CN108997500B - Anti-human PD-L1 antibody and application thereof - Google Patents

Abstract

The invention relates to the field of immunological technology, in particular to an anti-human PD-L1 antibody and application thereof, wherein the antibody comprises an isolated binding protein of an antigen binding structural domain, the antigen binding structural domain comprises at least one complementarity determining region selected from the following amino acid sequences, or the complementarity determining region has at least 80% sequence identity with the amino acid sequence, and the protein has Kd less than or equal to 8.0 × 10 and is PD-L1 protein^‑9(ii) a 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 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

Anti-human PD-L1 antibody and application thereof

Technical Field

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

Background

The incidence of lung cancer, especially female lung cancer, is on the rise worldwide, the lung cancer is the malignant tumor with the highest incidence in China, and the mortality rate is the first of the malignant tumors. At present, the international developed countries have achieved effect on the prevention and treatment of lung cancer, the death rate of lung cancer is decreasing year by year, besides smoking control and early discovery, the precise treatment means represented by targeted therapy also benefits lung cancer patients, especially in recent years, the immunotherapy of lung cancer makes substantial breakthrough, and the effective therapy of lung cancer is remarkably prolonged compared with the traditional chemotherapy. The lung cancer immunotherapy mainly aims at an expression molecule PD-1 (programmed death receptor-1) expressed in T lymphocytes in a failure state, utilizes a specific antibody of the molecule or a ligand (PD-L1) antibody thereof to block a negative regulatory signal of the T cells mediated by PD-1-PD-L1 so as to reverse the failure situation of the T cells and restore the activity of the T cells in killing tumor cells, except for the maximum indication of the lung cancer, the PD-1-PD-L1 blocks and treats MSI-H (highly microsatellite unstable) or mismatch DNA repair defect which is widely used for melanoma, kidney cancer, bladder cancer, Hodgkin lymphoma, head and neck cancer, cervical cancer, gastric cancer, intestinal cancer and the like, has broad-spectrum anti-tumor activity, and the current therapeutic antibody of the lung cancer PD-1 target mainly comprises Keytruda and Opdivo, and the therapeutic efficiency of the Keytruda and Opdivo to lung cancer patients is about 20 percent, the 5-year survival rate of the advanced lung cancer patient receiving the PD-1 antibody treatment is improved by 5-6 times, so that the screening of effective candidate patients for treatment has important clinical significance, and blind medication can be avoided. The current prediction methods with clinical guidance include tumor mutation load (TMB), MSI assay and PD-L1 expression assay, the PD-L1 assay is the most classical assay, two detection reagents are used internationally, namely Ventana PD-L1 rabbit monoclonal antibody (clone SP142) and PD-L1, IHC22C3pharmDx mouse monoclonal antibody atezolizumab (clone 22C3), for immunohistochemical companion assay, and Keytruda has been admitted as a standard detection antibody for clinical tumor tissue PD-L expression levels, which detects tumor cell expression rates above 50%, and Keytruda can be used for first-line non-small cell lung cancer treatment. In addition, PD-L1 expression correlates with a variety of tumor prognoses, the detection of which can predict survival.

In view of the potential prediction of clinical guidance value, the method is limited to tumor mutation load (TMB), MSI detection, tissue PD-L1 expression analysis and other methods, the former two methods involve molecular biology means and nucleic acid whole genome sequencing, are expensive, rely on surgery or puncture to obtain tumor tissue, and have limited application in the detection of patients who are not susceptible to obtaining tissue specimens from non-surgical or metastatic lesions, and in addition, TMB and MSI detection have not been determined as clinical standard detection methods. In recent years, the approved antibody preparation is subjected to immunohistochemical PD-L1 expression analysis (the only diagnostic partner approved by FDA is PD-L1IHC22C3 pharmDx-used for screening patients treated by pembrolizumab), is relatively simple and has more common clinical application, and at present, the two reagent approved antibodies are limited to Immunohistochemical (IHC) concomitant detection and are also acquired by depending on tissue specimens aiming at tumor tissues; in addition, the PD-L1 negative still has unreliability, the detection result may be different due to different antibodies and different tissue samples, meanwhile, tumor tissue heterogeneity, low expression and induced genes may also cause sampling errors or false negative, especially, the immunohistochemical staining analysis has subjective factors in clinical PD-L1 tissue expression critical value reading, therefore, the PD-L1-IHC positive still is an incomplete reaction biomarker at present, can not be used as a 'certainty' index for screening PD-1-PD-L1 inhibitor treatment patients, and an accurate, simple and feasible multi-component prediction marker with systematicness needs to be continuously discovered.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The present invention relates to a novel isolated binding protein comprising an antigen binding domain and studies on the preparation, use, etc. of the binding protein. The method can be efficiently used for supplementing the PD-L1 analysis method independent of a tissue sample of a tumor patient, an enzyme-linked immunosorbent assay (ELISA) technology suitable for determining the sPD-L1 level of a blood sample of a malignant tumor such as lung cancer is established, a small amount of blood samples of the patient need to be collected for detection during detection, blood collection is noninvasive or minimally invasive, repeated detection is facilitated, and the detection sensitivity is high.

The antigen binding domain comprises at least one complementarity determining region selected from the group consisting of amino acid sequences having at least 80% sequence identity with the complementarity determining region of the amino acid sequence and having a Kd of 8.0 × 10 to PD-L1 protein^-9Affinity of mol/L;

the amino acid sequences of the complementarity determining regions CDR-VL1, CDR-VL2 and CDR-VL3 are respectively shown in 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.

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

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a graph showing the results of identifying the specificity of PD-L1 antibody by ELISA in Experimental example 1 of the present invention;

FIG. 2 is a graph showing the result of identifying the specificity of the 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;

FIG. 3 shows the measurement of the affinity between the PD-L1 antibody and the PD-L1 protein in Experimental example 2 of the present invention;

FIG. 4 is a graph showing the detection of the transfection efficiency of pcDNA3.1-PD-L1 plasmid by flow cytometry in Experimental example 3 of the present invention; FITC-A: GFP; APC-A: PD-L1;

FIG. 5 is a graph showing the results of flow cytometry for detecting 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;

FIG. 6 is a graph showing the results of flow cytometry for detecting 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 a graph showing the standard curve of sandwich ELISA established by the PD-L1 antibody in Experimental example 4 of the present invention;

FIG. 8 is a graph showing the results of detection of sPD-L1 in serum of lung cancer patients and normal persons by the PD-L1 antibody in Experimental example 4 of the present invention;

FIG. 9 is a graph showing the results of detecting the expression of PD-L1 in 293 cells transfected by flow cytometry in Experimental example 5 of the present invention; APC-A: PD-L1;

FIG. 10 is a graph showing the results of flow cytometry for detecting the blocking of the binding of PD-L1 to PD-1 by PD-L1 antibody in Experimental example 5 of the present invention;

FIG. 11 is a graph showing the blocking results of the detection of the binding of PD-1 to PD-L1 by the PD-L1 antibody by ELISA in Experimental example 5 of the present invention.

Detailed Description

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

The term "isolated binding protein" is a protein that, due to its derivative origin or source, does not bind to the naturally associated component with which it is associated in its native state; substantially free of other proteins from the same species; expressed by cells from different species; or do not occur 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" broadly refers to all proteins/protein fragments that comprise a CDR region. The term "antibody" includes polyclonal and monoclonal antibodies and antigenic compound-binding fragments of these antibodies, including Fab, F (ab')2, Fd, Fv, scFv, diabodies and minimal recognition units of antibodies, as well as single chain derivatives of these antibodies and fragments. The type of antibody can be selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD. Furthermore, the term "antibody" includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, chimeric (chimeric), bifunctional (bifunctional) and humanized (humanized) antibodies, as well as related synthetic isomeric forms (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 the antibody. The variable domain of the heavy chain may be referred to as "VH". The variable domain of the light chain may be referred to as "VL". These domains are usually the most variable parts of an antibody and contain an antigen binding site. The light or heavy chain variable region (VL or VH) is made up of framework regions interrupted by three hypervariable regions, termed "complementarity determining regions" or "CDRs". The extent of the 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 (U.S. department of Health and human services), (1983), and Chothia. The framework regions of the antibody, which constitute the combination of the essential light and heavy chains, serve to locate and align the CDRs, which are primarily responsible for binding to the antigen.

As used herein, the "framework" or "FR" regions mean the regions of an antibody variable domain excluding those defined as CDRs. Each antibody variable domain framework can be further subdivided into adjacent regions separated by CDRs (FR1, FR2, FR3 and FR 4).

Typically, the variable domains VL/VH of the heavy and light chains are obtained by linking the CDRs and FRs numbered as follows in a combinatorial arrangement: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4.

As used herein, the term "purified" or "isolated" in relation to 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 that is removed from its original environment, e.g., from its natural environment if it is naturally occurring. For example, an isolated polypeptide is generally free of at least some proteins or other cellular components that are normally bound to or normally mixed with it or in solution. Isolated polypeptides include the naturally-produced polypeptide contained in a cell lysate, the polypeptide in purified or partially purified form, recombinant polypeptides, the polypeptide expressed or secreted by a cell, and the polypeptide in a heterologous host cell or culture. In connection with a nucleic acid, the term isolated or purified indicates, for example, that the nucleic acid is not in its natural genomic context (e.g., in a vector, as an expression cassette, linked to a promoter, or artificially introduced into a heterologous host cell).

Because PD-L1 in the in vivo tumor microenvironment is mainly expressed in tumor cells or other stroma cells of the tumor, the expression of PD-L1 can be digested and shed from the surfaces of cells such as the tumor and the like by interstitial protease or released into the blood circulation by different mRNA splicing proteins, the level of PD-L1 in the blood can potentially reflect the expression condition of PD-L1 in the tumor environment, the expression level is directly related to the size, necrosis and prognosis of a patient, and soluble PD-L1(sPD-L1) also plays a role in inhibiting the activity of T cells. sPD-L1 is more representative of PD-L1 levels in different organisms of origin, including tumor microenvironment tumor cells and other tumor stromal cells, and sPD-L1 is detectable in the sera of patients with various tumors and autoimmune diseases other than tumors and infectious diseases.

The present invention provides an isolated binding protein comprising an antigen binding domain comprising at least one complementarity determining region selected from the group consisting of amino acid sequences having at least 80% sequence identity with the complementarity determining region of amino acid sequence having a Kd of 8.0 × 10 with PD-L1 protein^-9Affinity of mol/L;

the amino acid sequences of the complementarity determining regions CDR-VL1, CDR-VL2 and CDR-VL3 are respectively shown in 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. It is well known in the art that both the binding specificity and avidity of an antibody are determined primarily by the CDR sequences, and that variants with similar biological activity can be obtained by readily altering the amino acid sequence of the non-CDR regions according to well-established, well-known techniques of the art. Thus, the invention also includes "functional derivatives" of the binding proteins. "functional derivative" refers to a variant of an amino acid substitution (which may be an antibody or fragment with the same or similar activity resulting from the substitution, deletion or addition of one or more amino acids), one functional derivative retaining detectable binding protein activity, preferably the activity of an antibody capable of binding cTnI. "functional derivatives" may include "variants" and "fragments" which have the exact same CDR sequences as the binding proteins of the invention and therefore 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%, or 96%, or 97%, or 98%, or 99% sequence to the complementarity determining region of an amino acid sequence described belowColumn identity with PD-L1 protein with Kd ≤ 8.0 × 10^-9The mol/L and Kd values can also be selected from 7.8 × 10^-9mol/L、7.5×10^-9mol/L、7×10^-9mol/L、6.96×10^-9mol/L、6.7×10^-9mol/L、6×10^-9mol/L、5.5×10^{- 9}mol/L、4×10^-9mol/L、2×10^-9mol/L、1.0×10^-9mol/L、9.0×10^-10mol/L、8.0×10^-10mol/L、7.0×10^-10mol/L、6.0×10^-10mol/L、5.0×10^-10mol/L、4.0×10^-10Affinity in mol/L, etc.

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

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

In some embodiments, the binding protein comprises light chain framework regions FR-L1, FR-L2, FR-L3 and FR-L4 in the sequence shown in SEQ ID Nos. 7-10, and/or heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4 in the sequence shown in SEQ ID Nos. 11-14.

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

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

In some embodiments, the constant region Fc comprises 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 IgG1, 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 the IgG2b constant region.

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

In some embodiments, the species of the constant region is derived from a cow, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, chicken fighting, or human.

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

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

In some embodiments, the composition of 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 code (Leader sequence is shown in SEQ ID NO: 20).

In some embodiments, the composition of heavy chain amino acids 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 code (Leader sequence is shown in SEQ ID NO: 21).

Constant region Fc.

In some embodiments, the binding protein is a whole antibody comprising a variable region and a constant region Fc.

In some embodiments, the binding protein is a "functional fragment" of an antibody, such as one of a nanobody, F (ab ')2, Fab', Fab, Fv, Fd, scFv-Fc chimeric fragment/diabody, and an antibody minimal recognition unit.

The term "functional fragment" as used herein refers in particular to an antibody fragment having the same specificity for PD-L1 as the parent antibody. In addition to the above functional fragments, any fragment having an increased half-life is also included. These functional fragments typically have the same binding specificity as the antibody from which they are derived. As the person skilled in the art deduces from the description of the invention, the antibody fragment of the invention may be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by chemical reduction cleavage of disulfide bonds. Antibody fragments can also be obtained by peptide synthesis by recombinant genetic 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 constant region Fc described above, the complementarity determining regions described above, the light chain framework regions and the heavy chain framework regions described above.

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.

The scFv (single chain antibody fragment) is a single-chain antibody fragment formed by connecting an antibody heavy chain variable region and a light chain variable region through an artificial flexible connecting peptide (Linker) with 15-20 amino acids.

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

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

In some embodiments, the artificial flexible linker peptide has a number of amino acids from 10 to 29, a sequence of (GGGGS) n, n being 2, 3, 4, 5, 6, or a sequence of GSTSGAGKSSEGKG. The artificial flexible connecting peptide also comprises other amino acid sequences with connecting effect.

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

or;

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

the Variable region of the heavy chain Variable region (FR-H1-CDR-VH1-FR-H2-CDR-VH2-FR-H3-CDR-VH 3-FR-H4).

The Variable region of light chain Variable region (FR-L1-CDR-VL1-FR-L2-CDR-VL2-FR-L3-CDR-VL 3-FR-L4).

The Signal peptide is the amino acid of the Signal peptide, and the sequence is shown as SEQ ID NO. 22.

It is also an aspect of the present invention to provide an isolated nucleic acid molecule which is DNA or RNA encoding a binding protein as described above.

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

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

Herein, a nucleic acid comprises conservatively substituted variants thereof (e.g., substitution of degenerate codons) and complementary sequences. The terms "nucleic acid" and "polynucleotide" are synonymous and encompass genes, cDNA molecules, mRNA molecules, and fragments thereof such as oligonucleotides.

In another aspect, the present invention provides a vector comprising the nucleic acid molecule described above.

The vector may contain a selectable marker, and an origin of replication compatible with the cell type specified by the cloning vector, while the expression vector contains the regulatory elements necessary to effect expression in a specified target cell. The vector can be a cloning vector and an expression vector, and comprises a plasmid vector, a phage vector, a virus vector and the like, when an antibody or a fragment is expressed or prepared, a prokaryotic expression vector and a eukaryotic expression vector are usually involved, a PET series and a pGEX series are usually used for the prokaryotic expression vector, pcDNA3.1, pcDNA3.4, pEGFP-N1, pEGFP-N1, pSV2 and the like are usually used for the eukaryotic expression vector, and the virus vector can be a lentivirus, a retrovirus, an adenovirus or an adeno-associated virus.

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

It is also a further aspect of the present invention to provide a host cell comprising a nucleic acid molecule as described above or a vector as described above.

The host cell mainly relates to eukaryotic cells, and the eukaryotic cells comprise mammalian cells, yeast cells and insect cells. Especially, the whole antibody or the full-length antibody is prepared, is commonly used for mammalian cells, and can be CHO, 293 and NSO cells.

In some embodiments, the host cell is a mammalian cell 293.

In some embodiments, the vector is introduced into the host cell by methods including lipofection and electroporation, such as Lipofectamine^TM、RNAiMAX、HiPerFect、DharmaFECT、X-tremeGENEsiLentFect^TMAnd TransIntro EL transformation Reagent. Viral vectors are introduced into mammalian cells by their natural mode of infection, e.g., retroviruses or lentiviruses, by preparing whole viral particles and adding them directly to cultured cells to infect mammalian cells.

In another aspect, the present invention provides a method for producing the above-described binding protein, comprising the steps of: the host cell described above is cultured in a medium, and the binding protein thus produced is recovered from the medium or from the cultured host cell.

Yet another aspect of the invention is to provide the use of a binding protein as described above for the preparation of a medicament for the diagnosis of a disease; the disease includes cancer and/or immune-related diseases.

In some embodiments, the cancer comprises one or more of lung cancer, melanoma, NSCLC, classical hodgkin's lymphoma, HNSCC, renal cell carcinoma, urothelial cancer, head and neck cancer, gastric cancer, hematological malignancies, prostate cancer, cervical cancer, brain cancer, hepatocellular carcinoma, and colorectal cancer.

In some embodiments, the immune-related disease comprises one or more of a viral infection, a bacterial infection, a fungal infection, a parasitic and rheumatoid arthritis, ulcerative colitis, pemphigus, dermatomyositis, alzheimer's disease.

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

It is also another aspect of the present invention to provide an antibody combination product comprising the above-described binding protein and a second antibody; the second antibody specifically recognizes PD-L1 and recognizes an epitope 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 separate containers (e.g., EP tubes).

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

In some embodiments, the antibody combination further comprises PBS, BSA, ddH₂One or more of O, glycerol, sodium azide and gentamicin.

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

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

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

In some embodiments, a second antibody that binds to the PD-L1 protein or the binding protein is also included in the immune complex.

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 article is blood, a cell, or a tissue.

In some embodiments, the binding protein, as a non-blocking antigen capture antibody, can form an antibody pair with an anti-PD-L1 antibody that is captured against PD-L1 antigen or has a different epitope, such as PD-1-PD-L1 anti-PD-L1 capture antibody, detected using a sandwich ELISA.

The binding protein or second antibody may be labeled with an indicator that indicates the strength of the signal so that the complex is readily detected.

In some embodiments, the indicator that shows signal intensity comprises any one of a fluorescent substance, a quantum dot, a digoxigenin-labeled probe, biotin, a radioisotope, a radiocontrast agent, a paramagnetic ion fluorescent microsphere, an electron-dense substance, a chemiluminescent label, an ultrasound contrast agent, a photosensitizer, colloidal gold, or an enzyme.

In some embodiments, the fluorescent species include 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-carboxytetramethylrhodamine, Cascade Blue, Cy2, Cy3, Cy5, Cy7, 6-FAM, dansyl chloride, fluorescein, HEX, 6-JOE, NBD (7-nitrobenz-2-oxa-1, 3-diazole), Any one of Oregon Green 488, Oregon Green 500, Oregon Green514, Pacific Blue, phthalic acid, terephthalic acid, isophthalic acid, cresol fast violet, cresol Blue violet, brilliant cresol Blue, p-aminobenzoic acid, erythrosine, phthalocyanine, azomethine, cyanine, xanthine, succinyl fluorescein, rare earth metal cryptate, europium tripyridyldiamine, europium cryptate or chelate, diamine, bispyanine, La Jolla Blue dye, allophycocyanin, allocyanonin B, phycocyanin C, phycocyanin R, thiamine, phycoerythrin R, REG, rhodamine Green, rhodamine isothiocyanate, rhodamine red, ROX, TAMRA, TET, TRIT (tetramethylrhodamine isothiol), tetramethylrhodamine, and Texas red.

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

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

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

It is also an aspect of the present invention to provide a kit comprising one or more of the binding protein as described above, the nucleic acid molecule, the vector and the antibody combination product.

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

In some embodiments, the antibody comprising the binding protein can be labeled with a detectable label to produce a labeled detection tracer reagent; or, as the second antigen, the antigen or the first antibody for capturing the antigen is combined to detect the antigen under the combined action, so that the sensitivity and the accuracy of the detection are improved; or, the antigen is combined with the antigen or captured by the first antibody and is combined with the second antibody to detect the antigen, so that the detection sensitivity and accuracy are improved.

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

In some embodiments, the anti-human PD-L1 monoclonal antibody is specifically a monoclonal antibody secreted by mouse-derived anti-human PD-L1 hybridoma, which specifically binds to the extracellular region of human PD-L1 by its specific antibody nucleic acid sequence, and the antibody protein completely blocks the binding of PD-1-PD-L1, and has a definite basic characteristic such as binding affinity to PD-L1.

In some embodiments, the invention relates to the combination of the antibody and other anti-human PD-L1 antibodies with different epitopes, a sensitive sandwich ELISA method is established, the method is used for evaluating the concentration of blood soluble PD-L1(sPD-L1) in patients with tumors such as lung cancer, melanoma, kidney cancer, bladder cancer, Hodgkin lymphoma, head and neck cancer, cervical cancer, gastric cancer, intestinal cancer and the like MSI-H (highly microsatellite instability) or DNA mismatch repair deficiency (MSI/dMMR), and the concentration level of sPD-L1 in the patients is determined, and the sensitivity is high, and the single-digit magnitude effective detection concentration (5pg/mL) is reached. The antibody can also be used for detecting tumor tissue PD-L1, guiding the prognosis evaluation of a clinical PD-1-PD-L1 channel blocker, and potentially expanding the application of the antibody in analysis of other immune-related disease inflammatory conditions.

The antibody of the invention codes the whole nucleic acid sequence, is convenient for PD-L1 antibody engineering, such as immunoglobulin class, subclass conversion and various subunit functional small molecule antibody transformation, the antibody sequence has effective expression strategy in a mammal cell system, and is suitable for the antibody production with stable genetic character.

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

EXAMPLE 1 construction of PD-L1 Single chain antibody

The design of the PD-L1scFv-Fc chimeric fragment, namely the chimeric form of the single-chain antibody, sequentially comprises the following steps: heavy chain variable region + connecting flexible peptide + light chain variable region + human Fc fusion protein tag + stop codon.

It encodes 486 amino acids, wherein 259 amino acids of a murine antibody, 138 heavy chain variable regions (FR-H1-CDR-VH1-FR-H2-CDR-VH2-FR-H3-CDR-VH3-FR-H4) including 19 Signal peptide amino acids (Signal peptide) with the sequence shown in SEQ ID NO:22, 15 artificial flexible Linker peptide (Linker) amino acids are encoded, and then 106 amino acids of light chain variable regions (FR-L1-CDR-VL1-FR-L2-CDR-VL2-FR-L3-CDR-VL3-FR-L4) are encoded, and the amino acids of the light chain variable regions do not include leader sequence amino acids. Then the 227 Human Ig-Fc amino acid sequence (Human Fc tag) with the sequence shown in SEQ ID NO:17 is joined, the tag sequence can be used for expressed protein purification and detection, and the amino acid design structure is as follows:

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

example 2 expression and purification of PD-L1 antibody in host cells

Cloning the total length of DNA containing light chain (shown as SEQ ID NO: 23) and the total length of DNA containing heavy chain (shown as SEQ ID NO: 24) into pcDNA3.1 or pcDNA3.4, respectively, transforming competent Escherichia coli, amplifying plasmids, identifying the sequence correctness by enzyme digestion and sequencing, and quantifying by using a spectrophotometer, wherein the following plasmids are used as the light chain vector and the heavy chain expression vector according to the ratio of 2: 1 DNA quality after mixing (. mu.g) for mammalian cells ExpicCHO-S^TM(ThermoFisher) transient transfection expression.

Day 1: fresh thawed ExpicHO-S^TMRecovering and expanding cells until the cell density reaches about 4-6 × 10⁶Viable cells/mL.

Day 2: seed separation ExpicHO-S^TMCulturing, adjusting cell density to 3-4 × 10⁶Viable cells/mL, cells were grown overnight.

Day 3 viable cell density and percent viability were determined cell density should reach about 7-10 × 10⁶Viable cells/mL. The survival rate should be 95-99%, so that the transfection can be continued.

ExpicHO freshly preheated to 37 ℃^TMExpression medium, cell dilution to final density 6 × 10⁶The culture flask was gently shaken for each viable cell/mL, and the cells were mixed well.

Day 4: and (4) transfection. Expifeacmine was prepared using cold reagent (4 ℃ C.)^TMCHO and cold plasmid solution are fully and evenly mixed to prepare Expifeactamine^TMCHO/plasmid. The concentration of the total plasmid in the light chain variable region is 0.5-1.0. mu.g DNA/mL as the transfection culture volume (for example, the culture volume is 280mL in a 1L culture flask system, the plasmid DNA volume is 8mL, and the total plasmid amount needs 4.0-8.0. mu.g).

The method specifically comprises the following operations: (i) mixing Expifeacamine^TMThe CHO reagent bottle was gently inverted 4-5 times and mixed well. Using cold OptiPRO^TMThe plasmid DNA was diluted in medium (ThermoFisher), shaken or inverted in the tube, mixed with DNA at room temperature to give a total plasmid DNA of 0.5-1.0. mu.g/mL culture volume, and mixed well. (ii) Using OptiPRO^TM7.4mL of Expifeacylamine diluted in Medium^TMCHO reagent (ThermoFisher) 640. mu.L, shaking or inverting the tube, and mixing. (iii) Diluting Expifeacmine^TMThe CHO reagent was added to the diluted DNA, the tube was shaken or inverted upside down, and mixed well. (iV) Expifeacylamine incubation at Room temperature^TMCHO/plasmid DNA Complex 1-5 min (Expifeacamine added immediately)^TMCHO/DNA complexes to cells, mixing for no more than 5 minutes, then slowly transfer the solution to the step 4 flask, gently shaking the flask during the addition. The cells were placed on a orbital shaker at 37 ℃ in an incubator containing 8% CO₂The cultivation is carried out under the condition of humidified air, and the shaking speed of a shaking table (25-mm orbit) is 120 +/-5 rpm.

Day 5: the following day after transfection (day 1, 18-22 hours after transfection), the addition of Expifeacylamine was performed according to the chosen protocol^TMCHO enhancer 1.2mL and ExpicCHO^TM48mL of auxiliary materials: transfer the flasks to a 32 ℃ incubator containing 5% CO₂The culture was performed under shaking in a humidified atmosphere.

Day 12-14: the culture supernatant was collected. The optimal collection time for antibody proteins is typically 8-10 days after transfection.

Antibody purification: centrifuging at 4000g for 20min, removing cell particles or debris, filtering with 0.22 μ M or 0.45 μ M membrane filter, measuring pH of supernatant, and adding 0.2M Na₂HPO₄Or 0.2M NaH₂PO₄Adjusting pH to 7.0-7.4 (1M NaOH or 1M HCl), completely equilibrating protein A purification column (Sigma) with buffer solution (PBS, pH7-7.4), and subjecting the cells to AKTA system or peristaltic pumpLoading the culture supernatant onto a completely balanced column at 4 ℃, and balancing the column by using 3 CV-5 CV combined buffer solution; the antibody/protein was eluted from the column from the elution buffer using the AKTA system. Collecting eluate at 1-3mL/min according to UV280 absorbance, desalting with desalting column, quantifying with spectrophotometer, packaging, and storing at-80 deg.C or freeze drying.

EXAMPLE 3 preparation of anti-human PD-L1 monoclonal antibody

1. Immunization of animals

The immunogen was the soluble antigen PD-L1 tag (Fc) protein (Yinqiao). 4-6 weeks old inbred BALB/c mice were selected, primary immunization was started 2 months before fusion, and adjuvants used in this experiment included Freund's complete adjuvant, Freund's incomplete adjuvant (Sigma) in order to increase the immunogenicity of soluble proteins.

On day 0, priming was performed, antigen was emulsified in complete adjuvant, 0.25mL, 10-100. mu.g per mouse, and immunized by intraperitoneal injection, for a total of 5 mice. On day 14, immunization was performed by intraperitoneal injection, using 0.25mL of antigen emulsified in incomplete adjuvant, 10-50. mu.g per mouse. On day 24, tail vein blood was collected, centrifuged at 37 ℃ for 1 hour and 4 ℃ for 2 hours, and the supernatant was collected and subjected to primary assay for serum titer. On day 35, immunization was performed by intraperitoneal injection, with 0.25mL and 10-50 μ g of antigen emulsified in incomplete adjuvant per mouse.

On day 45, tail vein blood was collected, centrifuged at 37 ℃ for 1 hour and 4 ℃ for 2 hours, and the supernatant was collected and assayed for serum titer by ELISA. The titer of 5 immunized mice reaches or exceeds 1:512000, 2 mice with titer exceeding 1:512000 are selected, and on day 56, the mice are finally immunized, and the antigen is dissolved in PBS, 0.25mL of antigen is dissolved in PBS, 10-50 μ g of antigen is dissolved in PBS, and tail vein and intraperitoneal injection are carried out. One week later (day 60), cell fusion was performed.

2. Cell fusion

(1) Preparing a feeder cell layer: mouse abdominal cavity macrophages are selected. A mouse of the same strain as the immunized mouse, which is a BALB/c mouse, is 6-10 weeks old, is killed by pulling the neck, soaked in 75% alcohol for 3-5 min, the skin is cut off by sterile scissors, the peritoneum is exposed, 5-6 mL of precooled culture solution is injected by a sterile syringe, the washing is repeatedly carried out, the washing solution is sucked out and put into a 10mL centrifuge tube,separating at 1200rpm/min for 5-6 min, suspending with culture medium of 20% calf serum (NCS) or Fetal Calf Serum (FCS), and adjusting cell number to 1 × 10⁵Perml, add to 96 well plate, 100. mu.L/well, put in CO at 37 ℃₂And (5) incubator culture.

(2) Preparing immune spleen cells: after 3 days of the last booster immunization, the mice were killed by cervical draining, spleens were aseptically removed, and the cultures were washed once. Grinding spleen, sieving with stainless steel sieve, centrifuging, washing cells with culture solution for 2 times, counting, and collecting 10⁸Individual spleen lymphocyte suspensions were ready for use.

(3) Preparing myeloma cells by centrifuging logarithmic growth myeloma cells, washing with serum-free culture solution for 2 times, counting, and collecting 1 × 10⁷The cells are ready for use.

(4) Fusing:

combining myeloma cells and spleen cells according to the following ratio of 1: 10 or 1:5, washing the mixture with a serum-free incomplete culture solution for 1 time in a 50mL centrifuge tube, and centrifuging the mixture for 8min at 1200 rpm/min; discard the supernatant, and use the pipette to clean the residual liquid so as to avoid affecting the concentration of polyethylene glycol (PEG). Lightly flick the bottom of the tube to loosen the cell pellet.

② 1mL of 45 percent PEG (molecular weight 4000) solution pre-warmed at 37 ℃ is added in 90s while slight shaking is carried out. The reaction was carried out in a water bath at 37 ℃ for 90 s.

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

Fourthly, centrifuging for 6min under the condition of 800 rpm/min.

Fifthly, removing supernatant, and resuspending the supernatant by HAT selection culture solution containing 20% calf serum.

Sixthly, adding the cells into a 96-well plate with a feeder cell layer, adding 100 mu L of the cells into each well, and inoculating 10 96-well plates into one immune spleen, wherein 20 plates are counted.

⑦ the culture plate was placed at 37 ℃ in 5% CO₂Culturing in an incubator.

3. Hybridoma prescreening and secreted antibody detection thereof

(1) HAT selection of hybridomas. After the spleen cells and myeloma cells are treated by PEG, a mixture of various cells is formed, and after the mixture is maintained in HAT selection culture solution for 7-10 days, HT culture solution is used for maintenance for 2 weeks, and common culture solution is used instead. During the selective culture, when the hybridoma cells spread over the area of the well bottom 1/10, the detection of specific antibodies is started, and the desired hybridoma cell line is selected. During the selective culture period, half of the culture solution is generally changed every 2 to 3 days.

(2) The antibody is detected by enzyme-linked immunosorbent assay (ELISA), human PD-L1 (including PD-L1-Fc and PD-L1-his) fusion protein with the concentration of 2 mu g/mL is coated, hybridoma culture supernatant is detected, anti-mouse secondary antibody marked by enzyme and substrate are used for developing color, OD value is detected, 40 positive holes (PD-L1-Fc negative and PD-L1-his positive) are preliminarily screened in the assay, and cloning is further carried out.

4. Hybridoma cloning

Clones which are detected as positive are cloned to avoid being inhibited by cells which do not secrete the antibody, so that cells which secrete the antibody are lost, and the cloned hybridoma cells also need to be periodically re-cloned to prevent the hybridoma cells from being mutated or losing chromosomes, so that the capacity of producing the antibody is lost. In the experiment, a limiting dilution method is adopted for cloning, and the antibody screened in the experiment is subcloned for more than 4 times.

Feeder cell layers (confluent with cells) were prepared 1 day before cloning. The hybridoma cells to be cloned were gently blown up from the culture well and counted. Adjusting the cell to 3-10 cells/mL. Taking cell culture plate of feeder cell layer prepared for the first day, adding diluted cells 100 μ L per well, incubating at 37 deg.C and 5% CO₂An incubator. Changing the liquid on the 7 th day, and changing the liquid 1 time every 2-3 days later. Cell clone formation can be seen in 8-9 days, and the activity of the antibody can be detected by ELISA in time.

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

Antibody detection, expanded culture, and repeated recloning as necessary.

5. Freezing and thawing hybridoma cells

(1) Loss of antibody secretion may occur during cell culture, and hybridoma cryopreservation may verify hybridoma stability and long-term storage of the hybridoma strains. Each ampoule for freezing hybridoma cellsContaining 1 × 10⁷Above, cell cryopreservation solution: 50% calf serum; 40% incomplete culture solution; 10% DMSO (dimethyl sulfoxide). When frozen, the temperature can be immediately reduced to 0 ℃ from room temperature, then the frozen. The cell freezing device can also be used for freezing. The frozen cells are periodically revived, examined for their viability and for the stability of the secreted antibodies, and stored in liquid nitrogen for several years or more.

(2) Cell recovery: carefully taking out glass ampoule from liquid nitrogen, placing in 37 deg.C water bath, thawing frozen cells within 1min, washing cells with whole culture solution twice, transferring into culture flask of feeder layer cells prepared for the first day, placing in 37 deg.C 5% CO₂Culturing in an incubator, and detecting the antibody activity of the supernatant by ELISA when the cells form colonies.

6. Hybridoma antibody nucleic acid full length sequencing

According to

The technical manual of reagents (AMBION, CAT, No. 15596-026) was used to isolate total RNA from hybridoma cells, reverse transcribing the total RNA into cDNA using isotype-specific antisense primers (or universal primers) according to the technical manual of PrimeSeCpTTM first strand cDNA Synthesis kit (Takara, CAT, No. 610A), amplifying antibody fragments of VH, VL, CH and CL according to the Rapid amplification of cDNA ends of GESTcript (SOP) Standard Operating Procedure (SOP), cloning the amplified antibody fragments into standard cloning vectors, sequencing, sequence analysis tools for variable regions: (i) NCBI Nucleotide BLAST; (ii) IMGT/V Quest program; (iii) screening inserts with correct sizes by colony PCR, sequencing each fragment of not less than five inserts with correct sizes, and comparing different clone sequences to obtain a consistent sequencing result.

Example 4 detection of sPD-L1 by Sandwich ELISA

The antibody of example 1 was used as a non-blocking antibody, and a sandwich ELISA experiment was performed using 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 shown in SEQ ID NO: 19) as an antigen-capturing antibody.

1. Antibody coating

The X monoclonal antibody was diluted to a final concentration of 2. mu.g/mL into the coating solution, and 100. mu.L was added to each well in an ELISA-96 well plate. Cover or protect film, 4 ℃ overnight. The next day, the coated plate was removed, the supernatant was discarded, washed 3 times with 1 × Wash Buffer, the supernatant was discarded, and the wells were blotted dry on filter paper or toilet paper.

2. Sealing of

200 μ L/well of 5% milk was added to block non-specific binding sites. Cover or protect film, 37 deg.C for 2 hours or 4 deg.C overnight. The following day, the supernatant was discarded, washed 1 time with 1 × Wash Buffer, discarded, and the wells were blotted dry on filter paper or toilet paper.

3. Incubation of standards and samples

Diluted standards (or samples), 100. mu.L/well, were added. Cover or protect film, and stand at room temperature for 2 hours. The supernatant was discarded, washed five times with 1 × Wash Buffer, discarded, and the liquid in the wells was blotted on filter paper or toilet paper and patted dry.

4. Secondary antibody and HRP-Strep incubation

The bio-labeled PD-L1 antibody of example 1 was added to a final concentration of 2. mu.g/mL and 100. mu.L/well, and the mixture was covered with a cap or a protective film and allowed to stand at room temperature for 1 hour. The supernatant was discarded, washed five times with 1 × Wash Buffer, discarded, and the liquid in the wells was blotted on filter paper or toilet paper. HRP-Streptavidin (Cell signaling 3999S,1:5000), 100. mu.L/well, cap or protective membrane, and left at room temperature for half an hour. The supernatant was discarded, washed five times with 1 × Wash Buffer, discarded, and the liquid in the wells was blotted on filter paper or toilet paper.

5. Detection of

Adding 100 mu L/hole A + B mixed solution, and developing for 5-10 minutes at room temperature in a dark place. Add 50. mu.L/well stop solution and mix well. OD values of 450nm were read by a plate reader (Thermo scientific Multiskan GO). And establishing a standard curve and calculating the concentration of the sample.

Experimental example 1 identification of specificity of PD-L1 antibody

1.1 ELISA identification

Experiments were carried out by referring to the method of example 4, which were coated with PD-L1, EGFR, CTLA-4, CD137, Epcam fusion proteins (all extracellular domain proteins) 1. mu.g/mL, all of which are his-tag proteins, the antibody of example 1 was added, the HPR-labeled goat anti-mouse IgG was used as a secondary antibody, color was developed by adding a substrate, and the OD450 value was determined. The results of the experiment are shown in FIG. 1, and the results show that the antibody in example 1 of the present invention has strong specificity for PD-L1-his.

1.2 Western blot identification

Experiments were carried out using the conventional Western blot method, and the samples to be tested were PD-L1-Fc, PD-1-his, PD-L1-his, respectively, and were stained with ponceau Red after running through SDS-PAGE followed by incubation with the antibody of example 1 and development of ECL (FIG. 2 right). The results show that there are protein bands in lanes PD-L1-his and PD-L1-Fc, indicating that the antibody of the present invention binds only to PD-L1 and not to PD-1.

Experimental example 2 affinity assay for PD-L1 antibody

The specific method comprises the following steps:

AMC sensors (PALL, USA) in the balance (0.1% BSA + 0.02% TWEEN20 PBS) pre-wet for 10 minutes, PD-L1 antibody with the balance diluted to 20 u g/mL, added to the light-shielding 96 plate second column, 200 u L/hole.

2. PD-L1-his was diluted from 500nM to 7.8nM starting fold, added to the fourth column of a light-shielded 96-well plate, 200. mu.L/well, well H4 was blank, and 200. mu.L of the equilibration buffer was added.

3. The equilibration solution was added to the first and third columns at 200. mu.L/well.

4. The sensor was equilibrated in the first column for 120 seconds to obtain a basal equilibration curve and then in the second column for 300 seconds for antibody immobilization, using a Fortebio octet96 instrument for detection. Re-equilibration was performed for 120 seconds in the third column, binding curves were obtained in the fourth column for 180 seconds in binding the antigen, and dissociation curves were obtained by returning to the third column for 300 seconds in dissociation.

5. The affinity values were obtained by fitting analysis of the curves using Fortebio Octet96 analysis software.

TABLE 1 Fortebio Octet analysis of the binding and dissociation results of PD-L1 antibody to PD-L1

Experimental results referring to FIG. 3 and Table 1, the Fortebio Ocet molecular interaction Analyzer determined that the antibody of example 1 binds to PD-L1-his protein with an affinity of 6.96 × 10^-9mol/L, i.e. can reach 10^-9mol/L, and the smaller the affinity, the stronger the binding capacity.

Experimental example 3 validation of PD-L1 antibody in flow cytometry applications

3.1 detection of PD-L1 on cell membranes by PD-L1 antibody

Referring to the method of example 2, pcDNA3.1-PD-L1 plasmid with pGFP tag was transferred into 293 cells, and the expression of GFP (FITC) in the recombinant cells was detected by flow cytometry, thereby obtaining the transfection efficiency, and the experimental results are shown in FIG. 4. As can be seen from FIG. 4, 37.1% of the cells were GFP positive, that is, the transfection efficiency was 37.1%.

Detecting the expression condition of PD-L1 in the recombinant cell by a flow cytometer, wherein the specific method comprises the following steps:

total number of 1 × 10⁶The cells were suspended in 100. mu.L of 1% bovine serum-containing PBS buffer, 1. mu.g of 1-2. mu.L PD-L1 antibody was added, stained at room temperature for 30min, resuspended in 1mL of 1% bovine serum-containing PBS buffer, gently mixed, centrifuged at 1000rpm, washed 2 times, and then resuspended in 100. mu.L of 1% bovine serum-containing PBS buffer, goat anti-mouse IgG-APC (about 1. mu.g) was added, stained for 30min, washed 2 times in 1% bovine serum-containing PBS buffer, the cells were suspended in 1mL of 1% bovine serum-containing PBS buffer, and the cell surface PD-L1 expression level was analyzed by flow cytometry.

The experimental results are shown in FIG. 5, which shows that the staining of PD-L1 antibody in GFP positive cells is also positive, and thus the PD-L1 antibody of the invention can detect PD-L1 on the cell membrane with higher accuracy.

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

Referring to experimental example 3.1, the sample to be detected is H2009 human lung adenocarcinoma cells, the H2009 cell line is incubated with or without PD-L1 antibody (blank control), and then incubated with goat anti-mouse IgG-PE secondary antibody for flow analysis, the left peak is not incubated with antibody, and the right peak is incubated with antibody. The results of the experiment are shown in FIG. 6. The results show that the PD-L1 antibody can be used as a flow antibody for detecting PD-L1 of cancer cells.

Experimental example 4 verification of PD-L1 antibody in ELISA application

4.1 Standard Curve for Sandwich ELISA established with the PD-L1 antibody

Referring to the experimental method of example 4, a diluted PD-L1-his protein standard was detected by establishing a sandwich ELISA using PD-L1 antibody as a non-blocking antibody, biotin labeling as a detection antibody, and antibody X as a capture antibody. The results show that the standard curve produced has a high degree of fitting, R²0.9998, The expected sensitivity (The expectedsensity) was 5pg/mL, in other words The PD-L1 antibody detection sensitivity was high with a minimum detection concentration of 5pg/mL (fig. 7, table 2).

Table 2 standard concentration in standard curve and corresponding OD450 readings

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

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

Referring to the experimental procedure of example 4, 40 sera from normal and advanced lung cancer patients were taken, and subjected to 1: after 25 dilutions PD-L1 assays were performed using a sandwich ELISA. The results showed significant differences (p <0.001) between the two groups of assayed serum concentrations of sPD-L1 (FIG. 8).

Experimental example 5 blocking Effect of PD-L1 antibody on binding of PD-1 to PD-L1

5.1 flow cytometry detection of blocking Effect of PD-L1 antibody on 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, which showed a positive rate of 22.7% (FIG. 9); the result of the incubation of 293 transfected cells of PD-L1 with PD-L1 antibody and then with PD-1-his and anti-his fluorescent secondary antibody respectively shows that the positive rate is 29.9% (FIG. 10), which indicates that the PD-L1 antibody does not block the binding of PD-1-his protein to the transfected cells.

5.2 ELISA detection of blocking Effect of PD-L1 antibody on the binding of PD-1 to PD-L1

PD-1-his 1. mu.g/ml was coated while PD-L1-Fc was mixed with PD-L1 antibody and left overnight at 4 degrees. The next day, the mixture was added to PD-1-his coated ELISA plates and incubated for 2h at room temperature for detection with HRP-streptavidin. Reading OD450 values (fig. 11); and calculating the blocking rate: the blocking rate (inhibition rate) was (OD1-OD0/OD 0).

The experiment shows that the PD-L1 antibody does 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 solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

<110> the university of capital medical department affiliated to the Beijing thoracic hospital; research institute of tuberculosis and breast tumor in Beijing

<120> anti-human PD-L1 antibody and application thereof

<130>20180910

<160>24

<170>PatentIn version 3.3

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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> Artificial Synthesis

<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> Artificial Synthesis

<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> Artificial Synthesis

<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> Artificial Synthesis

<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> Artificial Synthesis

<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> Artificial Synthesis

<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> Artificial Synthesis

<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 said antigen binding domain comprises the complementarity determining regions of the amino acid sequences;

the amino acid sequences of the complementarity determining regions CDR-VL1, CDR-VL2 and CDR-VL3 are respectively shown in SEQ ID NO. 1-3; and

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.

2. The antibody of claim 1, wherein the antibody comprises light chain framework regions FR-L1, FR-L2, FR-L3 and FR-L4 as shown in the sequence of SEQ ID NO 7-10, and/or heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4 as shown in the sequence of SEQ ID NO 11-14.

3. The antibody of claim 1, further comprising an antibody constant region Fc.

4. The antibody of claim 3, wherein said 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 set forth in SEQ ID NO. 15.

6. The antibody of claim 4, wherein the heavy chain constant region sequence is set forth in SEQ ID NO 16.

7. The antibody of claim 4, wherein the constant region Fc has a sequence selected from the group consisting of sequences of any one of the constant regions IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD.

8. The antibody of claim 4, wherein the sequence of the constant region Fc is selected from the group consisting of the sequences of the IgG2b constant regions.

9. The antibody of claim 4, wherein the sequence of the constant region Fc is selected from the group consisting of sequences of the IgG2b/kappa constant region.

10. The antibody of claim 4, wherein the species of the constant region Fc is derived from a cow, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, or human.

11. The antibody of claim 2, wherein the antibody is one of F (ab ')2, Fab', Fab, Fv, Fd, scFv-Fc chimeric fragment, and diabody.

12. The antibody of claim 2, wherein the antibody is a scFv-Fc chimeric fragment comprising the constant region Fc of claim 3, the complementarity determining region of any one of claims 1-3, 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 set forth in SEQ ID NO 17.

15. The antibody of claim 12, wherein said scFv-Fc chimeric fragment further comprises an artificial flexible linker peptide.

16. An isolated nucleic acid molecule which is DNA or RNA encoding 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 comprising the nucleic acid molecule of claim 16 or the vector of claim 17.

19. Use of an antibody according to any one of claims 1 to 15 in the manufacture of a medicament for the diagnosis of a disease which is cancer.

20. Use of an antibody according to claim 19 in the manufacture of a medicament for the diagnosis of a disease, said cancer comprising one or more of lung cancer, melanoma, NSCLC, classical hodgkin's lymphoma, HNSCC, renal cell carcinoma, urothelial cancer, head and neck cancer, gastric cancer, hematologic malignancies, prostate cancer, cervical cancer, brain cancer, hepatocellular carcinoma and colorectal cancer.

21. An antibody combination comprising an antibody of any one of claims 1 to 15 and a second antibody;

the second antibody specifically recognizes PD-L1 and recognizes an epitope different from that of the antibody of any one of claims 1 to 15.

22. The antibody product according to claim 21, wherein the amino acid sequences of the light chain variable region VL of the second antibody are shown in SEQ ID NO: 18; the amino acid sequences of VH in the heavy chain variable region are respectively shown in SEQ ID NO: 19.

23. The antibody product of claim 21, said antibodyThe combination product further comprises PBS, BSA, ddH₂One or more of O, glycerol, sodium azide and gentamicin.

24. A kit comprising one or more of the antibody of any one of claims 1 to 15, the nucleic acid molecule of claim 16, the vector of claim 17 and the antibody combination of claims 21 to 23.

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