CN111363039A

CN111363039A - anti-PD-L1 antibody suitable for immunohistochemical detection

Info

Publication number: CN111363039A
Application number: CN201811587248.7A
Authority: CN
Inventors: 侯盛; 郭怀祖; 徐进
Original assignee: Shanghai Biomabs Pharmaceuticals Co Ltd
Current assignee: Shanghai Biomabs Pharmaceuticals Co Ltd
Priority date: 2018-12-25
Filing date: 2018-12-25
Publication date: 2020-07-03

Abstract

The invention provides a recombinant anti-PD-L1 monoclonal antibody suitable for immunohistochemical detection, wherein the anti-PD-L1 antibody provided by the invention is used for immunohistochemical detection, and compared with the existing commercialized anti-PD-L1 monoclonal antibody for immunohistochemical detection, the antibody provided by the invention has stronger staining effect signals on PD-L1 positive tissue sections under relatively low working concentration, so that the detection sensitivity and resolution can be improved, the antibody dosage can be reduced, and the detection cost can be reduced.

Description

anti-PD-L1 antibody suitable for immunohistochemical detection

Technical Field

The invention relates to the technical field of biological medicines, and particularly discloses a recombinant anti-PD-L1 monoclonal antibody suitable for immunohistochemical detection.

Background

PD-L1 (Programmed Cell Death 1 ligand 1), also known as surface antigen cluster of differentiation 274 (CD 274) or B7 homolog 1 (B7 homolog 1, B7-H1), is a transmembrane Protein of 40kDa in size and is one of the 2 ligands of PD-1 (Programmed Cell Death Protein 1). In mice, PD-L1 is widely expressed in various organs such as heart, lung, thymus, spleen, kidney, and almost all mouse tumor cell lines, and is highly expressed in various human tumor cells. PD-L1 can transmit immunosuppressive signal and reduce T cell proliferation after being combined with PD-1.

The "Immune checkpoint" (Immune checkpoint) mechanism formed by PD-1 and PD-L1 can be used by cancer cells to realize Immune escape, so PD-1 and PD-L1 become targets for cancer immunotherapy, and a plurality of anti-PD-1 or anti-PD-L1 monoclonal antibodies are approved by the U.S. Food and Drug Administration (FDA) and European drug administration (EMA) for treating malignant solid tumors such as melanoma, non-small cell lung cancer, head and neck squamous cell carcinoma, renal cell carcinoma, gastric cancer, and the like, such as anti-PD-1 antibodies Nivolumab, Pembrolizumab, and anti-PD-L1 antibodies Atezolizumab, Avelumab, Durvamab, and the like.

Clinically, malignant solid tumor patients who need to use PD-1 or PD-L1 immunotherapy drugs need to receive Immunohistochemical (IHC) detection of tumor tissues in advance to determine whether the tumor tissues express PD-L1 molecules and the proportion of cells expressing PD-L1, so that the anti-PD-1/PD-L1 drugs can be used more specifically, and doctors and patients can be assisted to reasonably select a treatment mode.

Immunohistochemistry is a technique of detecting the distribution of an antigen (or antibody) within a tissue section in situ using a labeled specific antibody (or antigen). Tissue sections used for immunohistochemistry included both paraffin sections and frozen sections. The paraffin section is the most common and basic method for manufacturing the tissue specimen, the tissue shape is well preserved, and the paraffin section can be used as a continuous section, thereby being beneficial to various staining contrast observations; and can also be archived for long term for retrospective study.

The preparation process of the paraffin section has certain influence on the conformation of the antigen in the tissue and the exposure of the epitope, and the epitope exposed by the antigen in the tissue section may be different from that in the natural state. Monoclonal antibodies are directed against specific epitopes only, and therefore, in order to improve the detection efficiency and consistency of immunohistochemistry, it is necessary to select monoclonal antibodies that specifically bind to antigen-exposed epitopes in paraffin sections.

In the development process of the immunohistochemical detection method, because the influence of paraffin embedding on the epitope is difficult to predict, and non-specific binding with a detection antibody may exist in various normal tissues and tumor tissues of a human body, a large number of tests are required to screen appropriate antibodies from a plurality of candidate antibodies, and indexes such as sensitivity, specificity, accuracy and the like of the candidate antibodies in immunohistochemical detection of paraffin sections of different tissues are evaluated.

The current commercial PD-L1 immunohistochemical detection methods for international Clinical application use paraffin sections, and these kits include Dako PD-L1 IHC 22C3 pharmDx, Dako PD-L1 IHC 28-8 pharmDx, Ventana PD-L1(SP263) Assay, Ventana PD-L1(SP 142) Assay, and it is reported that PD-L1 detected by 28-8, 22C3 and SP263 has higher uniformity in tumor Cell membrane expression, while PD-L1 expression level obtained by SP142 is lower (programmed Death-Ligand 1 immunological Testing: A Review of Analytical analysis and Clinical evaluation in Non-Smart-cancer Cell-Journal of Clinical diagnosis 35, 12034, 3876). At present, no domestic commercial PD-L1 immunohistochemical kit for clinical use exists.

Antibodies (antibodies) are immunoglobulins (igs) that can be classified into various classes, such as IgG, IgM, IgA, IgE, IgD, etc., where IgG is a class of antibodies commonly used in the biomedical field. Human and mouse IgG is composed of 4 peptide chains, including 2 identical heavy chains (H chain) and 2 identical light chains (L chain), the peptide chains are linked by disulfide bonds, and can be divided into subtypes of IgG1, IgG2, IgG3, IgG4 and the like according to the slight difference of the heavy chain structure, and the light chains can also be divided into subtypes of Lamda (lambda), Kappa (Kappa) and the like. Each peptide chain comprises a variable region (V-region) called VL and VH, respectively, and a constant region (C-region) called CL and CH, respectively, for the light and heavy chains, and the constant region for the heavy chain can be divided into three parts, CH1, CH2, and CH3, respectively. VL and VH each contain a highly variable region of 3 amino acid composition and arrangement, called hypervariable region (HVR) or Complementarity Determining Region (CDR), designated HVRl (or CDRl), HVR2 (or CDR 2) and HVR3 (or CDR 3), which determine the specificity of the antibody, the site where the antibody recognizes and binds antigen. In the V region, the region other than the CDR is relatively conserved in amino acid composition and arrangement order and is called a Framework Region (FR). Between CH1 and CH2 is a hinge region rich in proline, which is easily stretched and bent, and also a region where 2 heavy chains are linked by disulfide bonds, and which is sensitive to papain and pepsin. IgG can form 2 Fab fragments and 1 Fc fragment after papain hydrolysis, the Fab fragment is monovalent and can be combined with antigen without agglutination reaction or precipitation reaction; the Fc fragment consists of CH2 and CH3 and is the site of IgG interaction with effector molecules or cells. IgG can form 1F (ab ')2 fragment and a plurality of small fragments pFC' after being hydrolyzed by pepsin, the F (ab ')2 fragment is bivalent, can be simultaneously combined with two antigen epitopes, can generate agglutination reaction and precipitation reaction when being combined with antigen, and can form 2 Fab' fragments after being further hydrolyzed. The Fv fragment is the smallest fragment of the IgG-type antibody that is enzymatically analyzed and consists of the VH and VC regions, including only the antigen-binding region.

Disclosure of Invention

The invention provides an anti-PD-L1 antibody suitable for immunohistochemical detection, the anti-PD-L1 antibody provided by the invention is prepared by a preparation method of a preferred embodiment, and the anti-PD-L1 antibody has higher binding capacity with PD-L1.

Compared with the existing commercialized anti-PD-L1 monoclonal antibody for immunohistochemistry, the anti-PD-L1 antibody provided by the invention is used for immunohistochemistry detection, and compared with the existing commercialized anti-PD-L1 monoclonal antibody for immunohistochemistry, the antibody provided by the invention has stronger staining effect signals on PD-L1 positive tissue sections under relatively low working concentration, so that the detection sensitivity and resolution can be improved, the antibody dosage can be reduced, and the detection cost can be reduced.

In a preferred embodiment, the anti-PD-L1 antibody provided by the invention is used for immunohistochemical detection of paraffin sections of human tissues, and the detection result of the immunohistochemical detection is carried out by using 0.1ug/mL anti-PD-L1 antibody, so that the staining result of the 0.1ug/mL anti-PD-L1 antibody provided by the invention is obviously better than that of the commercial anti-PD-L1 antibody at the working concentration of 3ug/mL, and the sensitivity of the anti-PD-L1 antibody provided by the invention in immunohistochemical detection is obviously better than that of the existing product.

In order to improve the signal intensity of PD-L1 immunohistochemical detection in a tumor tissue paraffin section, improve the detection sensitivity and reduce the detection cost, the invention provides an anti-PD-L1 antibody suitable for immunohistochemical detection, and the technical scheme is as follows:

an anti-PD-L1 antibody (No. mADL 107) suitable for immunohistochemical detection, wherein a heavy chain CDR1, a CDR2 and a CDR3 respectively have sequences of SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10; the light chain CDR1, CDR2 and CDR3 have the sequences of SEQ ID NO 5, SEQ ID NO 6 and SEQ ID NO 7, respectively.

Preferably, the antibody heavy chain variable region has the sequence of SEQ ID NO 2; the light chain variable region has the sequence of SEQ ID NO 1. Preferably, the variable region of the antibody heavy chain is encoded by the nucleotide sequence of SEQ ID NO. 4; the light chain variable region is encoded by the nucleotide sequence of SEQ ID NO. 3.

In addition, it is predicted that similar technical effects can be achieved when the heavy chain variable region sequence is 95% or more identical to the sequence of SEQ ID NO:2 and the light chain variable region sequence is 95% or more identical to the sequence of SEQ ID NO: 1.

Preferably, the antibody is a mouse IgG-type antibody; the antibody may be a Fab fragment, a F (ab ')2 fragment, a Fv fragment, a Fab' -SH fragment, or a single-chain antibody, a single-domain antibody, a bispecific antibody, or a multispecific antibody.

The antibody may carry a detectable label, such as a fluorescent label, an enzymatic label, a radioactive label, a biotin label or an avidin label.

The anti-PD-L1 antibody for immunohistochemical detection, which is described above, and the amino acid sequence of the heavy chain variable region of the anti-PD-L1 antibody is SEQ ID NO. 2; the amino acid sequence of the light chain variable region is SEQ ID NO. 1, the amino acid sequence of the heavy chain of the anti-PD-L1 antibody is SEQ ID NO. 14, and the amino acid sequence of the light chain is SEQ ID NO. 13.

The anti-PD-L1 antibody for immunohistochemical detection is applied to an immunohistochemical method for detecting the expression of PD-L1 in human tissues. Use in an immunohistochemical method for detecting expression of PD-L1 in human tissue at a concentration of 0.1ug/mL to 3ug/mL of anti-PD-L1 antibody.

The use as described above, wherein the tissue is a human tumor tissue, a gastric tissue, a placental tissue, a lung tissue or a lymphoid tissue.

A method for immunohistochemical detection using the above anti-PD-L1 antibody as a primary antibody, comprising the steps of: 1) making a tissue slice; 2) using an anti-PD-L1 antibody as a first antibody, acting on the prepared tissue section, and incubating overnight; 3) adding a second antibody combined with the first antibody, labeling, developing, washing and sealing; 4) and (5) observing and detecting through a mirror. One preferred embodiment the concentration of the first anti-PD-L1 antibody is 0.1ug/mL to 6 ug/mL; preferably, the concentration of the first anti-PD-L1 antibody is 0.1ug/mL to 3 ug/mL.

In a most preferred embodiment, the first anti-PD-L1 antibody is present at a concentration of 0.1 ug/mL.

Table 1 summarizes the possible amino acid sequences of mADL 107 and its encoding nucleic acid sequences.

TABLE 1 sequence of mouse anti-human PD-L1 monoclonal antibody mADL 107

Since the affinity of an antibody to an antigen in paraffin sections is related to the degree of exposure of the epitope (epitope) bound by it in the sections, an antibody having the same binding epitope as mADL 107 on human PD-L1 molecule, or an antibody that competitively binds to human PD-L1 molecule with mADL 107, can also achieve similar technical effects.

In practical applications, the mADL 107 can be structurally and sequentially engineered as needed using known antibody engineering protocols to achieve desired properties. These modifications include:

(1) labeled antibodies

The antibody may be labeled for detection by fluorescent, chemiluminescent, radioactive, enzyme-linked (e.g., horseradish peroxidase (HRP)), biotin/avidin, magnetic bead, nanoparticle, etc.

(2) Structural improvement

The antibody structure can be modified, and molecules with similar antigen affinity but different structures, such as Fab, F (ab ')2, Fab' -SH and Fv fragments, single-chain antibodies (such as scFv), single-domain antibodies (sdAb, also called Nanobody), antibody conjugates, bifunctional antibodies or multispecific antibodies, and the like, can be constructed according to the CDR sequence or the variable region sequence. Wherein Fab ' fragment refers to Fab with a small number of amino acid residues (including one or more cysteines from the antibody hinge region) added at the carboxy terminus of the heavy chain CH1 domain, and Fab ' -SH refers to Fab ' with a free thiol group at the cysteine residue in the constant region.

(3) Antibody humanization

Antibody humanization strategies can be used to humanize the antibody to reduce immunogenicity while maintaining substantially unchanged antigen affinity. The mutation site for humanization selection may be in the constant region or framework region, or in the CDR region or HVR region.

(4) Amino acid substitution

Amino acids with similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, aromaticity, backbone configuration, rigidity) can replace amino acids in an antibody without substantially altering the biological activity of the antibody, commonly referred to as Conservative substitutions (Conservative substitutions), are commonly used: mutual substitution among aromatic amino acids Phe, Trp and Tyr; mutual replacement among hydrophobic amino acids Leu, Ile and Val; mutual replacement between polar amino acids Gln and Asn; mutual substitution among basic amino acids Lys, Arg, His; mutual replacement of acidic amino acids Asp and Glu; mutual replacement between amino acids Ser and Thr of hydroxyl; mutual replacement among small side chain amino acids Ala, Gly and Ser.

There are also cases where amino acid substitutions are made for the purpose of improving antibody properties (e.g., improving antibody stability), and the following are commonly used: deamidation in the Asn-Gly or Asp-Gly sequence may result in the production of isoaspartic acid residues, which may cause kinking of the polypeptide chain, reduced stability (isoaspartic acid effect), and may even inactivate the binding activity of the antibody. In this case, Asn may be replaced by Gln or Ala to reduce isoaspartic acid formation; deamidation of Gln may also occur, and sometimes the occurrence of deamidation can be reduced by substituting an amino acid adjacent to Gln or Asn (particularly, an amino acid having a short side chain). Met can be replaced with Lys, Leu, Ala or Phe to reduce the probability of sulfur oxidation, which can reduce the affinity of antigen binding and lead to heterogeneity of the antibody product. In order to reduce the Asn-Pro sequence which may cause cleavage, the sequence may be replaced with Gln-Pro, Ala-Pro or Asn-Ala. Cys may cause a wrong disulfide bond or be oxidized or reduced, and may be substituted with Ala or Ser.

(5) Affinity maturation

Known antibody sequences can be optimized using methods of affinity maturation to produce more avidity mutants. In many cases, substitution, insertion or deletion of 1-2 or even 3 amino acids in the antibody CDR1, CDR2, or CDR3 regions does not cause significant changes in antigen binding activity. Through the combination of artificial serial mutants with affinity screening technology or through the selective mutation of some amino acids in the structure analysis of antigen-antibody compound, antibody mutants with excellent affinity may be obtained.

The mouse anti-human PD-L1 monoclonal antibody mAPL 107 can be used for immunohistochemical detection of paraffin sections of human tissues, such as paraffin sections of normal human stomach tissues, paraffin sections of normal human placenta tissues, paraffin sections of lung cancer tissues and paraffin sections of lymph cancer tissues, the working concentration is as low as 0.1ug/mL, and the staining image result with the staining intensity at the working concentration is better than that of a commercial antibody at the concentration of 3 ug/mL. Preferably, the lung cancer tissue is non-small cell lung cancer tissue and the lymph cancer tissue is hodgkin lymph cancer tissue.

Compared with the existing commercialized anti-PD-L1 monoclonal antibody for immunohistochemistry, the antibody provided by the invention has stronger staining effect signals on PD-L1 positive tissue sections under relatively low working concentration, so that the detection sensitivity and resolution can be improved, the antibody dosage can be reduced, and the detection cost can be reduced.

Drawings

FIG. 1 shows the results of flow cytometry for the binding ability of mouse anti-PD-L1 monoclonal antibody (mAPL 101) to cell surface PD-L1;

FIG. 2 shows the results of flow cytometry to detect the binding ability of mouse anti-PD-L1 monoclonal antibody (mAPL 102) to cell surface PD-L1;

FIG. 3 shows the results of flow cytometry to detect the binding ability of mouse anti-PD-L1 monoclonal antibody (mAPL 103) to cell surface PD-L1;

FIG. 4 shows the results of flow cytometry to detect the binding ability of mouse anti-PD-L1 monoclonal antibody (mAPL 104) to cell surface PD-L1;

FIG. 5 shows the results of flow cytometry to detect the binding ability of mouse anti-PD-L1 monoclonal antibody (mAPL 105) to cell surface PD-L1;

FIG. 6 shows the results of flow cytometry to detect the binding ability of mouse anti-PD-L1 monoclonal antibody (mAPL 106) to cell surface PD-L1;

FIG. 7 shows the results of flow cytometry for the binding ability of mouse anti-PD-L1 monoclonal antibody (mAPL 107) to cell surface PD-L1;

FIG. 8 shows the results of ELISA detection of the binding ability of each mouse anti-PD-L1 monoclonal antibody (mAPL 101) to soluble PD-L1;

FIG. 9 shows the results of ELISA detection of the binding ability of each mouse anti-PD-L1 monoclonal antibody (mAPL 102) to soluble PD-L1;

FIG. 10 shows the results of ELISA detection of the binding ability of each mouse anti-PD-L1 monoclonal antibody (mAPL 103) to soluble PD-L1;

FIG. 11 shows the results of ELISA detection of the binding ability of each mouse anti-PD-L1 monoclonal antibody (mAPL 104) to soluble PD-L1;

FIG. 12 shows the results of ELISA detection of the binding ability of each mouse anti-PD-L1 monoclonal antibody (mAPL 105) to soluble PD-L1;

FIG. 13 shows the results of ELISA detection of the binding ability of each mouse anti-PD-L1 monoclonal antibody (mAPL 106) to soluble PD-L1;

FIG. 14 shows the results of ELISA detection of the binding ability of each mouse anti-PD-L1 monoclonal antibody (mAPL 107) to soluble PD-L1;

FIG. 15, surface plasmon resonance detection of the affinity detection profile of mAPL 106 with PD-L1;

FIG. 16, surface plasmon resonance detection of the affinity detection profile of mAPL 107 with PD-L1;

FIG. 17, comparison of staining effects of different concentrations of mADL 107 on paraffin-embedded tissue sections;

figure 18, mAPDL107 compared to the effect of commercial antibody staining on paraffin-embedded tissue sections.

Detailed Description

Example 1 construction and screening of hybridoma cells

(1) Preparation of immunogens

The gene coding sequence of 128-bit 239 peptide segment of PD-L1 extracellular region (382-717 of GenBank: AF 177937.1), the gene coding sequence of killifish enterokinase light chain restriction site (amino acid sequence is DDDDK) (nucleotide sequence is gatgacgatgacaag), the gene coding sequence of IgG1 type antibody Fc segment (759-1457 of GenBank: Y14735.1), the codons of the first three cysteines are respectively mutated into serine codons agt and agc by tgt and tgc, then inserted into pcDNA3.1 vector, transfected CHO-K1 cells for transient mass expression, purified protein is extracted by a recombinant protein A affinity chromatography column, the Fc segment is cut by killifish enterokinase light chain protease, the PD-L1 extracellular region peptide segment is separated and collected by anion exchange chromatography, labeled by biotin, and mixed magnetic beads coated by streptavidin with 1 um diameter and streptavidin are mixed with Thermohr Onla magnetic beads (Thermoh One Onex) 001, Inc Coupling; after the coupling is finished, washing and processing the magnetic beads by using 70% ethanol, absolute ethanol and xylene in sequence, embedding the magnetic beads into paraffin according to the proportion of 0.1 mL of paraffin to 1 mg of magnetic beads, dewaxing by using the xylene, the absolute ethanol and the 70% ethanol in sequence, putting the magnetic beads into an EDTA antigen retrieval solution (10 mM Tris alkali, 1mM EDTA and 0.05% Tween 20) with the concentration of 5 mg/mL for 5 minutes in a boiling water bath, and naturally cooling to be used as immunogen of immune animals.

(2) Immunizing animals

Injecting the immunogen prepared in the last step into an abdominal cavity of a Balb/c mouse for 3 times for immunization, wherein the dosage of the magnetic bead used in each time is 0.5mg/100 uL, and injecting the immunogen into a tail vein for 1 time of boosting immunization 3-7 days before fusion.

(3) Cell fusion

Fusing a mouse myeloma cell NS-1 (1-2 × 10^ 7/mL) with an immunized Balb/c mouse spleen cell, then placing the fused cell on a 96-well cell culture plate, screening by using a complete culture medium containing HAT, changing the culture medium for half a day for 3-5 days, and cloning to form the hybridoma after about 2 weeks.

(4) Hybridoma clone culture supernatant identification

Respectively adding 0.1 mg of PD-L1 extracellular segment coated magnetic beads (experimental wells) and 0.1 mg of blank magnetic beads (control wells) prepared in the step (1) into a 96-well plate, respectively adding 200 uL of culture supernatants of different hybridoma clones into the experimental wells and the control wells, sequentially adding HRP (horseradish peroxidase) -labeled anti-mouse IgG antibody (enzyme-labeled secondary antibody) and TMB substrate for color development, reading OD (OD) values of the wells by using an enzyme-labeling instrument, selecting a monoclonal antibody of the experimental well, namely a mouse anti-human PD-L1 monoclonal antibody, and selecting a monoclonal positive clone to culture by using a complete culture solution containing HT (hypoxanthine and thymidine).

(5) Subcloning

Cloning the single positive clones screened in the previous step by using the complete culture medium, and selecting single cloning wells by using the complete culture medium for culturing.

(6) Antibody subtype detection

The culture supernatants of 7 monoclonal antibodies (accession number mADL 101-mADL 107) were tested with a mouse monoclonal antibody subtype test kit (purchased from Western Bao Biotechnology (Shanghai) Co., Ltd., product number ISO 2) according to the instructions to determine the light and heavy chain subtypes, and the test results are shown in Table 2.

TABLE 2 detection results of mouse antibody subtypes

Serial number	Clone name	Heavy chain subtype	Light chain subtype
				1	mAPDL101	IgG1	kappa
2	mAPDL102	IgG2a	kappa
				3	mAPDL103	IgG1	kappa
4	mAPDL104	IgG1	kappa
				5	mAPDL105	IgG1	kappa
6	mAPDL106	IgG1	kappa
				7	mAPDL107	IgG1	kappa

Example 2 detection of the binding Capacity of mouse anti-PD-L1 monoclonal antibody to cell surface PD-L1

The full-length human PD-L1 coding sequence (GenBank: AF 177937.1) is inserted into pcDNA3.1 expression vector, CHO-K1 host cell is transfected by liposome method, and CHO-K1 engineering cell (CHO-K1/hmPD-L1) with PD-L1 stably expressed on the surface of cell membrane is obtained by pressure screening. CHO-K1/hmPD-L1 in the logarithmic growth phase was collected, 7 of the murine anti-human PD-L1 monoclonal antibodies mADLD L101-mADLD L107 screened in example 1 were added, and each antibody was diluted in a 2-fold gradient from 10ug/ml or 100 ug/ml to 15 concentrations and allowed to bind to PD-L1 on the cell surface of CHO-K1/hmPD-L1 in 45 min ice. Wash 2 times with 1% FBS, add FITC-labeled rabbit anti-mouse IgG (H + L) (fluorescently labeled secondary antibody), ice-wash for 45 min, wash 2 times with 1% FBS. The pellet was resuspended in 1% FBS and analyzed by flow cytometry. MFI (mean fluorescence intensity) measured by flow cytometry was plotted against antibody concentration, and the results are shown in fig. 1 to 7. The EC50 (half maximal effect concentration) values for each monoclonal antibody were obtained by calculation from the data in the figure, see table 3.

TABLE 3 EC50 binding of mouse anti-PD-L1 monoclonal antibody to cell surface PD-L1

Serial number	Clone name	EC50(ng/ml)
			1	mAPDL101	274.7112
2	mAPDL102	338.21233
			3	mAPDL103	275.14833
4	mAPDL104	291.75626
			5	mAPDL105	247.06005
6	mAPDL106	457.96678
			7	mAPDL107	665379.2182

The data in Table 3 show that mADL 101-mADL 106 can bind to cell surface PD-L1 at a very low effective concentration, while mADL 107 binds to cell surface PD-L1 only weakly.

Example 3 detection of the binding Capacity of the mouse anti-PD-L1 monoclonal antibody to soluble PD-L1

The gene coding sequence of 19 th-239 th peptide segment of human PD-L1 extracellular region (55 th-717 th position of GenBank: AF 177937.1) and IgG1 type antibody Fc segment gene coding sequence (759 rd 1457 th position of GenBank: Y14735.1), the codons of the first three cysteines are respectively mutated into serine codons agt and agc from tgt and tgc, then inserted into pcDNA3.1 vector, transfected CHO-K1 cell for instantaneous mass expression, and purified protein is extracted by recombinant protein A affinity chromatography column to obtain recombinant human PD-L1-Fc fusion protein.

The recombinant human PD-L1-Fc fusion protein is diluted to 10ug/ml, added into an enzyme label plate at 100 ul/well and coated for 1-2 hours at 37 ℃. Adding sealing liquid, sealing at 37 deg.C for 1-2 hr, and washing for 7 times. Diluting a mouse anti-human PD-L1 monoclonal antibody mADL 101-mADL 107 by PBS, from initial concentration of 10000-50000ng/ml, diluting 15 concentrations in a gradient manner by 2-4 times, adding 100 ul/hole to an enzyme label plate, and reacting for 1-2 hours at 37 ℃. After washing the plate, the enzyme-labeled secondary antibody, goat anti-mouse IgG (H + L) -HRP, was added to the plate and reacted at 37 ℃ for 1 hour. After washing the plate, adding TMB substrate solution to the ELISA plate, shading and developing for 10 minutes, adding stop solution, reading OD450nm value by an ELISA reader, and taking OD570nm as reference wavelength. The results are shown in FIGS. 8-14, where the ratio of OD450/OD570 is plotted against the antibody concentration. The EC50 (half maximal effect concentration) values for each monoclonal antibody were obtained by calculation from the data in the figure, see table 4.

TABLE 4 EC50 of mouse anti-PD-L1 monoclonal antibody binding to soluble PD-L1

Serial number	Clone name	EC50(ng/ml)
			1	mAPDL101	1.471
2	mAPDL102	1.601
			3	mAPDL103	1.663
4	mAPDL104	2.733
			5	mAPDL105	2.462
6	mAPDL106	5.779
			7	mAPDL107	9.325

The data in Table 4 show that mADL 101-mADL 107 can be combined with soluble PD-L1 at a very low effective concentration.

Example 4 Surface Plasmon Resonance (SPR) detection of the affinity of the mouse anti-PD-L1 monoclonal antibody to PD-L1

The recombinant human PD-L1-Fc fusion protein is labeled by biotin, is coupled to an SA (SCBS SAHC 200M) chip of BIACORE equipment at a concentration of 15 ug/mL (both the equipment and the chip are purchased from GE Healthcare), reaches 336.3RU, a mouse anti-human PD-L1 monoclonal antibody mADL 101-mADL 107 to be detected is subjected to gradient dilution by using a running buffer (namely HBS-EP buffer) and then is injected respectively, the flow rate of the whole experiment process is set to be 30uL/min and is carried out at 25 ℃, the refractive index change caused by the combination of protein coupled with the surface of the chip in a flow phase is measured by an instrument, kinetic parameters such as Ka, Kd, KD values and the like are obtained by calculation of matched software of the instrument, and detection data are shown in Table 5, wherein the affinity detection spectra of mADL 106 and PDL107 are shown in figure 15 and figure 16.

TABLE 5 EC50 of mouse anti-PD-L1 monoclonal antibody binding to soluble PD-L1

Serial number	Clone name	Ka(1/Ms)	Kd(1/s)	KD(M)
					1	mAPDL101	3.748E6	3.062E-6	8.170E-13
2	mAPDL102	2.032E6	3.623E-5	1.783E-11
					3	mAPDL103	1.209E6	5.597E-5	4.631E-11
4	mAPDL104	1.477E6	6.074E-7	4.113E-13
					5	mAPDL105	1.283E6	4.245E-6	3.308E-12
6	mAPDL106	5.912E5	3.462E-6	5.855E-12
					7	mAPDL107	5.3910E5	2.588E-5	4.801E-11

The data in Table 5 show that the mouse anti-human PD-L1 monoclonal antibody mAPL 101-mAPL 107 can bind to the soluble PD-L1 with high affinity of 10^ 11-10^ -13M.

Example 5 enzyme-linked immunosorbent assay (ELISA) to test the ability of the mouse anti-PD-L1 monoclonal antibody to compete with PD-1 for binding to PD-L1

The recombinant human PD-1-Fc fusion protein is diluted to 10ug/ml, and 100 ul/well is coated on an enzyme label plate. The mouse anti-human PD-L1 monoclonal antibody mADL 101-mADL 107 with the concentration of 1000ug/ml is respectively diluted in a 2-fold ratio gradient, mixed with the recombinant human PD-L1-Fc fusion protein coupled with Biotin (Biotin) in equal volume, added into an enzyme label plate at 100 uL/hole, reacted for 1 hour at 37 ℃, and washed. Horseradish peroxidase (Avidin-HRP) conjugated with Avidin was added, reacted at 37 ℃ for 1 hour, and the plate was washed. Adding TMB substrate solution to an ELISA plate, performing dark color development for 15 minutes, adding stop solution, reading OD450nm by an ELISA reader, and taking 570nm as reference wavelength. The ratio of OD450/OD570 was plotted against the antibody concentration, and the EC50 value of each monoclonal antibody was obtained by calculation based on the data in the graph, and the ability of each monoclonal antibody to compete with PD-1 for binding to PD-L1 was judged, as shown in Table 6.

TABLE 6 ability of mouse anti-PD-L1 monoclonal antibody to compete with PD-1 for binding to PD-L1

Serial number	Clone name	EC50 (ug/mL)	Whether to compete
				1	mAPDL101	6.298	Competition
2	mAPDL102	6.071	Competition
				3	mAPDL103	8.424	Competition
4	mAPDL104	243.3	Weak competition
				5	mAPDL105	302.3	Weak competition
6	mAPDL106	N/A	Do not compete
				7	mAPDL107	N/A	Do not compete

Example 6 enzyme-linked immunosorbent assay (ELISA) to test the ability of the mouse anti-PD-L1 monoclonal antibody to compete with Tecnriq for binding to PD-L1

The recombinant human PD-L1-Fc fusion protein is diluted to 10ug/ml, and 100 ul/well is coated on an enzyme label plate. The mouse anti-human PD-L1 monoclonal antibody mADL 101-mADL 107 at a concentration of 1000ug/ml was diluted in a 2-fold gradient, mixed with a Biotin (Biotin) -labeled Tecntriq (general name Atezolizumab, Genentech) in equal volume, added to an ELISA plate at 100 ul/well, reacted at 37 ℃ for 1 hour, and the plate was washed. Horseradish peroxidase (Avidin-HRP) conjugated with Avidin was added, reacted at 37 ℃ for 1 hour, and the plate was washed. Adding TMB substrate solution to an ELISA plate, performing dark color development for 15 minutes, adding stop solution, reading OD450nm by an ELISA reader, and taking 570nm as reference wavelength. The ratio of OD450/OD570 was plotted against the antibody concentration, and the EC50 value of each monoclonal antibody was obtained by calculation based on the data in the graph, and the ability of each monoclonal antibody to compete with Tecntriq for binding to PD-L1 was judged, as shown in Table 7.

TABLE 7 ability of mouse anti-PD-L1 monoclonal antibody to compete with Tecntriq for binding to PD-L1

Serial number	Clone name	EC50 (ug/mL)	Whether to compete
				1	mAPDL101	4.917	Competition
2	mAPDL102	6.251	Competition
				3	mAPDL103	9.805	Competition
4	mAPDL104	6.954	Competition
				5	mAPDL105	6.939	Competition
6	mAPDL106	54.73	Weak competition
				7	mAPDL107	N/A	Do not compete

Example 7 measurement of mouse PD-L1 monoclonal antibody variable region cDNA and amino acid sequence

Extracting total RNA of a mouse hybridoma clone mADL 107, carrying out reverse transcription to synthesize cDNA, selecting conserved sequences near variable regions of a mouse antibody heavy chain (IgG 1 subtype) and a mouse antibody light chain (Kappa subtype) to respectively design primers, carrying out PCR (polymerase chain reaction) amplification on nucleic acid fragments of the variable regions, separating PCR products by using 1% agarose gel electrophoresis, recovering and purifying the PCR products, cloning the PCR products into pGEM-T easy (purchased from Promega company), carrying out DNA sequencing, and translating the DNA sequencing result into an amino acid sequence by using software. The result shows that the DNA coding sequences of the light chain and heavy chain variable regions of the mAPL 107 are the sequences of SEQ ID NO. 3 and SEQ ID NO. 4 respectively, and the amino acid sequences are the sequences of SEQ ID NO. 1 and SEQ ID NO. 2 respectively.

The antibody variable region sequences were encoded using the Kabat encoding rules, and the antibody variable region CDRs (complementarity determining regions) were divided according to the Kabat definition, and the light chain CDR1, CDR2, and CDR3 amino acid sequences of mADL 107 were SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, and the heavy chain CDR1, CDR2, and CDR3 amino acid sequences were SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively.

Example 8 immunohistochemical assay method for mADL 107 detection of PD-L1 in Paraffin-embedded tissue sections

1) Preparation of tissue sections

Paraffin sections were dewaxed to water:

placing a paraffin-embedded tissue section specimen (hereinafter referred to as a specimen) in a 60 ℃ oven, and taking out the specimen after baking for 30 minutes; soaking in xylene solution for 10 min, draining, and soaking for 3 times; soaking in anhydrous ethanol for 5 min, draining, and soaking for 1 time; and then soaking in 95% ethanol, 85% ethanol and 70% ethanol for 5 minutes respectively, draining, and soaking in purified water for later use.

Antigen retrieval:

the specimen is put into a staining jar containing 200 mL of pH9.0 EDTA antigen retrieval solution (10 mM Tris alkali, 1mM EDTA, 0.05% Tween 20), put into an antigen retrieval instrument, put into a water bath, retained pressure and heat at 120 ℃ for retrieval for 5 minutes, and naturally cooled to room temperature.

Blocking of endogenous peroxidase:

the residual liquid on the sections was removed, and Hydrogen Peroxide blocking solution (Hydrogen Peroxide Block) 100-150uL in DAB Kit (Mouse Specific HRP/DAB (ABC) DetectionIHC Kit, available from abcam, cat # ab 64259) was added to cover the tissues, followed by incubation at room temperature for 10 minutes. Washing with TBST washing solution (Tris buffer (pH7.4), containing 0.05% Tween20, 50 mM Tris, and 150 mM NaCl), draining for 10 s, soaking in TBST washing solution for 3 min, and repeating for 2 times.

And (3) sealing:

the residual liquid on the sections was removed, and 10% goat serum 100-150uL was added to cover the tissues and incubated for 60 min at room temperature. And (4) washing the samples with a TBST cleaning solution, taking out the samples one by one, removing residual liquid, completely soaking the samples in the TBST cleaning solution for 3 minutes, and repeating the soaking for 3 times.

Taking out the section, removing residual liquid, adding 150uL of Protein blocking liquid (Protein Block, DAB kit) 100-.

2) First antibody

The primary antibody (anti-PD-L1 monoclonal antibody, or control antibody) was diluted with 1% BSA (bovine serum albumin) in TBS buffer (1% (m/v) BSA, 50 mM Tris, 150 mM NaCl in water) to working concentration, the specimen was removed, the residual liquid was removed, 150uL of the diluted primary antibody was added dropwise to each chip to cover the tissue, and the chip was incubated overnight at 4 ℃ in a wet box.

Rewarming and washing:

the wet box was left at room temperature for 45 minutes, the slides were rinsed with TBST rinse, drained for 10 seconds and then soaked in TBST rinse for 3 minutes, and the procedure was repeated 5 times.

3) Secondary antibody

The residual liquid on the sections was removed, 150uL of HRP-labeled goat anti-mouse IgG (Invitrogen, cat. 62-6520) diluted in 1/2000 was added dropwise, and the mixture was applied to the tissues and incubated at room temperature for 60 minutes. The slide was rinsed with TBST rinse, drained for 10 seconds and then immersed in TBST rinse for 3 minutes, and the procedure was repeated 5 times.

Color development:

DAB Chromogen (dye) and DAB Substrate (Substrate) in a DAB kit are uniformly mixed in a ratio of 1:50 to prepare DAB developing solution for later use.

Removing residual liquid on the slices, dropwise adding 150uL of prepared DAB color development solution, covering the tissues, and incubating for 1.5 minutes at room temperature.

Washing:

the slide was rinsed with TBST rinse, drained for 10 seconds and then immersed in TBST rinse for 3 minutes, and the procedure was repeated 5 times.

Counterstaining, differentiation and bluing:

the sections were immersed in hematoxylin stain (purchased from Shanghai leaf Biotech Co., Ltd., product No. R20568) and incubated for 4 minutes. Washed with tap water, then immersed in 1% ethanol hydrochloride differentiation solution for 3 seconds, washed with tap water, and rinsed with running water to turn blue for 80 minutes.

Dehydrating and transparent:

taking out the specimen, draining, sequentially soaking in 70%, 85%, 95% and 100% ethanol for 5 min, and repeatedly soaking in 100% ethanol for 1 time; then soaking the substrate in a dimethylbenzene solution for treatment for 2 times, 10 minutes each time; draining before each soaking.

Sealing:

the specimens were taken out, drained, sealed with neutral gum (purchased from national pharmaceutical group chemical reagents ltd., cat # 10004160), horizontally placed in a dark place, and dried for about 1 week.

4) Mirror viewing, photographing and detecting

The observation and photography were carried out with an upright microscope.

And a result judgment method comprises the following steps:

selection of negative and positive tissues and quality control standard:

the experiment uses a normal human stomach tissue section theoretically without PD-L1 expression as a negative control tissue, the specimen has no specific binding reaction with mouse serum (irrelevant antibody control) and anti-PD-L1 antibody, if inevitable non-specific binding occurs in the experiment, the staining degrees of the sample group treated specimen and the mouse serum treated specimen are compared, and the non-specific staining can be judged only when the staining intensities of the two are the same.

The experiment used a normal human placental tissue section with high expression of PD-L1 as a positive control tissue, which should have no specific binding reaction with murine serum (irrelevant antibody control) and specific binding reaction with anti-PD-L1 antibody.

The two organizations are used in the experiment and can be used as quality standard control in a system, so that the results in batch experiments are ensured to be true and reliable.

And (4) judging a result standard:

after the experiment is finished, the negative and positive tissues should be analyzed firstly, and only when the dyeing result accords with the expected result, the result of the experiment is reliable, and other samples can be analyzed.

Furthermore, ideally the murine serum (irrelevant antibody control) should not bind specifically to human tissue, and therefore no staining of tissue sections treated with murine serum should occur. When non-specific binding occurs, the staining of the corresponding area under different antibody treatments is compared, and the positive judgment can be made only when the stained area of the sample treatment group does not stain at the corresponding position of the mouse serogroup; otherwise, it should be non-specific staining.

Determining the expression level of PD-L1 only requires calculating the staining ratio of tumor cell membranes in the tumor area, without considering immune cell staining.

The staining pattern of the cell membrane is acceptable as discontinuous linear, circular, or basolateral staining.

The intensity of cell membrane staining is acceptable at any intensity after background color interference is excluded.

According to the above test procedure, immunohistochemical detection tests were performed using different concentrations of the first antibody, and 0.1ug/mL, 1ug/mL, 3ug/mL, and 6ug/mL of the anti-PD-L1 antibody were used as the first antibody, and the results are shown in FIG. 17, and can be analyzed from FIG. 7: the final staining effect of the anti-PD-L1 antibody of 0.1ug/mL is already obvious, and can be used for judging the result of the immunohistochemical detection test; although the staining effect is increased with the increase of the concentration of the anti-PD-L1 antibody, the staining effect is not obvious, and the staining results are similar at low concentration, final concentration and high concentration.

Example 9 verification of the Effect of mADL 107 detection of PD-L1 in Paraffin sections of Normal stomach tissue and placental tissue

For further comparison and verification of the staining effect, paraffin-embedded placental tissue sections (PD-L1 positive tissue), gastric tissue sections (PD-L1 negative tissue), non-small cell lung cancer tissue and hodgkin's lymphoma tissue were examined as described in example 8 using self-made antibody mAPDL107 (working concentration 0.1 ug/mL), commercial anti-PD-L1 monoclonal antibody 22C3 (working concentration 3 ug/mL) and normal Balb/C mouse serum (irrelevant control) as primary antibodies, and the staining images are shown in fig. 18 (each cancer tissue section is selected from 2 different fields under the microscope), and the results of staining are shown in table 8.

TABLE 8 comparison of the staining of paraffin-embedded tissue sections with mAPL L107 and commercial antibodies

From the staining results it can be seen that:

(1) the home-made antibody mADL 107, the commercial antibody 22C3, and the irrelevant control mouse serum were not stained for PD-L1 negative gastric tissue sections.

(2) The self-made antibody mAPL 107 can specifically stain the placenta tissue positive to PD-L1 at the working concentration of 0.1ug/mL, and the staining intensity is higher than that of the commercial antibody 22C3 at the working concentration of 3 ug/mL.

(3) Mouse serum has no staining on non-small cell lung cancer tissue slices; 22C3 and mADL 107 stained lung cancer tissue sections to different degrees, there was a staining of mADL 107 in some fields and no staining of 22C3, and the staining intensity of mADL 107 was slightly higher than 22C 3.

(4) Mouse serum was not stained on hodgkin lymphoma tissue sections; 22C3 and mADL 107 both stained differently, with comparable intensities, but the working concentration of mADL 107 was only 1/30 at 22C 3.

Claims

1. An anti-PD-L1 antibody for immunohistochemical detection is characterized in that the sequences of a heavy chain CDR1, a CDR2 and a CDR3 of the anti-PD-L1 antibody are respectively SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10; the light chain CDR1, CDR2 and CDR3 have the sequences of SEQ ID NO 5, SEQ ID NO 6 and SEQ ID NO 7, respectively.

2. The anti-PD-L1 antibody for use in immunohistochemical detection according to claim 1, wherein the amino acid sequence of the heavy chain variable region of the anti-PD-L1 antibody is SEQ ID NO 2; the amino acid sequence of the light chain variable region is SEQ ID NO 1.

3. The anti-PD-L1 antibody for use in immunohistochemical detection of claim 1, wherein the anti-PD-L1 antibody heavy chain amino acid sequence is SEQ ID NO 14 and the light chain amino acid sequence is SEQ ID NO 13.

4. The anti-PD-L1 antibody for use in immunohistochemical detection according to claim 1, wherein the antibody is a Fab fragment, a F (ab ')2 fragment, a Fv fragment or a Fab' -SH fragment of an antibody, or is a single chain antibody, a single domain antibody, a bispecific antibody, a multispecific antibody.

5. An anti-PD-L1 antibody for use in an immunohistochemical assay according to claims 1 and 4, in an immunohistochemical method for detecting expression of PD-L1 in human tissue.

6. The use of the anti-PD-L1 antibody for immunohistochemical detection according to claim 5 in an immunohistochemical method for detecting expression of PD-L1 in human tissues, characterized in that the concentration of the anti-PD-L1 antibody is 0.1. mu.g/mL to 3. mu.g/mL.

7. The use of claim 5, wherein said tissue is human tumor tissue, gastric tissue, placental tissue, lung tissue, or lymphoid tissue.

8. A method for immunohistochemical detection using the anti-PD-L1 antibody of claim 1 as a first antibody, comprising the steps of: 1) making a tissue slice; 2) using an anti-PD-L1 antibody as a first antibody, acting on the prepared tissue section, and incubating overnight; 3) adding a second antibody combined with the first antibody, labeling, developing, washing and sealing; 4) and (5) observing and detecting through a mirror.

9. The method of claim 7, wherein the concentration of the first anti-PD-L1 antibody is 0.1 μ g/mL to 6 μ g/mL.

10. The method of claim 8, wherein the concentration of the first anti-PD-L1 antibody is 0.1 μ g/mL to 3 μ g/mL.

11. The method of claim 9, wherein the concentration of the first anti-PD-L1 antibody is 0.1 μ g/mL.