Anti-CD8 antibody or antigen binding fragment thereof, CD8 + cell-enriched product and application thereof

Abstract

The invention discloses an anti-CD8 antibody or an antigen binding fragment thereof, a CD8 + cell-enriched product and application thereof, and relates to the technical field of antibodies. The heavy chain complementarity determining region comprises a heavy chain complementarity determining region in a heavy chain variable region as shown in SEQ ID NO: 16 and a light chain complementarity determining region in the heavy chain variable region as shown in SEQ ID NO: 15. According to the invention, more protein choices are provided for detection of CD8 and detection of CD8 + cells. The antibody specifically combined with the CD8 polypeptide provided by the invention can be used for preparing a reagent or a kit for detecting, separating or enriching CD8 + cells, and preparing a reagent or a kit for monitoring disease progression in a cancer subject; and preparing a reagent or a kit for predicting responsiveness of a subject with cancer to immunotherapy or a cancer vaccine.

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, and in particular to an anti-CD8 antibody or an antigen-binding fragment thereof, a product for enriching CD8+ cells, and applications thereof. Background Art

[0002] The CD8 molecule is a leukocyte differentiation antigen and a glycoprotein on the surface of some T cells. It is used to assist the T cell receptor (TCR) in recognizing antigens and participate in the transduction of T cell activation signals. It is also called the co-receptor of TCR. T cells expressing CD8 (CD8+T cells) usually differentiate into cytotoxic T cells (CTLs) after activation and can specifically kill target cells.

[0003] CD8 cells are a type of T cell cytotoxic cell that mainly reflects the body's immunity; the higher the number of CD8 cells, the stronger the body's immunity. CD8 cells are mainly found in the spleen, lymph nodes, and tonsils; they play a positive role in anti-virus, anti-tumor, and AIDS testing standards.

[0004] In clinical practice, immunohistochemistry (IHC) pathology experiments are often used to detect protein expression in tumor cells. The accuracy and sensitivity of IHC experiments are determined by the quality of monoclonal antibodies that specifically bind to proteins. Therefore, developing a monoclonal antibody against CD8 with high binding specificity is of great significance for IHC experiments to detect CD8 expression levels.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The object of the present invention is to provide an anti-CD8 antibody or an antigen-binding fragment thereof, a product for enriching CD8+ cells and its application to solve the above-mentioned technical problems.

[0007] The present invention is achieved in that:

[0008] In a first aspect, the present invention provides an anti-CD8 antibody or an antigen-binding fragment thereof, which comprises a heavy chain complementary determining region in the heavy chain variable region as shown in SEQ ID NO: 16 and a light chain complementary determining region in the heavy chain variable region as shown in SEQ ID NO: 15.

[0009] In a second aspect, the present invention further provides use of an anti-CD8 antibody or an antigen-binding fragment thereof in any of the following:

[0010] (1) Enrichment of CD8+ cells;

[0011] (2) preparing reagents or kits for detecting, separating or enriching CD8+ cells;

[0012] (3) Use of a reagent or kit for preparing a test for predicting the responsiveness of a subject with cancer to immunotherapy or a cancer vaccine;

[0013] (4) Use of a reagent or kit for preparing a test kit for monitoring disease progression in a subject with cancer;

[0014] (5) preparing CD8 protein detection products;

[0015] (6) Preparing CD8 protein separation products or enrichment products.

[0016] In a third aspect, the present invention further provides a product for enriching CD8+ cells, comprising: the above-mentioned anti-CD8 antibody or its antigen-binding fragment.

[0017] In a fourth aspect, the present invention further provides a CD8 protein detection product, separation product or enrichment product, which comprises: the above-mentioned anti-CD8 antibody or antigen-binding fragment thereof.

[0018] In a fifth aspect, the present invention further provides a cell, which is a non-plant cell and expresses the above-mentioned anti-CD8 antibody or antigen-binding fragment thereof.

[0019] In a sixth aspect, the present invention also provides a nucleic acid molecule encoding the above-mentioned anti-CD8 antibody or antigen-binding fragment thereof.

[0020] In a seventh aspect, the present invention also provides a recombinant vector comprising the above-mentioned nucleic acid molecule.

[0021] The present invention has the following beneficial effects:

[0022] The present invention finds that antibodies with specific six complementary determining regions have good activity and can maintain high binding specificity and affinity with CD8 antigen. This means that the binding protein can be used to detect CD8, which helps to improve the sensitivity and specificity of the detection. The present invention provides more protein options for the detection of CD8 and the detection of CD8+ cells.

[0023] The antibodies specifically binding to CD8 polypeptides provided by the present invention can be used to prepare reagents or kits for detecting, separating or enriching CD8+ cells, preparing reagents or kits for monitoring disease progression in subjects with cancer, and preparing reagents or kits for predicting the responsiveness of subjects with cancer to immunotherapy or cancer vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0025] Figure 1 This is a picture of the results of immunohistochemistry experiments on lymphoma samples;

[0026] Figure 2 This is the result of immunohistochemistry experiment on spleen samples. DETAILED DESCRIPTION

[0027] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0028] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc., 1987). Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0029] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0030] Definition

[0031] The term "antigen binding fragment" refers to all proteins / protein fragments containing CDR regions, especially antibodies or antibody functional fragments. "Antigen binding fragments" include antigen compound binding fragments of the above-mentioned antibodies, including Fab, F(ab')2, Fd, Fv, scFv, bispecific antibodies, multispecific antibodies and antibody minimum recognition units, 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, etc. In addition, the term "antibody" includes naturally occurring antibodies and non-naturally occurring antibodies, including, for example, chimeric, bifunctional and humanized antibodies, and related synthetic isoforms. The term "antibody" can be used interchangeably with "immunoglobulin".

[0032] The term "antibody" herein is used in the broadest sense and may include full-length monoclonal antibodies, bispecific or multispecific antibodies, chimeric antibodies, and antibody fragments, so long as they exhibit the desired biological activity, such as specific binding to CD8 protein.

[0033] In the present invention, the term "complementarity determining region or complementarity determining region", "CDR" refers to the highly variable region of the heavy and light chains of immunoglobulins, and refers to the region containing one or more or even all of the major amino acid residues that contribute to the binding affinity of the antibody or antigen-binding fragment with its recognized antigen or epitope. In a specific embodiment of the present invention, CDRs refer to the highly variable regions of the heavy and light chains of the antibody.

[0034] In the present invention, the heavy chain complementary determining region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementary determining region is represented by LCDR, which includes LCDR1, LCDR2 and LCDR3. Commonly used CDR labeling methods in the art include: Kabat numbering scheme, IMGT numbering scheme, Chothia and Lesk numbering scheme, and the new standardized numbering system introduced by Lefranc et al. for all protein sequences of the immunoglobulin superfamily in 1997. Kabat et al. were the first to propose a standardized numbering scheme for immunoglobulin variable regions. Over the past few decades, the accumulation of sequences has led to the creation of the KABATMAN database, and the Kabat numbering scheme is generally considered to be a widely used standard for numbering antibody residues. The present invention uses the Kabat annotation standard to mark the CDR region, but CDR regions marked by other methods also fall within the scope of protection of the present invention.

[0035] Typically, the variable region VH of the heavy chain of an antibody can be obtained by arranging and connecting the following numbered CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4. HCDR1 is synonymous with CDR-H1.

[0036] The variable region VL of the antibody light chain can be obtained by arranging and connecting the CDRs and FRs with the following numbers in the following combination: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0037] LCDR1 is CDR-L1, LCDR2 is CDR-L2, and LCDR3 is CDR-L3.

[0038] The subject in the present invention is a human, a non-human primate, a mouse, or a rat.

[0039] In a first aspect, the present invention provides an anti-CD8 antibody or an antigen-binding fragment thereof, which comprises a heavy chain complementary determining region in the heavy chain variable region as shown in SEQ ID NO: 16 and a light chain complementary determining region in the heavy chain variable region as shown in SEQ ID NO: 15.

[0040] Light chain variable region SEQ ID NO: 15

[0041] DILMTQFPLSLPVSFGDQASISCRSSQSIVHSNGNTYLEWYLQ KPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLG VYYCFQGSHVPWT FGGGTKPENK.

[0042] Heavy chain variable region SEQ ID NO: 16

[0043] QAQLQQSGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQTP EKRLEWVAYISNGGGSTYYPDTVKGRFTVSRDNAKNILYLQMSR LRSDDTAMYYCGRHGGDGDY WGQGTTVTVSS.

[0044] In a preferred embodiment of the present invention, the heavy chain complementary determining region includes: CDR-H1, CDR-H2 and CDR-H3, whose amino acid sequences are shown in SEQ ID NOs: 9-11, respectively, and the light chain complementary determining region includes CDR-L1, CDR-L2 and CDR-L3, whose amino acid sequences are shown in SEQ ID NOs: 12-14, respectively.

[0045] CDR-H1: DYYMY; SEQ ID NO: 9.

[0046] CDR-H2: YISNGGGSTYYPDTVKG; SEQ ID NO: 10.

[0047] CDR-H3: HGGDGDY; SEQ ID NO: 11.

[0048] CDR-L1: RSSQSIVHSNGNTYLE; SEQ ID NO: 12.

[0049] CDR-L2: KVSNRFS; SEQ ID NO: 13.

[0050] CDR-L3: FQGSHVPWT. SEQ ID NO: 14.

[0051] The inventors have found that antibodies or antigen-binding fragments thereof having the above-mentioned six complementary determining regions have good activity and can maintain high binding specificity and affinity with the CD8 antigen. This means that the antibody can be used to detect CD8, which helps to improve the sensitivity and specificity of the detection. The present invention provides more protein options for the detection of CD8 and for the detection of CD8+ cells. The antibodies that specifically bind to the CD8 polypeptide provided by the present invention can be used to prepare reagents or kits for detecting, separating or enriching CD8+ cells, preparing reagents or kits for monitoring disease progression in subjects with cancer, and preparing reagents or kits for predicting the responsiveness of subjects with cancer to immunotherapy or cancer vaccines.

[0052] In a preferred embodiment of the present invention, the antibody and CD8 polypeptide are K D ≤2.478×10 8 L / mol affinity binding.

[0053] In a preferred embodiment of the present invention, the antibody or its antigen-binding fragment further comprises a heavy chain framework region, and / or a light chain framework region; the heavy chain framework region comprises HFR1, HFR2, HFR3 and HFR4, which have at least 80% homology to the amino acid sequences shown in SEQ ID NOs:5-8, respectively; the light chain framework region comprises LFR1, LFR2, LFR3 and LFR4, which have at least 80% homology to the amino acid sequences shown in SEQ ID NOs:1-4, respectively.

[0054] The sequences of SEQ ID NO: 1-8 are shown in the following table:

[0055]

[0056] In an optional embodiment, the heavy chain framework region includes HFR1, HFR2, HFR3 and HFR4, which are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous to the amino acid sequences shown in SEQ ID NO:5-8.

[0057] The light chain framework region includes LFR1, LFR2, LFR3 and LFR4, which are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous to the amino acid sequences shown in SEQ ID NOs: 1-4, respectively.

[0058] In a preferred embodiment of the present invention, the antibody or antigen-binding fragment thereof further comprises a constant region, the constant region comprises a heavy chain constant region, and / or a light chain constant region, the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the κ type or λ type light chain constant region;

[0059] In a preferred embodiment of the present invention, the species of the constant region is cattle, horse, sheep, goat, rat, mouse, pig, dog, cat, donkey, deer, mink, chicken, duck, rabbit, goose or human;

[0060] In a preferred embodiment of the present invention, the species of origin of the constant region is mouse;

[0061] In a preferred embodiment of the present invention, the antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv, Fab'-SH and scFv of an antibody.

[0062] The antigen-binding fragments of the above antibodies generally have the same binding specificity as the antibodies from which they are derived. It is easy for those skilled in the art to understand based on the contents described in the present invention that the functional fragments of the above antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction to split disulfide bonds.

[0063] The antigen-binding fragments of the above antibodies can also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, an automatic peptide synthesizer, such as those sold by Applied BioSystems and the like.

[0064] In a second aspect, the present invention further provides use of an anti-CD8 antibody or an antigen-binding fragment thereof in any of the following:

[0065] (1) Enrichment of CD8+ cells;

[0066] (2) preparing reagents or kits for detecting, separating or enriching CD8+ cells;

[0067] (3) Use of a reagent or kit for preparing a test for predicting the responsiveness of a subject with cancer to immunotherapy or a cancer vaccine;

[0068] (4) Use of a reagent or kit for preparing a test kit for monitoring disease progression in a subject with cancer;

[0069] (5) preparing CD8 protein detection products;

[0070] (6) Preparing CD8 protein separation products or enrichment products.

[0071] Application (1) includes but is not limited to: coating an anti-CD8 antibody or its antigen-binding fragment on a solid phase and incubating it with a cell sample to enrich CD8+ cells. The solid phase includes but is not limited to substrates such as magnetic beads, plates, membranes, and glass.

[0072] In a preferred embodiment of the application of the present invention, application (3) comprises: administering a labeled anti-CD8 antibody to the subject; and wherein the detection of the binding of the labeled anti-CD8 antibody to CD8+T cells in the tumor tissue of the subject indicates that the subject is likely to respond to the immunotherapy or the cancer vaccine. The above-mentioned detection of the binding of the labeled anti-CD8 antibody to CD8+T cells in the tumor tissue of the subject comprises imaging the CD8+T cells in the subject, and the imaging of the CD8+T cells in the subject is performed, for example, by performing a positron emission tomography / computed tomography (PET / CT) scan on the subject.

[0073] In a preferred embodiment of the application of the present invention, application (4) includes: administering a labeled anti-CD8 antibody to the subject, and the monitoring includes detecting the binding of the labeled anti-CD8 antibody to CD8+T cells in the tumor tissue of the subject at a first time point and a second time point. The above-mentioned detection of the binding of the labeled anti-CD8 antibody to CD8+T cells in the tumor tissue of the subject includes imaging the CD8+T cells in the subject, and imaging the CD8+T cells in the subject is performed, for example, by performing a positron emission tomography / computed tomography (PET / CT) scan on the subject.

[0074] In a preferred embodiment of the present invention, the product in application (5) is a reagent, a test kit, a test strip, an antibody chip, an antibody probe or a detector; the separation product or enrichment product in application (6) is a magnetic bead, a test kit or a separation column;

[0075] In a preferred embodiment of the present invention, in applications (1)-(6), the anti-CD8 antibody or antigen-binding fragment thereof is labeled with a detectable marker.

[0076] Detectable markers refer to a class of substances that have properties that can be directly observed by the naked eye or detected or detected by instruments, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.

[0077] In an alternative embodiment, the detectable labels include, but are not limited to, fluorescent dyes, enzymes that catalyze the color development of substrates, radioactive isotopes, chemiluminescent agents, and nanoparticle labels.

[0078] In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter which marker is used, it belongs to the protection scope of the present invention.

[0079] Fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5 .5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), prephycocyanin-chlorophyll protein (preCP), etc.).

[0080] In an alternative embodiment, the enzyme that catalyzes the color development of the substrate includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxidase.

[0081] In an alternative embodiment, the radioactive isotopes include but are not limited to 212 Bi, 131 I. 111 In, 90 Y. 186 Re, 211 At 125 I. 188 Re,153 Sm, 213 Bi, 32 P. 94 mTc, 99 mTc, 203 Pb, 67 Ga, 68 Ga, 43 Sc, 47 Sc, 110 mIn、 97 Such as 62 Cu, 64 Cu, 67 Cu, 68 Cu, 86 Y. 88 Y. 121 Sn, 161 Tb, 166 Ho, 105 Rh, 177 Lu, 172 Lu and 18 F.

[0082] In an optional embodiment, the chemiluminescent reagent includes, but is not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxyoxalates and their derivatives.

[0083] In an optional embodiment, the nanoparticle markers include but are not limited to nanoparticles and colloids; the nanoparticles include but are not limited to organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles and rare earth complex nanoparticles.

[0084] In an alternative embodiment, the colloid includes, but is not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex. In an alternative embodiment, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0085] In a preferred embodiment of the present invention, in applications (2)-(5), tissues and / or cells are labeled with anti-CD8 antibodies or antigen-binding fragments thereof, and the labeled tissues and / or cells are detected.

[0086] In a preferred embodiment of the present invention, the tissues are selected from tonsils, lymphoma and spleen.

[0087] In a third aspect, the present invention further provides a product for enriching CD8+ cells, comprising: the above-mentioned anti-CD8 antibody or antigen-binding fragment thereof, for example, an antibody coated with the anti-CD8 antibody or antigen-binding fragment thereof.

[0088] In a fourth aspect, the present invention further provides a CD8 protein detection product, separation product or enrichment product, which comprises: the above-mentioned anti-CD8 antibody or antigen-binding fragment thereof.

[0089] For example, the above-mentioned antibody is coated on magnetic beads for separation and enrichment of CD8 protein. In one embodiment, the antibody is coated on a filler and filled in a separation column; it is used for affinity separation and enrichment of CD8 protein. Therefore, the above-mentioned antibody or its antigen-binding fragment has a good application prospect in the preparation of CD8 protein enrichment products.

[0090] In a fifth aspect, the present invention further provides a cell, which is a non-plant cell and expresses the above-mentioned anti-CD8 antibody or antigen-binding fragment thereof.

[0091] The host cell is selected from mammalian cells; the mammalian cell is selected from any one of 293 cells, 293T cells, 293FT cells, CHO cells, COS cells, mouse L cells, LNCaP cells, 633 cells, Vero, BHK cells, CV1 cells, Hela cells, MDCK cells, Hep-2 cells and Per6 cells. Among them, 293 series cells, Per6 cells and CHO cells are commonly used mammalian cells for producing antibodies or recombinant proteins, and are well known to those of ordinary skill in the art.

[0092] In a sixth aspect, the present invention also provides a nucleic acid molecule encoding the above-mentioned anti-CD8 antibody or antigen-binding fragment thereof.

[0093] Taking into account the degeneracy of codons, the gene sequence encoding the above-mentioned antibody can be modified in its coding region without changing the amino acid sequence to obtain a gene encoding the same antibody amino acid sequence; the gene can also be artificially synthesized and modified according to the codon preference of the host expressing the antibody to improve the expression efficiency of the antibody.

[0094] In a seventh aspect, the present invention also provides a recombinant vector comprising the above-mentioned nucleic acid molecule.

[0095] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0096] Example 1

[0097] This example is about the preparation of specific anti-CD8 monoclonal antibody.

[0098] 1. Animal (mice) immunization.

[0099] Immunize mice with immunogens using a common method. The immunogen is a human CD8 polypeptide synthesized by Gill Biotech, and is also used as a detection antigen to measure and screen serum titers and hybridomas. High-purity antigens can increase the chance of obtaining the desired monoclonal antibody and reduce the amount of screening. Immunize 5 mice, each with 50ug CD8 antigen. Prepare the antigen protein solution with PBS. Put an appropriate amount of antigen protein, PBS, and Freund's adjuvant into a syringe, plug the outlet of the syringe with a stopper, and place it on an emulsifier to stir and fully emulsify, so that the antigen adjuvant forms a stable oil-in-water solution. The first tail blood serum titer test is performed 7-10 days after the primary and secondary immunizations, and a good titer is obtained after 2 to 4 booster immunizations. Select immunized mice with high serum titers for final intraperitoneal immunization and perform cell fusion.

[0100] 2. Hybridoma cell fusion and screening.

[0101] Preparations need to be made before cell fusion: 1. Cultivate mouse myeloma cells SP2 / 0 to the logarithmic growth phase; 2. Sacrifice a negative mouse one day before fusion, inject HAT culture medium into the mouse peritoneal cavity in a sterile environment, take mouse peritoneal trophoblast cells and plate them on a 96-well plate, 100ul per well. These cells promote the growth of hybridoma cells. Sacrifice the immune mouse, take the spleen in a sterile environment, use PEG to chemically fuse spleen B cells and SP2 / 0 myeloma cells, add appropriate HAT culture medium according to the number of cells to be plated, and finally plate the fused cells on the trophoblast cell culture plate, 100ul per well.

[0102] After 7-10 days, the growth of surviving hybridoma cells can be observed under a microscope. Two weeks after plating, the supernatant of each well is collected and the hybridoma cells are screened by ELISA method using human CD8 polypeptide antigen. The method is as follows: Coat the enzyme-labeled plate with 100ul 2ug / ml PBS solution of human CD8 polypeptide antigen at 37℃ for two hours. After washing the plate three times with PBST, add 150ul / well of 3% skim milk powder in PBS solution and place it at 4℃ for blocking overnight. Wash the plate three times again, add 80ul / well of hybridoma supernatant, incubate at 37℃ for 1 hour, and then wash the plate three times. Add 1:8000 diluted horseradish peroxidase-labeled goat anti-mouse secondary antibody to 100ul / well, incubate at 37℃ for 45 minutes, wash the plate three times, and pat dry. Add 100ul / well of TMB colorimetric solution, color at room temperature for 5-10 minutes, stop with 2M sulfuric acid solution, and measure the absorbance of each well at 450nm. Select positive hybridoma cells.

[0103] Select the ELISA-positive fusion wells and then perform immunohistochemistry (IHC), and select the positive wells for subsequent experiments. The experimental steps are as follows:

[0104] (1) Slice processing: Place the tonsil slices in a 60°C constant temperature oven and bake for 60 minutes. Soak the slices in xylene I for 15 minutes, then replace with xylene II and soak for 15 minutes. Soak the slices in anhydrous ethanol ① for 5 minutes, anhydrous ethanol ② for 5 minutes, 95% ethanol for 5 minutes, 85% ethanol for 5 minutes, and 75% ethanol for 5 minutes. Soak in ddH2O for 5 minutes and wash 3 times. Perform antigen repair in a pressure cooker (boiling method). Add enough EDTA to cover the slices in the pressure cooker and heat to boiling. Place the slices on a heat-resistant slice rack and put them in the pot. Cover the pot with a lid, fasten the pressure valve, continue heating, set the pressure to maintain for 4 minutes, open the air valve to release air after the time is up, and open the lid to remove the inner pot after the pressure returns to zero and let it cool at room temperature. After the solution cooled to room temperature, take out the slices (about 40 minutes); soak in ddH2O for 5 minutes, wash twice, soak in PBST for 5 minutes, wash twice; place the slices in 20ml 3% H2O2-methanol solution, protect from light, and treat at room temperature for 10 minutes; soak in PBST for 5 minutes and wash 3 times; add one drop of goat serum blocking solution (about 25ul) to each tissue group, incubate in a wet box at room temperature for 45 minutes; soak in PBST for 5 minutes and wash 3 times.

[0105] (2) Incubation of tissue sections with antibodies:

[0106] The processed tissue sections were added with CD8 antibody of Maixin Company for comparison, and the remaining sections were added with the above-mentioned positive hybridoma cells. Incubate overnight in a 4℃ wet box; remove from a 4℃ refrigerator and incubate at room temperature for 60 minutes; rinse gently with PBST, soak for 5 minutes, and wash 3 times; add 25ul of HRP-labeled Long Island Company secondary antibody (CAT#: D-3004-0100) to each tissue group, incubate for 45 minutes at room temperature; wash; prepare DAB color developing solution, react in the dark for 10-15 minutes, then add it dropwise to the slices and color for 1-5 minutes; terminate the color development reaction with distilled water; add 50ul of hematoxylin staining solution to each tissue group, stain for 5-10 minutes, and rinse with distilled water; decolorize the slices in 1% hydrochloric acid-ethanol for 2-3 seconds, then quickly take them out and terminate them in distilled water, and then put them in PBST (pH8.0) for anti-blueing for 5-10 minutes; soak in 75% ethanol for 5 minutes: 85% ethanol for 5 minutes; soak in 95% ethanol for 5 minutes: and soak in anhydrous ethanol for 5 minutes. Soak in xylene for 10 minutes, then change to xylene and soak for another 10 minutes; add neutral gum to seal the slide, then cover with a coverslip; photograph the slide under a microscope.

[0107] The fusion cells with positive binding were selected by ELISA and IHC experiments, cloned by limiting dilution method, and each well of the positive strain was plated on 48 / 96 well plates for further culture. A second round of screening was performed by ELISA method to screen out hybridomas that specifically recognized CD8 polypeptide and could block CD8 binding, and subcloned by limiting dilution method to obtain a monoclonal cell line 1F1E1A10.

[0108] The monoclonal cell line was expanded and about 1×10 6 The cells were injected into selected mice (mice were injected with paraffin oil in the peritoneal cavity one week in advance), and then after 7-10 days of waiting, the mice produced ascites, which was collected for antibody purification. After purification, a mouse monoclonal antibody CD8-1F1E1A10 specific for CD8 polypeptide was obtained.

[0109] Figure 1 The test specimens are the immunohistochemistry results after incubation of lymphoma tissue sections with CD8 antibody (B) and 1F1E1A10 antibody (A) of Maixin Company. Figure 2 The results of immunohistochemistry experiments after the test specimens were incubated with CD8 antibody (B) and 1F1E1A10 antibody (A) of Maixin Company.

[0110] Depend on Figure 1 and Figure 2 The staining results show that there is no significant difference in the staining location and staining intensity between the CD8-1F1E1A10 antibody and the Maixin antibody. This proves that the CD8 provided by the present invention has an application prospect in preparing a kit.

[0111] Example 2

[0112] Perform DNA cloning and sequencing, and sequence the variable region gene of anti-CD8 monoclonal antibody.

[0113] Total RNA from mouse monoclonal cell line 1F1E1A10 was extracted using Trizol reagent. Collect cells cultured in a 9cm dish into a 1.5ml centrifuge tube and aspirate the supernatant. Add 1ml of Trizol reagent and blow the cells evenly to lyse the cells. Place the lysed sample or homogenate at room temperature for 5-10min to completely separate the nucleoprotein from the nucleic acid. Add 0.2ml of chloroform, shake vigorously for 15sec, and place at room temperature for 3min. Centrifuge at 12000rpm at 4℃ for 10min. Transfer the upper aqueous phase to a clean centrifuge tube, add an equal volume of isopropanol, mix well, and place at room temperature for 20min. Centrifuge at 12000rpm at 4℃ for 10min and discard the supernatant. Add 1ml of 75% ethanol to wash the precipitate. Centrifuge at 12000rpm at 4℃ for 3min and discard the supernatant. Dry at room temperature for 5-10min. Add 30-50ulRNase-free ddH2O. The obtained RNA solution was stored at -70°C or used in subsequent experiments.

[0114] The AMV first-strand cDNA synthesis kit was used to reverse transcribe total RNA into cDNA. The experimental system was configured as follows: 6ul total RNA + 1ul Oligo dT + 4ul RNase-free water (a total of 11ul). Gently mix and centrifuge for 3-5s. The reaction mixture was pre-denatured at 65°C for 5min, ice-bathed for 30s, then centrifuged for 3-5s, and then ice-bathed for 2min. In an ice bath, 4ul of 5x buffer + 1ul of dNTP mixture + 1ul of RNase inhibitor + 1ul of reverse transcriptase (a total of 20ul system) was added, gently mix and centrifuged for 3-5s, and cDNA synthesis was completed on a PCR instrument at 42°C for 50 minutes and 85°C for 5min. Random primers are suitable for the synthesis of short-chain cDNAs below 500bp. The transcribed RNA template does not require a poly(A) tail, and the 5' end region can be transcribed.

[0115] PCR amplification of light and heavy chains. For amplifying the variable region sequence of the antibody light chain, configure the PCR reaction system: 2x Taq enzyme buffer 25ul + FP-VL 1ul + RP-VL 1ul + cDNA 2ul + ddH2O 21ul. For amplifying the variable region sequence of the antibody heavy chain, configure the PCR reaction system: 2x Taq enzyme buffer 25ul + FP-VH 1ul + RP-VH 1ul + cDNA 2ul + ddH2O21ul. The temperature cycle for PCR amplification of the heavy and light chain variable regions is as follows (steps 2 to 4 are repeated 35 cycles):

[0116] Step 1 - pre-denaturation at 94°C for 4 min;

[0117] Step 2 - denaturation at 94°C for 30 seconds;

[0118] Step 3-annealing at 55°C for 45 seconds;

[0119] Step 4 - extension 72°C, 60 sec;

[0120] Step 5-72°C, 10 min;

[0121] Step 6 - Store at 4°C.

[0122] The PCR products were analyzed by 1% agarose gel electrophoresis, and the DNA segment bands of corresponding sizes (VH is about 375bp, VL is about 325bp) were cut out, and DNA was extracted using the SanPrep column DNA gel recovery kit. The process is briefly described as follows: cut the gel block containing the target fragment from the agarose gel and weigh it; add 3-6 times the weight of the gel block buffer B2, and sol it in a 50℃ water bath for 5-10 minutes; transfer the sol solution into the adsorption column, centrifuge at 8000g for 30 seconds; pour out the liquid in the collection tube; add 500ul wash solution to the column, centrifuge at 9000g for 30 seconds, and pour out the liquid in the collection tube; repeat the addition of wash solution once and pour out the liquid; centrifuge the adsorption column at 9000g for 1 minute; place the adsorption column in a clean 1.5ml centrifuge tube, add 15-40ul Elution Buffer to the center of the adsorption membrane, let it stand at room temperature for 1 minute, and centrifuge it for 1 minute. The prepared DNA solution is obtained, and the PCR product is purified and sequenced to obtain the variable region sequence of the antibody.

[0123] Experimental Example 1

[0124] This experimental example tests the affinity and sensitivity of the antibody prepared in Example 1 above.

[0125] 1. The antigen in Example 1 was coated on plates at 3 mg / L, 1.5 mg / L, 0.75 mg / L and 0.375 mg / L respectively.

[0126] 2. Adjust the antibody concentration to 10 -7 mol / L level (1*10 -7 Up to 5*10 -7 mol / L). Then dilute it in multiples of 1:2-1:256 and add it to the wells with different antigen coating amounts.

[0127] 3. Add secondary antibody and develop color with TMB. Measure the absorbance at 450nm. The measured data are shown in Table 1.

[0128] 4. According to the antigen-antibody binding S curve, calculate the antibody concentration at half absorbance under different antigen concentrations. In this way, there are four antibody concentrations (mol / L).

[0129] 5. Substitute the formula K = (N-1) / (N*AB'-AB) to calculate the affinity constant. AB', AB are the antibody concentrations that produce half the absorbance value at the corresponding antigen concentration AG (3mg / L, 1.5ml / L, 0.75mg / L, 0.375mg / L). N = AG / AG' (AG>AG').

[0130] 6. When N = 2, three K values ​​can be obtained: 0.134, 0.240, and 0.493. When N = 4, two K values ​​can be obtained: 0.157 and 0.289. When N = 8, one K value can be obtained: 0.174. The average value of the six K values ​​is 2.478×10 8 L / mol.

[0131] Table 1 Statistics of absorbance values ​​under different treatments.

[0132]

[0133] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An anti-CD8 antibody or an antigen-binding fragment thereof, characterized in that: It includes a heavy chain complementarity determining region in the heavy chain variable region as shown in SEQ ID NO:16 and a light chain complementarity determining region in the heavy chain variable region as shown in SEQ ID NO:

15.

2. The anti-CD8 antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The heavy chain complementary determining region includes: CDR-H1, CDR-H2 and CDR-H3, whose amino acid sequences are shown in SEQ ID NOs: 9-11 respectively, and the light chain complementary determining region includes CDR-L1, CDR-L2 and CDR-L3, whose amino acid sequences are shown in SEQ ID NOs: 12-14 respectively.

3. The anti-CD8 antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof further comprises a heavy chain framework region, and / or a light chain framework region; the heavy chain framework region comprises HFR1, HFR2, HFR3 and HFR4, which are at least 80% homologous to the amino acid sequences shown in SEQ ID NOs: 5-8, in sequence; the light chain framework region comprises LFR1, LFR2, LFR3 and LFR4, which are at least 80% homologous to the amino acid sequences shown in SEQ ID NOs: 1-4, in sequence; Preferably, the antibody or antigen-binding fragment thereof further comprises a constant region, wherein the constant region comprises a heavy chain constant region and / or a light chain constant region, wherein the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; and the light chain constant region is selected from the κ type or λ type light chain constant region; Preferably, the species of origin of the constant region is cattle, horse, sheep, goat, rat, mouse, pig, dog, cat, donkey, deer, mink, chicken, duck, rabbit, goose or human; Preferably, the species of origin of the constant region is mouse; Preferably, the antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv, Fab'-SH and scFv of the antibody.

4. Use of the anti-CD8 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 in any of the following: (1) Enrichment of CD8+ cells; (2) preparing reagents or kits for detecting, separating or enriching CD8+ cells; (3) Use of a reagent or kit for preparing a test for predicting the responsiveness of a subject with cancer to immunotherapy or a cancer vaccine; (4) Use of a reagent or kit for preparing a test kit for monitoring disease progression in a subject with cancer; (5) preparing CD8 protein detection products; (6) preparing CD8 protein separation products or enrichment products; Preferably, the application (3) comprises: administering a labeled anti-CD8 antibody to the subject; and wherein detecting binding of the labeled anti-CD8 antibody to CD8+ T cells in a tumor tissue in the subject indicates that the subject is likely to respond to the immunotherapy or the cancer vaccine; Preferably, the application (4) comprises: administering a labeled anti-CD8 antibody to the subject, and the monitoring comprises detecting the binding of the labeled anti-CD8 antibody to CD8+T cells in the tumor tissue of the subject at a first time point and a second time point.

5. The use according to claim 4, characterized in that: The product in the application (5) is a reagent, a test kit, a test strip, an antibody chip, an antibody probe or a detector; the separation product or enrichment product in the application (6) is a magnetic bead, a test kit or a separation column; Preferably, in the applications (1)-(6), the anti-CD8 antibody or antigen-binding fragment thereof is labeled with a detectable marker; Preferably, in the applications (2) to (5), the anti-CD8 antibody or antigen-binding fragment thereof is used to label tissues and / or cells, and the labeled tissues and / or cells are detected; Preferably, the tissue is selected from tonsil, lymphoma and spleen.

6. A product enriched with CD8+ cells, characterized in that: It includes: The anti-CD8 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

7. A CD8 protein detection product, separation product or enrichment product, characterized in that: It includes: The anti-CD8 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

8. A cell, characterized in that The cell is a non-plant cell, which expresses the anti-CD8 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

9. A nucleic acid molecule, characterized in that It encodes the anti-CD8 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

10. A recombinant vector, characterized in that: It comprises the nucleic acid molecule according to claim 9.

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