An anti-human cd8 antibody and a preparation method and application thereof

Abstract

The application provides an anti-human CD8 antibody, which comprises a heavy chain and a light chain, the heavy chain comprises a heavy chain variable region, and the light chain comprises a light chain variable region; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3. The application belongs to the field of antibodies, and provides a brand-new anti-human CD8 antibody, which has good binding capacity with CD8, can be used for preparing a gene engineering cell expressing the anti-human CD8 antibody, so that mass production is realized, and more convenient conditions are provided for detection of CD8.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of antibodies, and particularly relates to an anti-human CD8 antibody and a preparation method and application thereof. BACKGROUND

[0002] CD (Cluster of Differentiation): Cluster of differentiation or cluster of differentiation, also known as leukocyte differentiation antigen, refers to the cell surface markers appearing or disappearing during the different stages of normal differentiation and maturation of leukocytes of different lineages and the activation process. They are a class of proteins or glycoproteins on the cell membrane. In physiology, CD molecules have many uses, usually as important receptors or ligands of cells. Not only can be used as a surface marker for cell identification and separation, but also widely involved in cell growth, maturation, differentiation, development, migration and activation.

[0003] CD8 is a leukocyte differentiation antigen, also known as T8 / Leu-2, which is a glycoprotein on the surface of part of T cells, used to assist T cell receptor (TCR) to recognize antigen and participate in the transduction of T cell activation signal, also known as the co-receptor of TCR, and the T cells expressing CD8 (CD8+ T cells) are usually differentiated into cytotoxic T cells (CTL) after activation, which can specifically kill target cells. The CD8 molecule can promote the degree of cell-cell interaction by directly binding to the appropriate MHC molecule on the target cell.

[0004] The generation of antigen-specific CD8+ T cell responses can enhance anti-tumor immunity. Hyun-Il Cho (BiVax: a peptide / poly-IC subunit vaccine that mimics an acute infection elicits vast and effective anti-tumor CD8 T-cell responses [J]. Cancer Immunol Immunother 62, 787-799 (2013)) discloses a peptide / poly-IC subunit vaccine that mimics an acute infection, which can elicit a wide and effective anti-tumor CD8+ T cell response, and the CD8 TCR can recognize small peptides combined with MHC class I (MHC-I) products on APC. These peptides, known as CD8+ T cell epitopes, are usually derived from processed proteins corresponding to microbial components or tumor-associated antigens (TAAs). The identification of these peptides leads to the development of epitope-based vaccines to induce antigen-specific CD8+ T cell responses to prevent or treat various infections and malignancies.

[0005] Traditional diagnostic detection antibodies can be prepared by animal hybridoma method, but in the preparation of these antibodies, due to the instability of hybridoma, the antibody gene is easy to lose, in addition, the traditional hybridoma preparation technology does not identify the gene sequence and protein sequence of the target antibody, which is not conducive to the subsequent production of the target antibody. The genetic engineering method can well solve the problem in the hybridoma method, and the necessary condition for preparing the antibody by the genetic engineering method is to determine the sequence of the antibody, therefore, mining the monoclonal antibody more suitable for CD8 detection is the more appropriate research and development direction at present. SUMMARY

[0006] In order to solve the above problems, the present application provides a specific amino acid sequence of an anti-human CD8 antibody, through which a genetic engineering cell expressing the anti-human CD8 antibody can be obtained by the existing technology, and the yield and production stability of the CD8 antibody are improved.

[0007] Terms:

[0008] In the present application, CDR (complementarity-determining regions, CDR) is called complementarity-determining region or complementarity-determining cluster. It is located in the hypermutation region of immunoglobulin, and the hypermutation region is the antigen binding site of antibody, which is complementary to the structure of antigenic determinant. Generally includes CDR1, CDR2 and CDR3.

[0009] In the present application, the variable region refers to the region with large variation of amino acid sequence near the N-terminal of immunoglobulin light chain and heavy chain, which is called variable region.

[0010] In the present application, the heavy chain refers to the two longer and relatively larger molecular weight identical heavy chains (H chains) in the antibody; the light chain refers to the two shorter and relatively smaller molecular weight identical light chains (L chains) in the antibody.

[0011] In the present application, similarity refers to the proportion of identicalness and substitutable amino acids in the amino acid sequence of homologous proteins.

[0012] In the present application, monoclonal antibody refers to the antibody produced by a single B cell clone, which is highly uniform and only directed to a certain specific antigen epitope.

[0013] In the present application, murine antibody refers to the antibody secreted by the murine hybrid fusion cell obtained by fusing the B cell derived from the immunized mouse with myeloma cell.

[0014] In one aspect, the present application provides an anti-human CD8 antibody.

[0015] The anti-human CD8 antibody comprises a heavy chain and a light chain, the heavy chain comprises a heavy chain variable region, and the light chain comprises a light chain variable region; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein:

[0016] (1) the HCDR1 is an amino acid sequence shown in SEQ ID NO. 1;

[0017] (2) the HCDR2 is an amino acid sequence shown in SEQ ID NO. 2;

[0018] (3) the HCDR3 is an amino acid sequence shown in SEQ ID NO. 3;

[0019] (4) the LCDR1 is an amino acid sequence shown in SEQ ID NO. 4;

[0020] (5) the LCDR2 is an amino acid sequence shown in SEQ ID NO. 5;

[0021] (6) the LCDR3 is an amino acid sequence shown in SEQ ID NO. 6.

[0022] Specifically, the heavy chain variable region amino acid sequence of the anti-human CD8 antibody is SEQ ID NO. 7, or a sequence having more than 80% sequence similarity with SEQ ID NO. 7; the light chain variable region amino acid sequence of the anti-human CD8 antibody is selected from SEQ ID NO. 8, or a sequence having more than 80% sequence similarity with SEQ ID NO. 8; or the light chain variable region amino acid sequence of the anti-human CD8 antibody is SEQ ID NO. 13, or a sequence having more than 80% sequence similarity with SEQ ID NO. 13.

[0023] Preferably, the heavy chain variable region amino acid sequence of the anti-human CD8 antibody is SEQ ID NO. 7, and the light chain variable region amino acid sequence is SEQ ID NO. 8.

[0024] Specifically, the heavy chain amino acid sequence of the anti-human CD8 antibody is SEQ ID NO. 9, or a sequence having more than 65% sequence similarity with SEQ ID NO. 9; or the heavy chain amino acid sequence of the anti-human CD8 antibody is SEQ ID NO. 11, or a sequence having more than 65% sequence similarity with SEQ ID NO. 11.

[0025] The anti-human CD8 antibody light chain amino acid sequence is SEQ ID NO. 10, or a sequence having more than 65% sequence similarity with SEQ ID NO. 10, and the anti-human CD8 antibody light chain amino acid sequence is SEQ ID NO. 12, or a sequence having more than 65% sequence similarity with SEQ ID NO. 12.

[0026] Preferably, the anti-human CD8 antibody heavy chain amino acid sequence is SEQ ID NO. 9 or SEQ ID NO. 11, and the light chain amino acid sequence is SEQ ID NO. 10 or SEQ ID NO. 12.

[0027] Specifically, the anti-human CD8 antibody is a monoclonal antibody.

[0028] Preferably, the anti-human CD8 antibody is a murinized antibody.

[0029] In another aspect, the present application provides a nucleotide sequence for expressing the aforementioned anti-human CD8 antibody.

[0030] The nucleotide sequence can be a sequence optimized by codon degeneracy, and can be used for encoding the aforementioned anti-human CD8 antibody.

[0031] Preferably, the coding gene sequence corresponding to the heavy chain SEQ ID NO. 9 and SEQ ID NO. 11 is shown as SEQ ID NO. 14 and SEQ ID NO. 15, and the coding gene sequence corresponding to the light chain SEQ ID NO. 10 and SEQ ID NO. 12 is shown as SEQ ID NO. 16 and SEQ ID NO. 17.

[0032] In still another aspect, the present application provides an expression vector comprising the aforementioned nucleotide sequence.

[0033] The expression vector can be a plasmid, a bacteriophage, or a virus.

[0034] In yet another aspect, the present application provides a cell for expressing the aforementioned anti-human CD8 antibody or nucleotide sequence or expression vector.

[0035] The cell can efficiently express the anti-human CD8 antibody.

[0036] Specifically, the cell can be HEK293 or CHO.

[0037] In yet another aspect, the present application provides a method for preparing the aforementioned anti-human CD8 antibody, wherein the method comprises culturing the aforementioned cell.

[0038] The method can further comprise an antibody purification step.

[0039] The antibody purification step can be precipitation, broad spectrum affinity purification, antigen specific purification, ion exchange.

[0040] In another aspect, the present application provides a method for fluorescently labeling the aforementioned anti-human CD8 antibody, which comprises labeling the anti-human CD8 antibody with a macromolecular dye or a small molecule dye.

[0041] Preferably, the macromolecular dye is RPE and the small molecule dye is FITC.

[0042] In another aspect, the present application provides the use of the aforementioned anti-human CD8 antibody or nucleotide sequence or expression vector or cell in CD8 detection, which is non-disease diagnosis or treatment.

[0043] The use is achieved by antibody antigen binding.

[0044] The use can be detecting CD8 in T lymphocytes.

[0045] In another aspect, the present application provides the use of the aforementioned anti-human CD8 antibody or nucleotide sequence or expression vector or cell in the preparation of a CD8 detection kit.

[0046] Specifically, the kit further comprises other reagents for CD8 detection, such as buffers, sample processing agents.

[0047] Specifically, the kit can further comprise molecules for fluorescently labeling antibodies.

[0048] Further specifically, the molecules can be macromolecular dyes or small molecule dyes.

[0049] Preferably, the macromolecular dye is RPE and the small molecule dye is FITC.

[0050] In another aspect, the present application provides a CD8 detection method, which is detected by the aforementioned anti-human CD8 antibody.

[0051] Specifically, the detection method can further comprise a sample pretreatment step.

[0052] The pretreatment step is sample collection and processing.

[0053] Preferably, the detection method is a non-disease diagnosis or treatment method.

[0054] In another aspect, the present application provides a CD8 detection kit, which comprises the aforementioned anti-human CD8 antibody or nucleotide sequence or expression vector or cell.

[0055] Specifically, the kit also includes other reagents for CD8 detection, such as buffer solutions and sample processing agents.

[0056] Specifically, the kit may also include molecules for fluorescently labeled antibodies.

[0057] More specifically, the molecule can be a macromolecular dye or a small molecule dye.

[0058] Preferably, the macromolecular dye is RPE, and the small molecule dye is FITC.

[0059] The beneficial effects of this invention are:

[0060] This invention provides a novel anti-human CD8 antibody that has a good binding ability to CD8 and can be used to prepare cells expressing anti-human CD8 antibodies, thereby enabling large-scale preparation and providing more convenient conditions for CD8 detection. Attached Figure Description

[0061] Figure 1 This is an SDS gel electrophoresis image of anti-human CD8 antibody.

[0062] Figure 2 The results are obtained by flow cytometry detection of the commercially available FITC-CD8[SK1] and RPE-CD8[SK1] control fluorescent antibodies purchased from Biolegend.

[0063] Figure 3 Mark FITC and RPE respectively. Flow cytometry results of HCD8-06.

[0064] Figure 4 Mark FITC and RPE respectively. Flow cytometry results for HCD8-08.

[0065] Figure 5 The results are the affinity assay results for the commercially available CD8[SK1] control antibody purchased from Biolegend.

[0066] Figure 6 for Affinity test results for HCD8-06.

[0067] Figure 7 for Affinity test results for HCD8-08.

[0068] Figure 8 for Specific detection results of HCD8-06.

[0069] Figure 9 for Specificity detection results of HCD8-08. DETAILED DESCRIPTION

[0070] The application will be further described in conjunction with specific examples. The following examples are not intended to limit the application, but merely to illustrate the application. Unless otherwise specified, the experimental methods used in the following examples were carried out according to conventional conditions. Unless otherwise specified, the materials and reagents used in the following examples were obtained from commercial sources.

[0071] Example 1 Preparation and detection of antibodies

[0072] Two antibodies were prepared, one was anti-human CD8 mouse antibody, numbered HCD8-06, and the other was anti-human CD8 rabbit antibody, numbered HCD8-08. HCD8-06, and the other was anti-human CD8 rabbit antibody, numbered HCD8-08. HCD8-08.

[0073] The heavy chain amino acid sequence of HCD8-06 is SEQ ID NO. 9, and the light chain amino acid sequence is SEQ ID NO. 10, The heavy chain amino acid sequence of HCD8-08 is SEQ ID NO. 11, and the light chain amino acid sequence is SEQ ID NO. 12. The nucleotide sequences for expressing the corresponding antibodies were designed by conventional methods. Specifically, the coding gene sequences corresponding to the heavy chains SEQ ID NO. 9 and SEQ ID NO. 11 are shown in SEQ ID NO. 14 and SEQ ID NO. 15; and the coding gene sequences corresponding to the light chains SEQ ID NO. 10 and SEQ ID NO. 12 are shown in SEQ ID NO. 16 and SEQ ID NO. 17. The nucleotide sequences were cloned into CHO stable strains for antibody stable expression, and the constructed vectors were introduced into CHO cells.

[0074] Transfection and screening:

[0075] (1) CHO cells were cultured using CHO CD medium, inoculated at 2 x 10 5 cells / mL, and added with 4 mM glutamine. When the cell density reached 4 x 10 6 cells / mL and the viability was greater than 95%, the cells were collected by centrifugation, resuspended with an electrotransfection buffer, and the density was adjusted to 1 x 10 7 cells / mL.

[0076] (2) Take a sterile EP tube, add 0.8 mL cell suspension, add 5 μg plasmid, mix gently, incubate for 15 min, ice bath for 5 min. Set the parameters of the electroporator to voltage 280 V, pulse number 3 times, pulse time 5 ms, pulse gap 0.784 s, and the inner diameter of the electroporation cup is 4 mm. Add 0.5 mL cell and plasmid mixture to each electroporation cup, place it in the electroporator and start the shock program. After completion, quickly aspirate the cells in the electroporation cup and transfer them to a 125 mL conical flask containing 20 mL CHO CD medium. Repeat the above steps to shock the second electroporation cup. Gently mix the cells in the flask, then tightly cap the flask and place it in a 37°C shaker.

[0077] (3) After 48 h, centrifuge the cells in a centrifuge tube at 1000 rpm for 5 min. Remove the supernatant and resuspend the cells in 20 mL CHO CD medium containing 2, 5, 20 μM MSX. Calculate the cell density and activity. The cell density is about 1.2 x 10 6 cells / mL.

[0078] (4) Transfer the cells to a new flask. From the seventh day, observe the cell density and activity every two days. The cell density decreases from the initial 1.2 x 10 6 cells / mL to a minimum value on days 5-7, and then increases from days 10-15. When the cell density increases to 1 x 10 6 cells / mL, use CD OPM medium containing 50 μM MSX to subculture the cells to a cell density of 3 x 10 5 cells / mL for continued culture.

[0079] (5) When the cell density increases to 4 x 10 6 cells / mL, use CD OPM medium containing 50 μM MSX to expand or subculture the cells, with a minimum inoculum density of 3 x 10 5 cells / mL. You can start preparing for freezing or expanding the culture.

[0080] (6) Antibody collection method: use protein A affinity chromatography for antibody purification.

[0081] Take 10 μg of commercial antibody CD8 [SK1] (manufacturer: Biolegend, item number: 344720), 10 μg of anti-human CD8 antibody [HCD8-06] prepared in Example 1, and 10 μg of anti-human CD8 antibody [HCD8-06] prepared in Example 2. HCD8-06] prepared in Example 1, and 10 μg of anti-human CD8 antibody [HCD8-06] prepared in Example 2. HCD8-08] 10 μg mixed with loading buffer (antibody and loading buffer volume ratio 4:1), 100 ℃ water bath 30 min, 10000 rpm room temperature centrifugal 1 min to obtain the sample to be tested. Take out the prefabricated glue installed in the electrophoresis tank, inject electrophoresis buffer, dial comb, sample. Among them, protein Marker sample 10 μL, sample 10 μg; electrophoresis instrument settings: voltage 80 V, electrophoresis time 90 min, bromophenol blue to the bottom when the end of electrophoresis; take out the gel to coomassie brilliant blue staining 30 min, placed in the decolorizing solution decolorization 1 h, repeated 5 times, take a photo.

[0082] The results show that the anti-human CD8 antibody prepared in example 1 is complete, the band is clear, and the purity reaches more than 95%, and the results are shown in Figure 1 .

[0083] Example 2 antibody labeling

[0084] Use FITC (fluorescein isothiocyanate) or RPE labeling HCD8-06 and HCD8-08.

[0085] 1, FITC labeling

[0086] (1) respectively take HCD8-06 0.2 mg and HCD8-08 0.2 mg each in the ultrafiltration tube, wash the antibody with 1 mM concentration PBS 3 times. 4000g, 2min inverted centrifugal ultrafiltration tube, collect the concentrated antibody, dilute to 10 mg / mL with 1 mM concentration PBS.

[0087] (2) take small molecule dye FITC (n (摩尔质量) antibody: n (摩尔质量) FITC = 1:15 add dye) to monoclonal antibody reaction solution, mix well.

[0088] (3) 25 ℃ room temperature, avoid light reaction on the oscillator for 45 min.

[0089] (4) transfer the reaction solution to the ultrafiltration centrifugal tube, wash the antibody with 1 mM concentration PBS 5 times. 4000g, 2min inverted centrifugal ultrafiltration tube, collect the concentrated antibody, dilute the antibody to a final concentration of 0.5 mg / mL with 1 mM concentration PBS.

[0090] The labeled antibody is recorded as HCD8-06 and HCD8-08.

[0091] 2, RPE labeling

[0092] According to the method of "fluorescent protein and / or conjugated protein monoclonal antibody labeling method and kit thereof (CN202010972671.X)", the antibodies are labeled respectively HCD8-06 and HCD8-08, and the labeled antibodies are marked as HCD8-06 and HCD8-08.

[0093] 3. Detection of the effect of labeled fluorescent antibodies

[0094] (1) Add 100 μL of cytoplasmic control (manufacturer: Beckman Coulter, item number: 6607077) to a 1.5 mL centrifuge tube.

[0095] (2) Take 1 μL of fluorescently labeled antibody HCD8-06,

[0096] HCD8-08, HCD8-06, HCD8-08, respectively.

[0097] (3) The above four samples are all reacted in the dark for 15 minutes.

[0098] (4) Add 1 mL of hemolysin and continue to react for 15 minutes. The hemolysin used is the hemolysin for positive flow cytometry (Zhejiang Medical Instrumentation Preparation 20200133). Hemolysin: Zhejiang Medical Instrumentation Preparation 20200133.

[0099] (5) Centrifuge at 4000 rpm for 2 min, discard the supernatant, and add 500 μL of PBS to resuspend the sample for detection.

[0100] (6) Detect the positive cell detection ratio, atlas grouping, and other indicators of the sample by flow cytometry (Agilent NovoCyte flow cytometry), and then determine HCD8-06 and HCD8-08 whether they can be labeled with fluorescent dyes FITC and RPE, and whether the labeled fluorescent antibodies detect the target antigens correctly.

[0101] FITC and RPE are successfully labeled HCD8-06 and HCD8-08, similar to the ratio of commercial Biolegend's control CD8 [SK1], see Figures 2-4 .

[0102] Antibody titer of Example 3

[0103] Antibody titer detection method:

[0104] The antibodies selected in this example are:

[0105] Antibody (1): positive control, commercial CD8 [SK1];

[0106] Antibody (2): CD8 antibody obtained in Example 1, denoted as anti-human CD8 HCD8-06] antibody;

[0107] Antibody (3): anti-human CD8 antibody obtained in Example 1, denoted as anti-human CD8 HCD8-08] antibody.

[0108] First, dilute the known concentration of antibodies (1), antibodies (2), and antibodies (3) to 1 mg / mL, and then dilute the antibodies again with PBS at a ratio of 1:100, 1:1000, 1:3000, 1:6000, 1:12000, 1:24000, 1:48000, 1:60000, and 1:96000. CD8 HCD8-06], and CD8 HCD8-08] antibodies as experimental groups, and anti-human CD8 [SK1] antibody as a control group.

[0109] The experimental group has two groups, and the control group has one group. Take 100 μL of healthy human blood enterprise reference product for each group, add 1 μL of anti-human CD8-RPE commercial flow cytometry antibody to each group, and incubate in the dark for 15 minutes. Then add 1 mL of hemolysin (hemolysin for positive flow cytometry (Zhejiang Huanye Preparation 20200133) to each of the three experimental groups, continue to react for 15 min. Centrifuge at 4000 rpm for 2 min, and discard the supernatant.

[0110] Add 100 μL of CD8 HCD8-06], and CD8 HCD8-08] antibodies diluted at a ratio to the experimental groups, and add 100 μl of commercial anti-human CD8 [SK1] to the control group. Mix and react at room temperature in the dark for 30 min, add 900 μL of PBS to resuspend and mix the sample, centrifuge at 4000 rpm for 2 min, discard the supernatant, add 500 μL of PBS to resuspend the sample for detection, and detect by flow cytometry. ​

[0111] The affinities of the three antibodies are respectively:

[0112] CD8 [SK1] antibody, the affinity (kd) is: 423.9 (1 mg / mL), see Figure 5 .

[0113] CD8[ HCD8-06] antibody, the affinity (kd) is: 257.4 (1 mg / mL), see Figure 6 .

[0114] CD8[ HCD8-08] antibody, the affinity (kd) is 622.6 (1 mg / mL), see Figure 7 .

[0115] The results show that the affinity of the prepared CHO secreted mouse antibody is weaker than that of the commercial antibody, and the affinity of the rabbit antibody is stronger than that of the commercial antibody.

[0116] Example 4 Antibody specificity

[0117] The antibodies selected in this example are:

[0118] Antibody (1): Anti-human CD8 antibody [HCD8-06] prepared in Example 1; HCD8-06];

[0119] Antibody (2): Anti-human CD8 antibody [HCD8-08] prepared in Example 1; HCD8-08];

[0120] Fluorescent antibody (1): labeled antibody HCD8-06 and HCD8-08 obtained in Example 2; Fluorescent antibody (2): labeled antibody HCD8-06 and

[0121] HCD8-08 obtained in Example 2; Fluorescent antibody (3): co-staining antibody commercial fluorescent antibody APC-CD3 (manufacturer: Longyang Zhengxi Biotechnology Co., Ltd., product number: 110610272).

[0122] Antibody specificity detection method:

[0123] The experiment is divided into two groups, lymphocyte acquisition, take 100 μL of cell quality control (manufacturer: Beckman Coulter, product number: 6607077). Add 1 mL of hemolysin, continue to react for 15 minutes, hemolysin is selected for Zhengxi flow cytometer hemolysin

[0124] The experiment is divided into two groups, lymphocyte acquisition, take 100 μL of cell quality control (manufacturer: Beckman Coulter, product number: 6607077). Add 1 mL of hemolysin, continue to react for 15 minutes, hemolysin is selected for Zhengxi flow cytometer hemolysin ​Hemolysin: Zhejiang Lake Machinery Reserve No. 20200133). Centrifuge at 4000 rpm for 2 min, discard the supernatant, and resuspend the sample with 100 μL PBS.

[0125] Experimental group: Add 3 μL of anti-human CD8 antibody HCD8-06] and CD8 antibody HCD8-08], respectively, and react in the dark for 15 minutes. Then, resuspend the sample with 500 μL PBS, centrifuge at 4000 rpm for 2 min, discard the supernatant, and resuspend the sample with 100 μL PBS. Prepare 2 samples for each pure antibody. In the 2 samples blocked by pure anti-CD8 HCD8-06], add HCD8-06, HCD8-06, respectively. In the 2 samples blocked by pure anti-CD8 HCD8-08], add HCD8-08, HCD8-08.

[0126] Control group: Add 1 μL of HCD8-06 and

[0127] HCD8-06 1 μL; and add 1 μL of HCD8-08 and HCD8-08 1 μL.

[0128] Add 1 μL of co-staining antibody APC-CD3 to both the control group and the experimental group.

[0129] After reacting in the dark for 15 minutes, add 500 μL PBS to resuspend the sample, centrifuge at 4000 rpm for 2 min, discard the supernatant, and resuspend the sample with 500 μL PBS.

[0130] Use a flow cytometer (Agilent NovoCyte flow cytometer) to detect the positive cell proportion and the graph grouping of the sample, and then determine whether HCD8-06 and HCD8-08 can specifically recognize the CD8 target antigen.

[0131] As can be seen from Figures 8-9 , the experimental group has an APC-CD3 signal, and there is no HCD8-06, HCD8-06, HCD8-08 and The signal of HCD8-08 fluorescent antibody. The control group, all have the signal of RPE-CD3 and CD8 fluorescent antibody. This shows that the CD8 antibody prepared by CHO / HEK293 can specifically recognize CD8 target antigen.

Claims

1. An anti-human CD8 antibody, characterized in that, comprises a heavy chain variable region, and the light chain comprises a light chain variable region; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein: (1) the amino acid sequence of HCDR1 is shown as SEQ ID NO. 1; (2) the amino acid sequence of HCDR2 is shown as SEQ ID NO. 2; (3) the amino acid sequence of HCDR3 is shown as SEQ ID NO. 3; (4) the amino acid sequence of LCDR1 is shown as SEQ ID NO. 4; (5) the amino acid sequence of LCDR2 is shown as SEQ ID NO. 5; (6) the amino acid sequence of LCDR3 is shown as SEQ ID NO.

6.

2. The anti-human CD8 antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the anti-human CD8 antibody is SEQ ID NO. 7 or a sequence with more than 80% sequence similarity with SEQ ID NO. 7; the amino acid of the light chain variable region of the anti-human CD8 antibody is SEQ ID NO. 8 or a sequence with more than 80% sequence similarity with SEQ ID NO. 8; or the amino acid of the light chain variable region of the anti-human CD8 antibody is SEQ ID NO. 13 or a sequence with more than 80% sequence similarity with SEQ ID NO.

13.

3. The anti-human CD8 antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain of the anti-human CD8 antibody is SEQ ID NO. 9 or a sequence with more than 65% sequence similarity with SEQ ID NO. 9; or the amino acid sequence of the heavy chain of the anti-human CD8 antibody is SEQ ID NO. 11 or a sequence with more than 65% sequence similarity with SEQ ID NO. 11; The amino acid sequence of the light chain of the anti-human CD8 antibody is SEQ ID NO. 10 or a sequence with more than 65% sequence similarity with SEQ ID NO. 10; or the amino acid sequence of the light chain of the anti-human CD8 antibody is SEQ ID NO. 12 or a sequence with more than 65% sequence similarity with SEQ ID NO.

12.

4. The anti-human CD8 antibody according to any one of claims 1 to 3, characterized in that, is a monoclonal antibody.

5. The anti-human CD8 antibody according to claim 4, characterized in that is a murinized antibody or a rabbitized antibody.

6. A nucleic acid expressing the anti-human CD8 antibody according to any one of claims 2 to 5, characterized in that, The nucleotide sequences corresponding to the heavy chain of SEQ ID NO. 9 and SEQ ID NO. 11 are shown as SEQ ID NO. 14 and SEQ ID NO. 15, respectively, and the nucleotide sequences corresponding to the light chain of SEQ ID NO. 10 and SEQ ID NO. 12 are shown as SEQ ID NO. 16 and SEQ ID NO. 17, respectively.

7. An expression vector comprising the nucleic acid of claim 6.

8. A cell comprising the anti-human CD8 antibody of any one of claims 1-5 or the nucleic acid of claim 6 or the expression vector of claim 7.

9. A method of CD8 detection, characterized in that, A method for detection by the anti-human CD8 antibody of any one of claims 1-5, wherein the method is a non-disease diagnosis or treatment method.

10. A CD8 detection kit, characterized by, An anti-human CD8 antibody according to any one of claims 1 to 5 or a nucleic acid according to claim 6 or an expression vector according to claim 7 or a cell according to claim 8.

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