An antibody or antigen-binding fragment thereof against CD8, a product enriched with CD8+ cells, and use thereof

Abstract

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

Description

Technical Field

[0001] This invention relates to the field of antibody technology, and more specifically, to an anti-CD8 antibody or its antigen-binding fragment, a product for enriching CD8+ cells, and their applications. Background Technology

[0002] CD8 is a leukocyte differentiation antigen, a glycoprotein present on the surface of some T cells. It assists the T cell receptor (TCR) in recognizing antigens and participates in the transduction of T cell activation signals; it is also known as a co-receptor of the TCR. T cells expressing CD8 (CD8+ T cells) usually differentiate into cytotoxic T cells (CTLs) after activation, which can specifically kill target cells.

[0003] CD8 cells are a type of cytotoxic T cell that primarily 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 the detection criteria for antiviral, antitumor, and HIV / AIDS treatments.

[0004] Immunohistochemistry (IHC) is a common clinical method for detecting protein expression in tumor cells. The accuracy and sensitivity of IHC testing depend heavily on the quality of the monoclonal antibody that specifically binds to the protein. Therefore, developing a monoclonal antibody with high binding specificity against CD8 is crucial for detecting CD8 expression levels using IHC.

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

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

[0007] This invention is implemented as follows:

[0008] In a first aspect, the present invention provides an anti-CD8 antibody or an antigen-binding fragment thereof, comprising 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.

[0009] Secondly, the present invention also provides the use of an anti-CD8 antibody or its antigen-binding fragment in any of the following:

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

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

[0012] (3) Use of reagents or kits for predicting the response of subjects with cancer to immunotherapy or cancer vaccines;

[0013] (4) Use in the preparation of reagents or kits for monitoring disease progression in subjects with cancer;

[0014] (5) Preparation of CD8 protein detection products;

[0015] (6) Prepare CD8 protein isolation or enrichment products.

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

[0017] Fourthly, the present invention also provides a CD8 protein detection product, isolation product or enrichment product, which includes: the above-mentioned anti-CD8 antibody or its antigen-binding fragment.

[0018] Fifthly, the present invention also provides a cell, which is a non-plant cell, expressing the above-mentioned anti-CD8 antibody or its antigen-binding fragment.

[0019] In a sixth aspect, the present invention also provides a nucleic acid molecule that encodes the aforementioned anti-CD8 antibody or its antigen-binding fragment.

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

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

[0022] This invention reveals that antibodies possessing specific six complementarity-determining regions exhibit superior activity and maintain high binding specificity and affinity to the CD8 antigen. This means that using this binding protein can be employed for CD8 detection, contributing to improved sensitivity and specificity. This invention provides more protein options for CD8 detection and for the detection of CD8+ cells.

[0023] The antibody that specifically binds to CD8 peptide provided by this invention can be used to prepare reagents or kits for detecting, isolating or enriching CD8+ cells, to prepare reagents or kits for monitoring disease progression in subjects with cancer, and to prepare reagents or kits for predicting the response of subjects with cancer to immunotherapy or cancer vaccines. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a diagram showing the results of immunohistochemical experiments on lymphoma samples.

[0026] Figure 2 This is a diagram showing the results of an immunohistochemical experiment on a spleen sample. Detailed Implementation

[0027] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0028] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (JEColigan et al., ed., 1991), each of which is explicitly incorporated herein by reference.

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0030] Definition of noun

[0031] The term "antigen-binding fragment" broadly refers to all proteins / protein fragments containing a CDR region, particularly antibodies or antibody functional fragments. "Antigen-binding fragment" includes antigen-binding fragments of the aforementioned antibodies, including Fab, F(ab')2, Fd, Fv, scFv, bispecific antibodies, multispecific antibodies, and the smallest antibody recognition unit, as well as single-chain derivatives of these antibodies and fragments. Antibody types can include IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD, etc. Furthermore, the term "antibody" includes both naturally occurring and non-naturally occurring antibodies, including, for example, chimeric, bifunctional, and humanized antibodies, as well as related synthetic isoforms. The term "antibody" is used interchangeably with "immunoglobulin."

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

[0033] In this invention, the terms "complementarity-determining region" or "CDR" refer to highly variable regions of the heavy and light chains of an immunoglobulin, specifically regions containing one or more, or even all, of the major amino acid residues that contribute to the binding affinity of an antibody or antigen-binding fragment to the antigen or epitope it recognizes. In specific embodiments of this invention, CDRs refer to highly variable regions of the heavy and light chains of the antibody.

[0034] In this invention, the heavy chain complementarity-determining region (CDR) is represented by HCDR, which includes HCDR1, HCDR2, and HCDR3; the light chain complementarity-determining region (LCDR) is represented by LCDR, which includes LCDR1, LCDR2, and LCDR3. Commonly used CDR labeling methods in the art include the Kabat numbering scheme, the IMGT numbering scheme, the Chothia and Lesk numbering scheme, and the new standardized numbering system introduced by Lefranc et al. in 1997 for all protein sequences of the immunoglobulin superfamily. 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 led to the creation of the Kabat database, and the Kabat numbering scheme is generally considered the widely adopted standard for numbering antibody residues. This invention uses the Kabat annotation standard to label CDR regions, but CDR regions labeled by other methods are also within the scope of this invention.

[0035] Typically, the variable region (VH) of the antibody heavy chain is obtained by linking the following 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 linking the following numbered CDRs with FRs 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 subjects in this invention are humans, non-human primates, mice, and rats.

[0039] In a first aspect, the present invention provides an anti-CD8 antibody or an antigen-binding fragment thereof, comprising 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.

[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 complementarity-determining region includes CDR-H1, CDR-H2 and CDR-H3, the amino acid sequences of which are shown in SEQ ID NO: 9-11, respectively, and the light chain complementarity-determining region includes CDR-L1, CDR-L2 and CDR-L3, the amino acid sequences of which are shown in SEQ ID NO: 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 discovered that antibodies or antigen-binding fragments possessing the aforementioned six complementarity-determining regions exhibit good activity and can maintain high binding specificity and affinity to the CD8 antigen. This means that the antibody can be used to detect CD8, helping to improve the sensitivity and specificity of detection. This invention provides more protein options for the detection of CD8 and CD8+ cells. Antibodies specifically binding to CD8 peptides provided by this invention can be used to prepare reagents or kits for detecting, isolating, or enriching CD8+ cells; reagents or kits for monitoring disease progression in subjects with cancer; and reagents or kits for predicting the response of subjects with cancer to immunotherapy or cancer vaccines.

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

[0053] In a preferred embodiment of the present invention, the antibody or its antigen-binding fragment further includes a heavy chain framework region and / or a light chain framework region; the heavy chain framework region includes HFR1, HFR2, HFR3 and HFR4, which have at least 80% homology with the amino acid sequences shown in SEQ ID NO:5-8; the light chain framework region includes LFR1, LFR2, LFR3 and LFR4, which have at least 80% homology with the amino acid sequences shown in SEQ ID NO:1-4.

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

[0055]

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

[0057] The light chain framework region includes LFR1, LFR2, LFR3 and LFR4, which have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology with the amino acid sequences shown in SEQ ID NO:1-4.

[0058] In a preferred embodiment of the present invention, the antibody or its antigen-binding fragment further includes a constant region, which includes a heavy chain constant region and / or a light chain constant region. The heavy chain constant region is selected from the heavy chain constant regions 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 source of the constant region is cattle, horses, sheep, goats, rats, mice, pigs, dogs, cats, donkeys, deer, mink, chickens, ducks, rabbits, geese, or humans;

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

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

[0062] The antigen-binding fragments of the aforementioned antibodies typically possess the same binding specificity as the antibodies from which they originate. Those skilled in the art will readily understand, based on the description herein, that the functional fragments of the aforementioned antibodies can be obtained, for example, by enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds.

[0063] The antigen-binding fragments of the aforementioned antibodies can also be obtained by recombinant genetic techniques known to those skilled in the art or by synthesizing, for example, automated peptide synthesizers sold by Applied BioSystems.

[0064] Secondly, the present invention also provides the use of an anti-CD8 antibody or its antigen-binding fragment in any of the following:

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

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

[0067] (3) Use of reagents or kits for predicting the response of subjects with cancer to immunotherapy or cancer vaccines;

[0068] (4) Use in the preparation of reagents or kits for monitoring disease progression in subjects with cancer;

[0069] (5) Preparation of CD8 protein detection products;

[0070] (6) Prepare CD8 protein isolation or enrichment products.

[0071] Application (1) includes, but is not limited to: coating an anti-CD8 antibody or its antigen-binding fragment onto a solid phase, incubating it with a cell sample, thereby enriching and obtaining 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 invention, application (3) includes: administering a labeled anti-CD8 antibody to the subject; and wherein detecting the binding of the labeled anti-CD8 antibody to CD8+ T cells in the subject's tumor tissue indicates that the subject is likely to respond to the immunotherapy or the cancer vaccine. Detecting the binding of the labeled anti-CD8 antibody to CD8+ T cells in the subject's tumor tissue includes imaging the subject's CD8+ T cells, such as performing a positron emission tomography / computed tomography (PET / CT) scan on the subject.

[0073] In a preferred embodiment 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 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, such as 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 reagent 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 reagent kit, or a separation column.

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

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

[0077] In optional embodiments, detectable markers include, but are not limited to, fluorescent dyes, enzymes that catalyze substrate color development, radioisotopes, chemiluminescent reagents, and nanoparticle markers.

[0078] In practical use, those skilled in the art can select appropriate markers according to the detection conditions or actual needs. Regardless of the marker used, it falls within the protection scope of this invention.

[0079] Fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5...). .5, Cy3, etc. or similar substances), Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or similar substances) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP), etc.).

[0080] In optional embodiments, the enzymes that catalyze substrate color development include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxygenase.

[0081] In optional embodiments, radioactive isotopes include, but are not limited to, those mentioned above. 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 Ru、 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 optional embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

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

[0084] In optional embodiments, the colloid includes, but is not limited to, colloidal metals, dispersed dyes, dye-labeled microspheres, and latexes. In optional embodiments, 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 steps (2)-(5), tissues and / or cells are labeled with an anti-CD8 antibody or its antigen-binding fragment, and the labeled tissues and / or cells are detected.

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

[0087] Thirdly, the present invention also provides a product for enriching CD8+ cells, comprising: the aforementioned anti-CD8 antibody or its antigen-binding fragment. For example, an anti-CD8 antibody or its antigen-binding fragment is coated onto an antibody.

[0088] Fourthly, the present invention also provides a CD8 protein detection product, isolation product or enrichment product, which includes: the above-mentioned anti-CD8 antibody or its antigen-binding fragment.

[0089] For example, the antibodies described above can be coated onto magnetic beads for the separation and enrichment of CD8 protein. In one embodiment, the antibodies are coated onto packing material and packed into a separation column for affinity separation and enrichment of CD8 protein. Therefore, the antibodies described above or their antigen-binding fragments have promising applications in the preparation of CD8 protein enrichment products.

[0090] Fifthly, the present invention also provides a cell, which is a non-plant cell, expressing the above-mentioned anti-CD8 antibody or its antigen-binding fragment.

[0091] The host cells are selected from mammalian cells; the mammalian cells are 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, the 293 series cells, Per6 cells, and CHO cells are commonly used mammalian cells for the production of antibodies or recombinant proteins and are well known to those skilled in the art.

[0092] In a sixth aspect, the present invention also provides a nucleic acid molecule that encodes the aforementioned anti-CD8 antibody or its antigen-binding fragment.

[0093] Considering the degeneracy of codons, the gene sequence encoding the above-mentioned antibodies can be modified in its coding region without changing the amino acid sequence to obtain a gene encoding the same antibody amino acid sequence; alternatively, the gene can 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-described nucleic acid molecules.

[0095] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0096] Example 1

[0097] This embodiment describes the preparation of a specific anti-CD8 monoclonal antibody.

[0098] 1. Animal (mouse) immunization.

[0099] Mice were immunized with an immunogen using a standard method. The immunogen was a human CD8 polypeptide synthesized by GIL Biotech, which also served as a detection antigen for serum titer and hybridoma screening. High-purity antigen increases the chance of obtaining the desired monoclonal antibody while reducing the screening workload. Five mice were immunized, each receiving 50 μg of CD8 antigen. An antigen-protein solution was prepared using PBS. Appropriate amounts of antigen protein, PBS, and Freund's adjuvant were placed in a syringe, the syringe outlet was plugged, and the solution was emulsified thoroughly on an emulsifier to form a stable water-in-oil solution. The first tail blood serum titer was measured 7-10 days after the primary and secondary immunizations. Good titers were obtained after 2-4 booster immunizations. Mice with high serum titers were selected for final intraperitoneal immunization followed by cell fusion.

[0100] 2. Hybridoma cell fusion and screening.

[0101] Preparations are required before cell fusion: 1. Culture mouse myeloma cells SP2 / 0 to the logarithmic growth phase; 2. One negative mouse is sacrificed the day before fusion, and peritoneal trophoblast cells are extracted by injecting HAT medium into the peritoneal cavity under sterile conditions and seeded onto 96-well plates at 100 μL per well. These cells promote hybridoma cell growth. Immunized mice are sacrificed, and spleens are harvested under sterile conditions. Splenic B cells and SP2 / 0 myeloma cells are chemically fused using PEG. Appropriate amounts of HAT medium are added according to the number of cells to be seeded, and finally, the fused cells are seeded onto trophoblast cell culture plates at 100 μL 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 from each well is collected, and hybridoma cells are screened using ELISA with human CD8 peptide antigen. The method is as follows: Coat the ELISA plate with 100 μL of PBS solution containing 2 μg / ml human CD8 peptide antigen and incubate at 37°C for two hours. After washing the plate three times with PBST, add 150 μL / well of PBS solution containing 3% skim milk powder and incubate overnight at 4°C. Wash the plate three more times, add 80 μL / well of hybridoma supernatant, incubate at 37°C for 1 hour, and then wash three more times. Add 100 μL / well of horseradish peroxidase-labeled goat anti-mouse secondary antibody diluted 1:8000, incubate at 37°C for 45 minutes, wash three times, and blot dry. Add 100 μL / well of TMB chromogenic solution, develop at room temperature for 5-10 minutes, stop with 2M sulfuric acid solution, and measure the absorbance at 450 nm for each well. Select positive hybridoma cells.

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

[0104] (1) Slice preparation: Place the tonsil slices in a 60℃ 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 in anhydrous ethanol ① for 5 minutes, anhydrous ethanol ② for 5 minutes, 95% ethanol for 5 minutes, 85% ethanol for 5 minutes, 75% ethanol for 5 minutes, and ddH2O for 5 minutes. Wash 3 times. Use a pressure cooker for antigen retrieval (boiling method). Add enough EDTA to submerge the slices in the pressure cooker and heat to boiling. Place the slices on a heat-resistant material slice rack and put them in the pot. Cover the pot, close the pressure valve, continue heating, and set the pressure to hold for 4 minutes. After the time is up, open the vent valve to release the gas. After the pressure returns to zero, open the pot lid and remove the inner pot to cool at room temperature. After the solution cools to room temperature, remove the sections (approximately 40 minutes); soak in ddH2O for 5 minutes, wash twice, soak in PBST for 5 minutes, wash twice; place the sections in 20 ml of 3% H2O2-methanol solution, protect from light, and treat at room temperature for 10 minutes; soak in PBST for 5 minutes, wash three times; add one drop (approximately 25 μl) of goat serum blocking solution to each tissue group, incubate in a humidified chamber at room temperature for 45 minutes; soak in PBST for 5 minutes, wash three times.

[0105] (2) Tissue sections are mixed with antibodies and incubated:

[0106] The processed tissue sections were compared with CD8 antibody from Maixin Company, while the remaining sections were mixed with the above-mentioned positive hybridoma cells. Incubate overnight in a humidified chamber at 4°C; remove from the refrigerator at 4°C and incubate at room temperature for 60 minutes; gently rinse with PBST and soak for 5 minutes, washing 3 times; add 25 μL of HRP-labeled Long Island Biotechnology secondary antibody (CAT#:D-3004-0100) to each tissue group and incubate at room temperature for 45 minutes; wash; prepare DAB staining solution, react in the dark for 10-15 minutes, then drop onto the sections and develop for 1-5 minutes; terminate the staining reaction with distilled water; add 50 μL of hematoxylin staining solution to each tissue group and stain for 5-10 minutes, then rinse thoroughly with distilled water; decolorize the sections in 1% hydrochloric acid-ethanol for 2-3 seconds, then quickly remove and place in distilled water to terminate the staining, then place in PBST (pH 8.0) for 5-10 minutes for inversion; soak in 75% ethanol for 5 minutes; soak in 85% ethanol for 5 minutes; soak in 95% ethanol for 5 minutes; soak in anhydrous ethanol for 5 minutes. Soak in xylene for 10 minutes, then replace with xylene and soak for another 10 minutes; add neutral resin to seal the slide, then cover with a coverslip; take a microscope image.

[0107] Fusion cells that showed positive binding were selected using ELISA and IHC assays, and cloned using the limiting dilution method. Each positive cell line was seeded into 48 / 96-well plates and cultured further. A second round of screening was performed using ELISA to identify hybridomas that specifically recognize the CD8 peptide and can block CD8 binding. These hybridomas were then subcloned using the limiting dilution method to obtain a single-clone cell line, 1F1E1A10.

[0108] The monoclonal cell line was expanded, and approximately 1 × 10⁻⁶ cells were collected. 6 One cell was injected into selected mice (the mice needed to be injected with paraffin oil into their peritoneum one week in advance), and after a waiting period of 7-10 days, the mice produced ascites. The ascites was collected for antibody purification. After purification, a mouse monoclonal antibody CD8-1F1E1A10 with specific anti-CD8 peptide was obtained.

[0109] Figure 1 The immunohistochemical results of lymphoma tissue sections incubated with CD8 antibody (B) and 1F1E1A10 antibody (A) from Maixin Company are shown in the figure. Figure 2 The images show the immunohistochemical results of spleen tissue samples after incubation with CD8 antibody (B) and 1F1E1A10 antibody (A) from Maixin Company.

[0110] Depend on Figure 1 and Figure 2 The staining results showed no significant difference in staining sites and intensity between the CD8-1F1E1A10 antibody and the Maixin antibody. This confirms that the CD8 antibody provided by this invention has promising applications in preparing kits.

[0111] Example 2

[0112] DNA cloning and sequencing were performed, including sequencing of the variable region gene of the anti-CD8 monoclonal antibody.

[0113] Total RNA was extracted from the mouse monoclonal cell line 1F1E1A10 using Trizol reagent. Cells cultured in 9cm dishes were transferred to 1.5ml centrifuge tubes, and the supernatant was aspirated. 1ml of Trizol reagent was added, and the cells were lysed by pipetting. The lysed sample or homogenate was incubated at room temperature for 5-10 minutes to allow complete separation of nucleoproteins and nucleic acids. 0.2ml of chloroform was added, and the mixture was vigorously vortexed for 15 seconds and incubated at room temperature for 3 minutes. The cells were centrifuged at 12000 rpm at 4°C for 10 minutes. The upper aqueous phase was transferred to a clean centrifuge tube, and an equal volume of isopropanol was added. The mixture was incubated at room temperature for 20 minutes. The cells were centrifuged at 12000 rpm at 4°C for 10 minutes, and the supernatant was discarded. The precipitate was washed with 1ml of 75% ethanol. The precipitate was centrifuged at 12000 rpm at 4°C for 3 minutes, and the supernatant was discarded. The cells were dried at room temperature for 5-10 minutes. 30-50ul of RNase-free ddH2O was added. Store the obtained RNA solution at -70°C or use it for subsequent experiments.

[0114] Total RNA was reverse transcribed into cDNA using the AMV first-strand cDNA synthesis kit. The experimental configuration was as follows: 6 μL total RNA + 1 μL Oligo dT + 4 μL RNase-free water (total 11 μL). After gentle mixing, centrifuge for 3-5 seconds. The reaction mixture was pre-denatured at 65°C for 5 minutes, then incubated on ice for 30 seconds, centrifuged for 3-5 seconds, and then incubated on ice for 2 minutes. While still on ice, 4 μL of 5X buffer + 1 μL of dNTP mixture + 1 μL of RNase inhibitor + 1 μL of reverse transcriptase (total 20 μL) was added. After gentle mixing, centrifuge for 3-5 seconds. The cDNA was synthesized at 42°C for 50 minutes, followed by 85°C for 5 minutes on a PCR instrument. Random primers are suitable for synthesizing short-strand cDNAs under 500 bp. The transcribed RNA template does not require a poly(A) tail and can transcribe the 5' end region.

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

[0116] Step 1 - Pre-denaturation: 94℃, 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 - Extend at 72°C for 60 seconds;

[0120] Step 5: 72℃, 10 min;

[0121] Step 6 - Store at 4℃.

[0122] PCR products were analyzed by 1% agarose gel electrophoresis, and DNA bands of corresponding sizes were excised (approximately 375 bp for VH and approximately 325 bp for VL). DNA extraction was performed using the SanPrep DNA Gel Extraction Kit. The procedure is briefly described as follows: A gel block containing the target fragment was excised from the agarose gel and weighed; 3-6 times the weight of the gel block was added to buffer B2, and the gel was incubated at 50°C for 5-10 minutes to dissolve; the solution was transferred to an adsorption column and centrifuged at 8000g for 30 seconds; the liquid in the collection tube was discarded; 500 μL of wash solution was added to the column, and the column was centrifuged at 9000g for 30 seconds, and the liquid in the collection tube was discarded; the wash solution was added again, and the liquid was discarded; the adsorption column was centrifuged at 9000g for 1 minute; the adsorption column was placed in a clean 1.5 ml centrifuge tube, and 15-40 μL of Elution Buffer was added to the center of the adsorption membrane. After standing at room temperature for 1 minute, the column was centrifuged for 1 minute. The prepared DNA solution was obtained, and the PCR product was 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 antigens in Example 1 were packaged into plates at concentrations of 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 is acceptable). Then serially dilute 1:2 to 1:256 and add to wells with different antigen coating amounts.

[0127] 3. Add secondary antibody and develop TMB colorimetric assay. Measure the absorbance at 450 nm; the data are shown in Table 1.

[0128] 4. Based on the antigen-antibody binding S-curve, determine the antibody concentration with the half-maximum absorbance at different antigen concentrations. This will result in four antibody concentrations (mol / L).

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

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

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

[0132]

[0133] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof against CD8, characterized in that, It comprises a heavy chain complementarity determining region and a light chain complementarity determining region, the heavy chain complementarity determining region comprises CDR-H1, CDR-H2 and CDR-H3, the amino acid sequences of which are shown in SEQ ID NO: 9-11 respectively, and the light chain complementarity determining region comprises CDR-L1, CDR-L2 and CDR-L3, the amino acid sequences of which are shown in SEQ ID NO: 12-14 respectively.

2. The anti-CD8 antibody or antigen-binding fragment thereof of claim 1, characterized in that, The antibody or antigen-binding fragment thereof further comprises a heavy chain framework region and a light chain framework region; the heavy chain framework region comprises HFR1, HFR2, HFR3 and HFR4 which have at least 80% homology with the amino acid sequences shown in SEQ ID NO: 5-8 respectively; and the light chain framework region comprises LFR1, LFR2, LFR3 and LFR4 which have at least 80% homology with the amino acid sequences shown in SEQ ID NO: 1-4 respectively.

3. The anti-CD8 antibody or antigen-binding fragment thereof of claim 2, characterized in that, The sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 16, and the sequence of the light chain variable region is shown in SEQ ID NO:

15.

4. The anti-CD8 antibody or antigen-binding fragment thereof of claim 1, wherein, The antibody or antigen-binding fragment thereof further comprises a constant region, and the constant region comprises a heavy chain constant region and 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; and the light chain constant region is selected from the kappa type or lambda type light chain constant region.

5. The anti-CD8 antibody or antigen-binding fragment thereof of claim 4, wherein, The species origin of the constant region is bovine, equine, sheep, goat, rat, mouse, pig, dog, cat, donkey, deer, mink, chicken, duck, rabbit, goose or human.

6. The anti-CD8 antibody or antigen-binding fragment thereof of claim 5, wherein, The species origin of the constant region is mouse.

7. The antibody or antigen-binding fragment thereof against CD8 according to any one of claims 1 to 6, characterized in that, The antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv, Fab'-SH and scFv of the antibody.

8. The use of the anti-CD8 antibody or antigen-binding fragment thereof of any one of claims 1-7 in any one of: (1) enriching CD8+ cells; (2) preparing a reagent or kit for detecting, isolating or enriching CD8+ cells; (3) preparing a reagent or kit for predicting the responsiveness of a subject with cancer to an immunotherapy or a cancer vaccine; (4) preparing a reagent or kit for monitoring disease progression in a subject with cancer; (5) preparing a CD8 protein detection product; (6) preparing a CD8 protein isolation product or enrichment product.

9. Use according to claim 8, characterized in that, The use (3) comprises: administering a labeled anti-CD8 antibody to the subject; and wherein detecting the binding of the labeled anti-CD8 antibody to CD8+ T cells in tumor tissue in the subject indicates that the subject is likely to respond to the immunotherapy or the cancer vaccine.

10. Use according to claim 8, characterized in that, The use (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 tumor tissue in the subject at a first time point and a second time point.

11. Use according to claim 8, characterized in that, The product in the application (5) is a reagent, a kit, a test strip, an antibody chip, an antibody probe or a detector; the separated product or enriched product in the application (6) is a magnetic bead, a kit or a separation column.

12. The use according to claim 8, characterized in that, In the applications (1)-(6), the anti-CD8 antibody or antigen-binding fragment thereof is labeled with a detectable label.

13. The use according to claim 8, characterized in that, In the applications (2)-(5), the tissue and / or cell is labeled with the anti-CD8 antibody or antigen-binding fragment thereof, and the labeled tissue and / or cell is detected.

14. Use according to claim 13, characterized in that, The tissue is selected from the group consisting of tonsil, lymphoma and spleen.

15. A product enriched for CD8+ cells, characterized in that, It comprises: The anti-CD8 antibody or antigen-binding fragment thereof of any one of claims 1-7.

16. A CD8 protein detection product, isolation product or enrichment product, characterized in that, It comprises: The anti-CD8 antibody or antigen-binding fragment thereof of any one of claims 1-7.

17. A cell, comprising: The cell is a non-plant cell expressing the anti-CD8 antibody or antigen-binding fragment thereof of any one of claims 1-7.

18. A nucleic acid molecule, characterized in that, It encodes the anti-CD8 antibody or antigen-binding fragment thereof of any one of claims 1-7.

19. A recombinant vector, characterized in that, It comprises the nucleic acid molecule of claim 18.

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