase the immunogenicity of soluble proteins, the adjuvants used in this experiment included Freund's complete adjuvant, Freund's incomplete adjuvant Adjuvant (Sigma).

On day 0, the primary immunization was carried out, the antigen was emulsified in complete adjuvant, 0.25 mL per mouse, 10-100 μg, and immunized by intraperitoneal injection, a total of 5 mice. On the 14th day, the second immunization, incomplete adjuvant emulsified antigen, 0.25mL per mouse, 10-50μg, was immunized by intraperitoneal injection. On the 24th day, blood was collected from the tail vein, centrifuged at 37°C for 1 hour and 4°C for 2 hours, the supernatant was collected, and the serum titer was tested for the first time. On the 35th day, three immunizations, emulsified antigen with incomplete adjuvant, 0.25mL per mouse, 10-50μg, were immunized by intraperitoneal injection.

On the 45th day, blood was collected from the tail vein, centrifuged at 37°C for 1 hour and 4°C for 2 hours, the supernatant was collected, and the serum titer was detected by ELISA. The titers of the 5 immunized mice all reached or exceeded 1:512000, and 2 mice with a titer of more than 1:512000 were selected. On the 56th day, the final immunization was performed, and the antigen was dissolved in PBS, 0.25 mL per mouse. , 10-50μg, tail vein and intraperitoneal injection immunization. One week later (day 60), cell fusion was performed.

2. Cell fusion

(1) Preparation of feeder cell layer: select mouse peritoneal macrophages. Mice of the same strain as the immunized mice, 6-10 weeks old BALB/c mice, were sacrificed by pulling the neck, immersed in 75% alcohol for 3-5 min, cut the skin with sterile scissors, exposed the peritoneum, and treated with sterile Inject 5-6mL of pre-cooled culture solution into the bacterial syringe, rinse repeatedly, aspirate the rinse solution, put the rinse solution into a 10mL centrifuge tube, separate at 1200rpm/min for 5-6min, and use 20% calf serum (NCS) or fetal calf serum (FCS). ), adjust the number of cells to 1×10 ⁵ /mL, add 100 μL/well to a 96-well plate, and culture in a 37°C CO ₂ incubator.

(2) Preparation of immune spleen cells: 3 days after the last booster immunization, the mice were sacrificed by necking, the spleen was aseptically removed, and the culture medium was washed once. The spleen was crushed, passed through a stainless steel mesh, and after centrifugation, the cells were washed twice with culture medium, counted, and 10 ⁸ spleen lymphocyte suspensions were taken for use.

(3) Preparation of myeloma cells: centrifuge logarithmically growing myeloma cells, wash twice with serum-free medium, count, and take 1×10 ⁷ cells for use.

(4) Fusion:

① Mix myeloma cells and spleen cells in a ratio of 1:10 or 1:5, wash once with serum-free incomplete culture medium in a 50mL centrifuge tube, and centrifuge at 1200rpm/min for 8min; discard the supernatant , and suck up the residual liquid with a pipette so as not to affect the polyethylene glycol (PEG) concentration. Gently flick the bottom of the centrifuge tube to loosen the cell pellet slightly.

② Add 1 mL of 45% PEG (molecular weight 4000) solution pre-warmed at 37° C. within 90 s, and shake slightly while adding. In a water bath at 37°C for 90s.

③ Add 37 ℃ pre-warmed incomplete culture medium to stop the PEG effect, add 1mL, 2mL, 3mL, 4mL, 5mL and 6mL respectively every 2min.

④ Centrifuge at 800rpm/min for 6min.

⑤ Remove the supernatant and resuspend with HAT selection medium containing 20% calf serum.

⑥ Add the above cells to the existing feeder cell layer 96-well plate, add 100 μL to each well, and inoculate 10 96-well plates with one immune spleen, totaling 20 pieces.

⑦Put the culture plate in a 37°C, 5% CO ₂ incubator.

3. Primary screening of hybridomas and detection of secreted antibodies

(1) HAT selects hybridomas. After spleen cells and myeloma cells were treated with PEG, a mixture of various cells was formed. After being maintained in the HAT medium for 7-10 days, the HT medium was used for 2 weeks, and the general medium was used. During the above selection and culture period, when the hybridoma cells cover 1/10 of the area of the bottom of the well, the detection of specific antibodies begins, and the desired hybridoma cell line is screened out. During the selection culture period, half of the culture medium is generally changed every 2 to 3 days.

(2) The antibody was detected by enzyme-linked immunosorbent assay (ELISA), which was coated with human PD-L1 (including PD-L1-Fc and PD-L1-his) fusion protein at a concentration of 2 μg/mL, and the hybridoma culture supernatant was detected. , 40 positive wells (PD-L1-Fc negative and PD-L1-his positive) were preliminarily screened in this experiment, and further cloned .

4. Hybridoma Cloning

The positive hybrid clones are cloned to avoid being inhibited by the non-secreting cells of the antibody, resulting in the loss of the cells secreting the antibody. The cloned hybridoma cells also need to be re-cloned regularly to prevent the hybridoma cells from mutating or chromosomal loss. , thereby losing the ability to produce antibodies. In this experiment, the limited dilution method was used for cloning, and the antibodies screened in the experiment were subcloned more than 4 times.

A layer of feeder cells (confluent with cells) was prepared 1 day before cloning. Gently pipette the hybridoma cells to be cloned from the wells and count. Adjust the cells to 3-10 cells/mL. Take the cell culture plate with the feeder cell layer prepared on the previous day, add 100 μL of diluted cells to each well, and incubate in a 37°C, 5% CO ₂ incubator. The medium was changed on the 7th day, and every 2 to 3 days thereafter. The formation of cell clones can be seen in 8 to 9 days, and the antibody activity can be detected by ELISA in time.

Cells from positive wells were transferred to 24-well plates for expansion.

Antibodies were detected, culture expanded, and recloning repeated if necessary.

5. Hybridoma cell cryopreservation and recovery

(1) The ability to secrete antibodies may be lost during cell culture. Hybridoma cryopreservation can verify hybridoma stability and long-term preservation of hybridoma strains. Cryopreservation of hybridoma cells Each ampoule contains more than 1×10 ⁷ , and the cell cryopreservation medium: 50% calf serum; 40% incomplete culture medium; 10% DMSO (dimethyl sulfoxide). During cryopreservation, it can be immediately dropped from room temperature to 0°C, then placed in -70°C ultra-low temperature refrigerator, and transferred to liquid nitrogen the next day. Cell cryopreservation devices can also be used for cryopreservation. Cryopreserved cells should be thawed regularly, and the viability of the cells and the stability of secreted antibodies should be checked. Cells can be stored in liquid nitrogen for several years or longer.

(2) Cell recovery: Carefully remove the glass ampoule from liquid nitrogen, put it in a water bath at 37°C, thaw the frozen cells within 1 min, wash the cells twice with complete culture medium, and then move them into the feeder prepared the previous day. Layer cells in a culture flask, and culture them in a 37°C, 5% _CO2 incubator. When the cells form colonies, ELISA detects the antibody activity of the supernatant. The present invention relates to hybridomas undergoing repeated freezing and thawing processes, and their antibody secretion activity and yield Stablize.

6. Full-length sequencing of hybridoma antibody nucleic acid

试剂(AMBION，CAT，编号：15596-026)技术手册，从杂交瘤细胞中分离出总RNA，依据PrimeSeCpTTM第一链cDNA合成试剂盒(Takara，CAT，号码：610A)技术手册，使用同种型特异性反义引物(或通用引物)将总RNA逆转录成cDNA，根据GESTcript的cDNA末端(RACE)快速扩增标准操作程序(SOP)扩增VH、VL、CH和CL的抗体片段，将扩增的抗体片段分别克隆到标准克隆载体中、测序，可变区的序列分析工具：(i)NCBI Nucleotide BLAST；(ii)IMGT/V Quest program；(iii)NCBI Ig BLAST.选用菌落PCR筛选具有正确大小插入物，不少于五个具有正确大小插入物的每个片段被测序，对不同克隆序列进行比对，得到一致测序结果。in accordance with

Reagent (AMBION, CAT, code: 15596-026) technical manual, isolation of total RNA from hybridoma cells, according to PrimeSeCpTTM first-strand cDNA synthesis kit (Takara, CAT, code: 610A) technical manual, using isotype Specific antisense primers (or universal primers) reverse-transcribe total RNA into cDNA, and amplify the antibody fragments of VH, VL, CH, and CL according to GESTscript's cDNA End (RACE) Rapid Amplification Standard Operating Procedure (SOP), and the amplified The increased antibody fragments were cloned into standard cloning vectors, sequenced, and sequence analysis tools for variable regions: (i) NCBI Nucleotide BLAST; (ii) IMGT/V Quest program; (iii) NCBI Ig BLAST. Correct-sized inserts, each fragment of no less than five inserts of the correct size was sequenced, and the sequences of different clones were aligned to obtain consistent sequencing results.

Example 4 Detection of sPD-L1 by sandwich ELISA

The antibody in Example 1 is used as a non-blocking antibody, with another antibody X (the amino acid sequence of the light chain variable region VL of antibody X is shown in SEQ ID NO: 18; the amino acid sequence of the heavy chain variable region VH is as shown in SEQ ID NO: 18). SEQ ID NO: 19) for antigen capture antibody sandwich ELISA experiments.

1. Antibody coating

The X mAb with a final concentration of 2 μg/mL was diluted into the coating solution, and 100 μL per well was added to the ELISA-96-well plate. Cover with a lid or protective film and leave overnight at 4°C. The next day, take out the coated plate, discard the supernatant, wash 3 times with 1× Wash Buffer, discard the supernatant, put the liquid in the dry well on filter paper or toilet paper, and pat dry.

2. Close

200 μL/well of 5% milk was added to block non-specific binding sites. Cover with a lid or protective film, 37°C for 2 hours or 4°C overnight. The next day, discard the supernatant, wash once with 1× Wash Buffer, discard the supernatant, dunk the liquid in the dry wells on filter paper or toilet paper, and pat dry.

3. Incubation of Standards and Samples

Add diluted standard (or sample), 100 μL/well. Cover with a lid or protective film and leave at room temperature for 2 hours. Discard the supernatant, wash five times with 1× Wash Buffer, discard the supernatant, dry the liquid in the wells on filter paper or toilet paper, and pat dry.

4. Secondary antibody and HRP-Strep incubation

Add bio-labeled PD-L1 antibody in Example 1 at a final concentration of 2 μg/mL, 100 μL/well, cover with a lid or protective film, and place at room temperature for 1 hour. Discard the supernatant, wash five times with 1× Wash Buffer, discard the supernatant, and dunk the liquid in the dry wells on filter paper or toilet paper. Add HRP-Streptavidin (Cell signaling 3999S, 1:5000), 100 μL/well, cover with a lid or protective film, and place at room temperature for half an hour. Discard the supernatant, wash five times with 1× Wash Buffer, discard the supernatant, and dunk the liquid in the dry wells on filter paper or toilet paper.

5. Detection

Add 100 μL/well of A+B mixture, and develop color at room temperature for 5-10 minutes in the dark. Add 50 μL/well of stop solution and mix well. OD values at 450 nm were read using a plate reader (Thermo scientific Multiskan GO). Build a standard curve and calculate the sample concentration.

Experimental example 1 PD-L1 antibody specific identification

1.1 ELISA identification

Experiments were carried out with reference to the method of Example 4, and PD-L1, EGFR, CTLA-4, CD137, and EpCam fusion proteins (all extracellular domain proteins) were coated at 1 μg/mL, all of which were his-tagged proteins. Antibody, HPR-labeled goat anti-mouse IgG was used as secondary antibody, and substrate was added to develop color, and OD450 value was determined. The experimental results are shown in FIG. 1 . The results show that the antibody in Example 1 of the present invention has strong PD-L1-his specificity.

1.2 Western blot identification

The conventional Western blot method was used for the experiment. The samples to be tested were PD-L1-Fc, PD-1-his, and PD-L1-his, which were stained with Ponceau red after SDS-PAGE electrophoresis and transferred to the membrane (left in Figure 2). The antibody of Example 1 was incubated, and ECL color was developed (Fig. 2 right). The results showed that there were protein bands in the PD-L1-his and PD-L1-Fc lanes, indicating that the antibody of the present invention only binds to PD-L1 but not to PD-1.

Experimental example 2 PD-L1 antibody affinity detection

The specific method is as follows:

1. The AMC sensor (PALL Corporation, USA) was pre-wetted in equilibration solution (0.1% BSA+0.02% TWEEN20 in PBS) for 10 minutes, and the PD-L1 antibody was diluted with equilibration solution to 20 μg/mL, and added to 96 wells protected from light. In the second column of the plate, 200 μL/well.

2. Dilute PD-L1-his from 500nM to 7.8nM, add 200μL/well to the fourth column of a 96-well plate protected from light, and add 200μL of equilibration solution to well H4 as blank control.

3. Add the equilibration solution to the first column and the third column, 200 μL/well.

4. Using a Fortebio octet96 instrument, equilibrate the sensor in the first column for 120 seconds to obtain a basic equilibrium curve, and then perform antibody immobilization in the second column for 300 seconds. Re-equilibrate in the third column for 120 seconds, bind the antigen in the fourth column for 180 seconds to obtain the binding curve, and return to the third column to dissociate for 300 seconds to obtain the dissociation curve.

5. Fit the curve with Fortebio Octet96 analysis software to obtain the affinity value.

Table 1 Fortebio Octet analysis of PD-L1 antibody binding and dissociation results of PD-L1

The experimental results are shown in Figure 3 and Table 1. The Fortebio Octet Molecular Interaction Analyzer determines that the binding affinity of the antibody of Example 1 to PD-L1-his protein is 6.96×10 ^-9 mol/L, that is, it can reach 10 ^-9 mol/ L, and the smaller the affinity, the stronger the binding ability.

Experimental Example 3 Validation of PD-L1 antibody in the application of flow cytometry

3.1 PD-L1 antibody detection of PD-L1 on cell membrane

Referring to the method of Example 2, the pcDNA3.1-PD-L1 plasmid with pGFP tag was transferred into 293 cells, and the expression of GFP (FITC) in this recombinant cell was detected by flow cytometry, and then the transfection rate was obtained. , and the experimental results are shown in Figure 4. As can be seen from Figure 4, 37.1% of cells were GFP positive, that is to say, the transfection rate was 37.1%.

The expression of PD-L1 in the recombinant cells was detected by flow cytometry, and the specific methods were as follows:

Take a total of 1×10 ⁶ cells and suspend them in 100 μL of PBS buffer containing 1% bovine serum, add 1 μg of PD-L1 antibody in a volume of 1-2 μL, and stain at room temperature for 30 min. Resuspend the cells, mix gently, centrifuge at 1000 rpm, wash twice, then resuspend the cells in 100 μL of 1% bovine serum in PBS buffer, add goat anti-mouse IgG-APC (about 1 μg), stain for 30 min, and then pass After washing twice with 1% bovine serum PBS buffer, the cells were suspended in 1 mL of 1% bovine serum PBS buffer, and the expression level of PD-L1 on the cell surface was analyzed by flow cytometry.

The experimental results are shown in Figure 5, which shows that PD-L1 antibody staining in GFP-positive cells is also positive, indicating that the PD-L1 antibody in the present invention can detect PD-L1 on the cell membrane with high accuracy.

3.2 PD-L1 antibody detection of PD-L1 in tumor cells

For the experimental method, refer to Experimental Example 3.1. The sample to be tested is H2009 human lung adenocarcinoma cells. The H2009 cell line was incubated with PD-L1 antibody or not (blank control), and then incubated with goat anti-mouse IgG-PE secondary antibody, and then flow cytometry analysis. , the left peak is not incubated with antibody, and the right peak is incubated with antibody. The experimental results are shown in Figure 6. The results show that the PD-L1 antibody of the present invention can be used as a flow antibody to detect PD-L1 in cancer cells.

Experimental Example 4 Validation of PD-L1 antibody in ELISA application

4.1 Standard curve of sandwich ELISA established by PD-L1 antibody

Referring to the experimental method of Example 4, the PD-L1 antibody was used as the non-blocking antibody, the biotin was used as the detection antibody, and the antibody X was used as the capture antibody to establish a paired sandwich ELISA to detect the diluted PD-L1-his protein. Standard. The results show that the standard curve has a high fit, R ² is 0.9998, and the expected sensitivity (The expected sensitivity) is 5 pg/mL. In other words, the detection sensitivity of PD-L1 antibody is high, and the minimum detection concentration is 5 pg/mL. (Figure 7, Table 2).

Table 2 Standard concentration and corresponding OD450 reading in the standard curve

Antigen concentration (pg/mL) 10000 2000 400 80 16 3.2 0.6 0 OD450 reading 3.7540 3.4865 2.7289 1.3016 0.4757 0.2005 0.1179 0.0912

4.2 PD-L1 antibody detection of PD-L1 in tumor patients

Referring to the experimental method of Example 4, 40 serum samples from normal patients and 40 patients with advanced lung cancer were taken, and after 1:25 dilution, sandwich ELISA was used for PD-L1 determination. The results showed that there was a significant difference in serum sPD-L1 concentration between the two groups (p<0.001) (Fig. 8).

Experimental Example 5 The blocking effect of PD-L1 antibody on the binding of PD-1 to PD-L1

5.1 Flow cytometry to detect the blocking effect of PD-L1 antibody on the binding of PD-1 to PD-L1

Referring to the experimental method of Experimental Example 3, 293 cells transfected with pcDNA-3.1-PD-L1 were incubated with PD-1-his protein, and anti-his fluorescent secondary antibody was added. The results showed that the positive rate was 22.7% (Figure 9); PD -L1 293 transfected cells were first incubated with PD-L1 antibody, and then incubated with PD-1-his and anti-his fluorescent secondary antibodies respectively. The results showed that the positive rate was 29.9% (Figure 10), indicating that PD-L1 antibody does not Block the binding of PD-1-his protein to transfected cells.

5.2 ELISA to detect the blocking effect of PD-L1 antibody on the binding of PD-1 to PD-L1

Coated with PD-1-his 1 μg/ml, and mixed PD-L1-Fc and PD-L1 antibody at 4 degrees overnight. The next day, the mixture was added to the ELISA plate coated with PD-1-his and incubated at room temperature for 2 h, and detected with HRP-streptavidin. Read the OD450 value (Figure 11); and calculate the blocking rate: blocking rate (inhibition rate)=(OD1-OD0/OD0).

Experiments showed that PD-L1 antibody did not block the binding of PD-1-his protein to transfected cells.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand: It is still possible to modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements to some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the embodiments of the present invention. range.

SEQUENCE LISTING

<110> Beijing Chest Hospital Affiliated to Capital Medical University; Beijing Institute of Tuberculosis and Thoracic Oncology

<120> An anti-human PD-L1 antibody and its application

<130> 20180910

<160> 24

<170> PatentIn version 3.3

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290 295 300

Lys Trp Glu Lys Thr Asp Ser Phe Ser Cys Asn Val Arg His Glu Gly

305 310 315 320

Leu Lys Asn Tyr Tyr Leu Lys Lys Thr Ile Ser Arg Ser Pro Gly Lys

325 330 335

<210> 17

<211> 227

<212> PRT

<213> Synthetic

<400> 17

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

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

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys

225

<210> 18

<211> 106

<212> PRT

<213> Synthetic

<400> 18

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

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Asp Thr Ser Pro Leu Ala Ser Gly Phe Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Ile Ile Ser Ser Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Phe Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 19

<211> 119

<212> PRT

<213> Synthetic

<400> 19

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Thr Ser Gly Phe Thr Phe Ser Ser Ser

20 25 30

Tyr Ile Ser Trp Leu Lys Gln Lys Pro Gly Gln Ser Leu Glu Trp Ile

35 40 45

Ala Trp Ile Phe Ala Gly Thr Gly Gly Thr Ser Tyr Asn Pro Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg His Glu Gly Lys Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 20

<211> 19

<212> PRT

<213> Synthetic

<400> 20

Met Ser Ser Ala Gln Phe Leu Gly Leu Leu Leu Leu Cys Phe Gln Gly

1 5 10 15

Thr Arg Cys

<210> 21

<211> 19

<212> PRT

<213> Synthetic

<400> 21

Met Glu Trp Thr Trp Leu Phe Leu Phe Leu Leu Ser Val Thr Ala Gly

1 5 10 15

Val His Ser

<210> 22

<211> 19

<212> PRT

<213> Synthetic

<400> 22

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser

<210> 23

<211> 702

<212> DNA

<213> Synthetic

<400> 23

atgtcctctg ctcagttcct tggtctcctg ttgctctgtt ttcaaggtac cagatgtgat 60

atccagatga cacagactac atcctccctg tctgcctctc tgggagacag agtcaccatc 120

acttgcagtg caagtcaggg cattagcaat tatttaaact ggtatcagca gaatccagat 180

ggaactgtta aactcctgat ctattacaca tcacatttac actcaggagt ctcatccagg 240

ttcagtggca gtgggtctgg gacagattat tctctcacca tcagcaacct ggaacctgaa 300

gatattgcca cttactattg tcaacagtat gtcaagcttc cgtggacgtt cggtggaggc 360

accaagctgg aaatcaaacg ggctgatgct gcaccaactg tatccatctt cccaccatcc 420

agtgagcagt taacatctgg aggtgcctca gtcgtgtgct tcttgaacaa cttctacccc 480

aaagacatca atgtcaagtg gaagattgat ggcagtgaac gacaaaatgg cgtcctgaac 540

agttggactg atcaggacag caaagacagc acctacagca tgagcagcac cctcacgttg 600

accaaggacg agtatgaacg acataacagc tatacctgtg aggccactca caagacatca 660

acttcaccca ttgtcaagag cttcaacagg aatgagtgtt ag 702

<210> 24

<211> 1413

<212> DNA

<213> Synthetic

<400> 24

atggaatgga cctggctctt tctcttcctc ctgtcagtaa ctgcaggtgt ccactcccag 60

gttcagctgc agcagtctgg aactgagctg atgaagcctg gggcctcagt gaagatatcc 120

tgcaaggcta ctggttacac attcagtaac tactggatag agtgggtaaa gcagaggcct 180

ggacatggcc ttgagtggat tggagagatt ttacctggaa gtggtaatac taactacaat 240

gagaacttca agggcaaggc cacattcact gcagatacat cctccaatac agcctacatg 300

caactcagca ggctgacatc tgaggactct gccgtctatt attgtgcaag agagagggct 360

tcgacttcgt ggggccaagg gactctggtc actgtctctg cagccaaaac aacaccccca 420

tcagtctatc cactggcccc tgggtgtgga gatacaactg gttcctccgt gactctggga 480

tgcctggtca agggctactt ccctgagtca gtgactgtga cttggaactc tggatccctg 540

tccagcagtg tgcacacctt cccagctctc ctgcagtctg gactctacac tatgagcagc 600

tcagtgactg tcccctccag cacctggcca agtcagaccg tcacctgcag cgttgctcac 660

ccagccagca gcaccacggt ggacaaaaaa cttgagccca gcgggcccat ttcaacaatc 720

aacccctgtc ctccatgcaa ggagtgtcac aaatgcccag ctcctaacct cgagggtgga 780

ccatccgtct tcatcttccc tccaaatatc aaggatgtac tcatgatctc cctgacaccc 840

aaggtcacgt gtgtggtggt ggatgtgagc gaggatgacc cagacgtcca gatcagctgg 900

tttgtgaaca acgtggaagt acacacagct cagacacaaa cccatagaga ggattacaac 960

agtactatcc gggtggtcag caccctcccc atccagcacc aggactggat gagtggcaag 1020

gagttcaaat gcaaggtcaa caacaaagac ctcccatcac ccatcgagag aaccatctca 1080

aaaattaaag ggctagtcag agctccacaa gtatacatct tgccgccacc agcagagcag 1140

ttgtccagga aagatgtcag tctcacttgc ctggtcgtgg gcttcaaccc tggagacatc 1200

agtgtggagt ggaccagcaa tgggcataca gaggagaact acaaggacac cgcaccagtc 1260

ctggactctg acggttctta cttcatatat agcaagctca atatgaaaac aagcaagtgg 1320

gagaaaacag attccttctc atgcaacgtg agacacgagg gtctgaaaaa ttactacctg 1380

aagaagacca tctcccggtc tccgggtaaa tga 1413

Claims (24)

1. an antibody comprising an antigen binding domain, wherein the antigen binding domain comprises the complementarity determining region of the following amino acid sequence; The amino acid sequences of the complementarity determining regions CDR-VL1, CDR-VL2, and CDR-VL3 are shown in SEQ ID NOs: 1-3, respectively; and The amino acid sequences of the complementarity determining regions CDR-VH1, CDR-VH2, and CDR-VH3 are shown in SEQ ID NOs: 4-6, respectively. 2. The antibody according to claim 1, wherein the antibody comprises the light chain framework regions FR-L1, FR-L2, FR-L3 and FR-L1, FR-L2, FR-L3 and FR- L4, and/or the heavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4 in sequence as shown in SEQ ID NOs: 11-14. 3. The antibody of claim 1, wherein the antibody further comprises an antibody constant region Fc. 4. The antibody of claim 3, wherein the constant region Fc comprises a light chain constant region and a heavy chain constant region. 5. The antibody of claim 4, wherein the light chain constant region sequence is shown in SEQ ID NO: 15. 6. The antibody of claim 4, wherein the heavy chain constant region sequence is shown in SEQ ID NO: 16. 7. The antibody of claim 4, wherein the sequence of the constant region Fc is selected from the sequence of any one of the constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD. 8. The antibody of claim 4, wherein the sequence of the constant region Fc is selected from the sequence of the constant region of IgG2b. 9. The antibody of claim 4, wherein the sequence of the constant region Fc is selected from the sequence of the IgG2b/kappa constant region. 10. The antibody of claim 4, wherein the species source of the constant region Fc is bovine, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey , deer, mink, chicken, duck, goose or human. 11. The antibody according to claim 2, wherein the antibody is one of F(ab')2, Fab', Fab, Fv, Fd, scFv, scFv-Fc chimeric fragment and bispecific antibody kind. 12. The antibody of claim 2, wherein the antibody is a scFv-Fc chimeric fragment, and the scFv-Fc chimeric fragment comprises the constant region Fc described in claim 3, claims 1 to 1 The complementarity determining region of any one of 3, the light chain framework region of claim 2 and the heavy chain framework region. 13. The antibody of claim 12, wherein the constant region Fc is human Ig-Fc. 14. The antibody of claim 13, wherein the human Ig-Fc amino acid sequence is shown in SEQ ID NO:17. 15. The antibody of claim 12, wherein the scFv-Fc chimeric fragment further comprises an artificial flexible linker peptide. 16. An isolated nucleic acid molecule, wherein the nucleic acid molecule is DNA or RNA, which encodes the antibody of any one of claims 1 to 15. 17. A vector comprising the nucleic acid molecule of claim 16. 18. A host cell, wherein the host cell comprises the nucleic acid molecule of claim 16 or the vector of claim 17. 19. Use of the antibody of any one of claims 1 to 15 in the preparation of a medicament for diagnosing a disease, the disease being cancer. 20. Use of the antibody of claim 19 in the preparation of a medicament for diagnosing diseases, the cancers comprising lung cancer, melanoma, NSCLC, classic Hodgkin's lymphoma, HNSCC, renal cell carcinoma, urothelial carcinoma, One or more of head and neck cancer, gastric cancer, hematological malignancies, prostate cancer, cervical cancer, brain cancer, hepatocellular cancer, and colorectal cancer. 21. An antibody combination product comprising the antibody of any one of claims 1 to 15 and a second antibody; The second antibody specifically recognizes PD-L1, and its recognition epitope is different from the antibody according to any one of claims 1 to 15. 22. The antibody product according to claim 21, wherein the amino acid sequence of the light chain variable region VL of the second antibody is respectively as shown in SEQ ID NO: 18; the amino acid sequence of the heavy chain variable region VH is respectively as shown in SEQ ID NO:19 shown. 23. The antibody product of claim 21, further comprising one or more of PBS, BSA, ddH2O, glycerol, _sodium azide, and gentamicin. 24. A test kit, characterized in that, the test kit comprises the antibody of any one of claims 1 to 15, the nucleic acid molecule of claim 16, the carrier of claim 17 and claims 21 to 21. One or more of the antibody combination products described in 23.

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