Anti-CD8 antibody and its application

Abstract

The present invention discloses an anti-CD8 antibody and its application. The antibody comprises a heavy chain variable region and a light chain variable region. The amino acid sequence of CDR-H1 of the heavy chain variable region is shown in SEQ ID NO: 4, the amino acid sequence of CDR-H2 is shown in SEQ ID NO: 5, and the amino acid sequence of CDR-H3 is GDL; the amino acid sequence of CDR-L1 of the light chain variable region is shown in SEQ ID NO: 6, the amino acid sequence of CDR-L2 is shown in SEQ ID NO: 7, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO: 8. The anti-CD8 antibody is a rabbit monoclonal antibody. The obtained antibody has high titer, clear specific localization in immunohistochemical staining, and no nonspecific background staining. The antibody has a wide range of uses and can also be used in Western blot and flow cytometry experiments.

Description

Technical Field

[0001] The present invention belongs to the field of antibodies, and in particular relates to an anti-CD8 antibody and applications thereof. Background Art

[0002] CD8 cells are an important type of immune cell in the human immune system, belonging to the T lymphocyte family and a key component of the adaptive immune system. In a normal body, CD8 is a subset of T lymphocytes, and some T lymphocytes have CD8 cells on their surface. These T lymphocytes, called CD8+ T lymphocytes, can assist receptors in recognizing antigens and participate in the transmission of T cell activation signals. Upon activation, CD8+ T lymphocytes can differentiate into cytotoxic T cells, which have the ability to specifically kill target cells. Like other types of T cells, CD8 originates in the bone marrow and matures in the thymus. After maturation, they travel through the lymphatic circulation throughout the body and are stored in organs such as the spleen, tonsils, and lymph nodes.

[0003] MHCⅠ (Major Histocompatibility Complex Ⅰ) is a key molecule in the immune system. It is expressed in many cells, especially on the cell membrane of nucleated cells. It is a membrane-bound protein and is mainly composed of a 45kDd α chain and a 12kD β-microglobulin.

[0004] MHC class I molecules are primarily responsible for presenting endogenous antigens, such as viral proteins or abnormal proteins on tumor cells, to T cells (especially CD8+ T cells). CD8 molecules are important markers on the surface of CTLs, recognizing specific antigens presented by their own MHC class I molecules through the TCR (T-cell receptor). Anti-CD8 antibodies can specifically bind to CD8 molecules on the surface of CTLs. The detection of CD8 molecules can assess the body's immune function and provide clinical guidance. It is also of great significance for the diagnosis of certain infectious diseases, immune diseases, and tumors. Summary of the Invention

[0005] To address the above-mentioned issues, the present invention provides an anti-CD8 antibody and its application. The antibody has high titer, clear specific localization in immunohistochemical staining, and no nonspecific background staining, and can also be widely used in Western blot detection and flow cytometry.

[0006] In order to achieve the above object, the present invention adopts the following technical means:

[0007] The first aspect of the present invention provides an anti-CD8 antibody, which includes a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of CDR-H1 of the heavy chain variable region is shown in SEQ ID NO: 4, the amino acid sequence of CDR-H2 is shown in SEQ ID NO: 5, and the amino acid sequence of CDR-H3 is: GDL; the amino acid sequence of CDR-L1 of the light chain variable region is shown in SEQ ID NO: 6, the amino acid sequence of CDR-L2 is shown in SEQ ID NO: 7, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO: 8.

[0008] In some embodiments, the antibody is analyzed using VBASE2 online software to include the following polypeptide sequences: CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3:

[0009] (1) CDR-H1 of at least 5 amino acid sequences; the sequence is SYDMT (SEQ ID NO: 4).

[0010] (2) CDR-H2 of at least 16 amino acid sequences; the sequence is YISYTGITHHASWAKG (SEQ ID NO: 5).

[0011] (3) CDR-H3 of at least 3 amino acid sequences; the sequence is GDL.

[0012] (4) CDR-L1 of at least 13 amino acid sequences; the sequence is QSSQSVYDNNYLS (SEQ ID NO: 6).

[0013] (5) CDR-L2 of at least 7 amino acid sequences; the sequence is AASTLAS (SEQ ID NO: 7).

[0014] (6) CDR-L3 of at least 10 amino acid sequences; the sequence is AGAYSGNIKT (SEQ ID NO: 8).

[0015] In some embodiments of the present invention, the antibody comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO: 2, and a light chain having an amino acid sequence as shown in SEQ ID NO: 3.

[0016] In some embodiments of the present invention, the antibody is a rabbit anti-human monoclonal antibody, and the rabbit immunogen comprises the amino acid sequence shown in SEQ ID NO:1.

[0017] In some specific embodiments of the present invention, a polypeptide fragment synthesized from aa217 to aa235 CPRPVVKSGDKPSLSARYV (SEQ ID NO: 1) in the amino acid sequence of human CD8 protein (Uniprot ID: P01732) is selected as the immunogen sequence for preparing the antibody.

[0018] In some embodiments of the present invention, the rabbit immunogen is formed by cross-linking the amino acid sequence shown in SEQ ID NO: 1 with the macromolecule KLH to form a complete antigen.

[0019] A nucleic acid encoding the antibody described in the first aspect. By isolating the nucleic acid encoding the antibody described in the first aspect, the antibody can be produced in a recombinant manner, the nucleic acid is isolated and inserted into a replicable vector, and then further cloned or further expressed. Based on this, the second aspect of the present invention provides an expression vector comprising the nucleic acid described above. The sequence of the nucleic acid encoding the heavy chain variable region and light chain variable region of the present invention can be changed, and such changes include the addition, deletion or non-conservative / conservative substitution of nucleotides. The DNA encoding the antibody can be easily isolated or synthesized using conventional procedures. A variety of vectors are available, and the vector components generally include but are not limited to one or more of the following components: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0020] Preferably, the expression vector is a prokaryotic expression vector or a eukaryotic expression vector.

[0021] A third aspect of the present invention provides a host cell comprising the expression vector described above. In some embodiments of the present invention, the expression vector is a prokaryotic expression vector and the host cell is a prokaryotic cell; in other embodiments, the expression vector is a eukaryotic expression vector and the host cell is a eukaryotic cell. In a specific embodiment of the present invention, the expression vector is Escherichia coli and the host cell is a HEK293 cell.

[0022] The fourth aspect of the present invention provides use of the antibody described in the first aspect in preparing an immunoassay tool for detecting and identifying CD8 protein.

[0023] In some embodiments of the present invention, the immunoassay tool is a reagent, a kit, a chip or a test paper.

[0024] In some embodiments of the present invention, the immunodetection tool is used for at least one of immunohistochemistry, flow cytometry, and immunoblotting.

[0025] The fifth aspect of the present invention provides a kit for detecting and identifying CD8 protein, wherein the kit comprises the antibody described in the first aspect.

[0026] The present invention also provides a method for obtaining the above-mentioned antibody, which comprises: culturing the host cell described in the fourth aspect, and isolating and purifying the antibody from the cultured cells.

[0027] Beneficial effects of the present invention

[0028] Compared to existing technologies, the present invention offers the following advantages: The anti-CD8 antibody provided herein is a rabbit monoclonal antibody with high titer, clear specific localization in immunohistochemical staining, and no nonspecific background staining. This antibody can be used not only in pathological immunohistochemical staining experiments but also in Western blot and flow cytometry experiments, demonstrating its wide range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 ELISA binding curves of the antigens in Example 1 of the present invention are shown;

[0030] Figure 2 The figures show the results of immunohistochemical staining of different tissue sections with anti-CD8 rabbit monoclonal antibody;

[0031] Figure 3 The figure shows the results of Western blot detection using anti-CD8 rabbit monoclonal antibody;

[0032] Figure 4 The graph shows the results of flow cytometry detection with anti-CD8 rabbit monoclonal antibody; the peak on the left indicates that the isotype control antibody does not recognize the CD8 protein on the Molt-4 wild cell line, and the peak on the right indicates that the anti-CD8 rabbit monoclonal antibody can recognize the CD8 protein on the Molt-4 wild cell line. DETAILED DESCRIPTION

[0033] The following examples are provided to illustrate preferred embodiments of the present invention. Those skilled in the art will appreciate that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to practice the present invention and, therefore, can be considered preferred embodiments of the present invention. However, those skilled in the art will appreciate from this disclosure that many modifications may be made to the specific embodiments disclosed herein while still achieving the same or similar results without departing from the spirit or scope of the present invention.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs, and the disclosures herein and the materials they cite are hereby incorporated by reference. Those skilled in the art will recognize or be able to ascertain, through routine experimentation, many technical equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0035] The technical solution of this patent is further described in detail below in conjunction with specific implementation methods.

[0036] Example 1 Preparation of anti-CD8 monoclonal antibodies

[0037] A peptide fragment synthesized from the amino acid sequence of human CD8 protein (Uniprot ID: P01732) spanning aa217-aa235 CPRPVVKSGDKPSLSARYV (SEQ ID NO: 1) was selected as the immunogen sequence for antibody preparation, and this immunogen was used to immunize New Zealand white rabbits. The peptide fragment was cross-linked with the macromolecular KLH to form a complete antigen.

[0038] The immunogen is used to immunize New Zealand white rabbits, and the steps are as follows: first, the polypeptide immunogen is coupled to the KLH macromolecular protein, and the New Zealand white rabbits are immunized after dialysis. For the first immunization, 1 mg / mL is used, and 0.5 mL is mixed with an equal amount of complete Freund's adjuvant for the first immunization; 2 weeks later, 0.5 mg / mL is used, and 0.5 mL is mixed with an equal amount of incomplete Freund's adjuvant for the second immunization, and the third and fourth immunizations are subsequently performed every 2 weeks according to the second immunization method.

[0039] After four immunizations, blood was collected from New Zealand white rabbits to test serum titers. B cells from qualified rabbits were cryopreserved. B cells were obtained using the method described in our patent: CN201610724209.1. The cells were then lysed using the method described in our patent: CN201910004299.0, and mRNA was extracted for RT-PCR. After RT-PCR, the resulting cDNA was subjected to PCR to obtain the antibody's light and heavy chain DNA. The light and heavy chain DNAs were ligated into the pBV vector and transformed into competent cells, which were then plated and cultured for 12 hours. Colonies were then picked and shake-cultured. After 24 hours of culture, the heavy and light chain plasmids were extracted. The extracted light and heavy chain plasmids were transfected into HEK293 cells and cultured for 6 days.

[0040] Six days after transfection, the cell culture supernatant was harvested and purified to obtain rabbit monoclonal antibodies, which were then sequenced.

[0041] The amino acid sequence of the CD8 rabbit monoclonal antibody H chain (heavy chain variable region) (SEQ ID NO: 2) is as follows:

[0042] METGLRWLLLVAVLKGVQCQSLEESGGGLVTPGTPLTLTCTASGFTISSYDMTWVRQAPGKGLEYIGYISYTGITHHASWAKGRFTISRTSTTVDLKMTSPTTEDTATYFCG RGDLWGPGTVVTVSSGQPKAPSVFPLAPCCGDTPSSAVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPS TCSKPTCPPPELLGGPSVFIFPPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEK TISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK

[0043] The amino acid sequence of the CD8 rabbit monoclonal antibody L chain (light chain variable region) (SEQ ID NO: 3) is as follows:

[0044] MDTRAPTQLLGLLLLWLPGATFAQVLTQTPASVSAAVGGTVTINCQSSQSVYDNNYLSWYQQKPGQPPKQLIYAASTLASGVPSRFSGSGSGTQFTLTISGVQCDDAATYYCAGAYSG NIKTFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC

[0045] The obtained anti-CD8 antibodies were analyzed using VBASE2 online software;

[0046] The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region are as follows:

[0047] (1) a CDR-H1 sequence of at least 5 amino acids; the sequence is SYDMT (SEQ ID NO: 4);

[0048] (2) a CDR-H2 of at least 16 amino acids; the sequence is YISYTGITHHASWAKG (SEQ ID NO: 5);

[0049] (3) CDR-H3 of at least 3 amino acids; the sequence is GDL;

[0050] The amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 of the light chain variable region are as follows:

[0051] (1) CDR-L1 of at least 13 amino acid sequences; the sequence is QSSQSVYDNNYLS (SEQ ID NO: 6);

[0053] (2) a CDR-L2 sequence of at least 7 amino acids; the sequence is AASTLAS (SEQ ID NO: 7);

[0054] (3) CDR-L3 of at least 10 amino acid sequences; the sequence is AGAYSGNIKT (SEQ ID NO: 8).

[0055] Example 2 Enzyme-linked immunosorbent assay of anti-CD8 monoclonal antibodies

[0056] The antibody can specifically bind to the immunogen synthetic polypeptide sequence designed for human CD8.

[0057] The procedure was carried out according to the conventional iELISA method. The steps were as follows:

[0058] (1) Coating: Coat the plate with a synthetic peptide designed to specifically bind to human CD8, 1 μg / mL, 50 μL / well, overnight at 4°C.

[0059] (2) Blocking: The next day, wash the plate three times with PBST and then add 5% skim milk for blocking, 100 μL / well, at 30°C for 1 h.

[0060] (3) Primary antibody incubation: After washing the plate, the obtained anti-CD8 rabbit monoclonal antibody was diluted 4-fold from 1 μg / mL, with a total of 7 gradients. The diluted antibody was added to the ELISA plate at 50 μL / well and incubated at 30°C for 1 h.

[0061] (4) Secondary antibody incubation: After washing the plate, add 1:30,000 diluted HRP-labeled goat anti-rabbit IgG (Jackson, Cat#111-035-144) to the ELISA plate at 50 μL / well and incubate at 30°C for 40-60 min.

[0062] Color development and termination: After washing the plate, add TMB color development solution at 50 μL / well and react at 30°C in the dark for 15 minutes. After the reaction is completed, add 50 μL 1M H2SO4 to each well to terminate the reaction and read the OD450 absorbance value on the microplate reader. Prepare a standard curve based on the reading. ELISA binding curve is shown in Figure 1 .

[0063] The results showed that there were significant differences in the readings between the dilutions. The above antibodies could specifically bind to the immunogen synthetic peptide sequence designed for human CD8. The ELISA binding curve of CD8 monoclonal antibody was as follows: Figure 1 As shown, the EC50 was 4.24 ng / mL.

[0064] Example 3 Application of anti-CD8 monoclonal antibodies in pathological immunohistochemistry

[0065] Normal tonsil tissue, endometrial tissue, brain tissue, stomach tissue, lung squamous cell carcinoma tissue and other tissue sections were selected for pathological immunohistochemical staining. The tissue sections were operated using conventional manual immunohistochemical operation procedures. After immunohistochemical staining was completed, the sections were placed under a microscope for reading and photographing. The immunohistochemical staining pictures taken under the microscope were as follows: Figure 2 shown.

[0066] The results show that: Figure 2 It can be clearly seen that the anti-CD8 rabbit monoclonal antibody has brown cell membrane-positive localization in T lymphocytes of normal tonsil tissue (indicated by arrows); there is brown positive staining in T lymphocytes of tonsil and endometrial tissue (indicated by arrows), and the subcellular localization is the cell membrane; the antibody has no nonspecific staining in other tissues such as brain, gastric tissue, and lung squamous cell carcinoma tissue. The staining results show that CD8 antibody is negative in all cells in brain tissue (indicated by arrows); negative in glandular epithelium in gastric tissue (indicated by arrows); and negative in tumor cells of squamous epithelium-derived in lung squamous cell carcinoma tissue (indicated by arrows); the expression spectrum of this antibody meets the theoretical and practical immunohistochemical application standards.

[0067] Example 4 Application of anti-CD8 monoclonal antibodies in Western blot

[0068] (1) Molt-4 cell lysate was used for polyacrylamide gel electrophoresis. The loading amount of Molt-4 lysate was 20 μg / lane. The electrophoresis conditions were: 80 V, 40 min; 120 V, 80 min.

[0069] (2) Transfer: Remove the gel from the glass plate after electrophoresis in step (1), soak it in transfer buffer for 30 minutes, and then perform the transfer experiment. The transfer conditions are: 0.4A horizontal flow transfer for 20 minutes.

[0070] (3) After the transfer is complete, remove the PVDF membrane, rinse it with ddH2O for 5 minutes, add 10 mL of Ponceau red for staining, and wash the membrane with methanol for 5 minutes. Let it dry and set aside. At this point, the appearance of a red area in the cell protein lysate on the PVDF membrane indicates that the protein lysate on the cells has been transferred to the PVDF.

[0071] (4) Blocking: Take out the PVDF membrane prepared in step (3), activate it with methanol for 30 seconds, and then wash the membrane with ddH2O for 5 minutes. Then, add 10 mL of 5% skim milk powder to block the nonspecific binding sites on the membrane, and incubate at room temperature at 60 rpm for 1 hour.

[0072] (5) Primary antibody incubation: After step (4), add anti-CD4 monoclonal antibody diluted 1:500 and incubate at room temperature and 60 rpm for 1 h.

[0073] (6) Secondary antibody incubation: After step (5), add 1:4000 diluted HRP-conjugated goat anti-rabbit IgG polyclonal antibody and incubate at room temperature at 60 rpm for 1 h.

[0074] (7) Development and photography: After step (6), add the prepared ECL developer to the membrane. Place the PVDF membrane in a UV exposure instrument and take a photo.

[0075] (8) The result after the photo is inverted is as follows Figure 3 shown.

[0076] The results showed that the anti-CD8 rabbit monoclonal antibody recognized CD8 protein in Molt-4 cell lysate with an apparent molecular weight of 35 kD and no obvious miscellaneous bands, which was consistent with theoretical and literature reports. This indicates that this antibody can be used in Western blot experiments.

[0077] Example 5 Application of anti-CD8 monoclonal antibodies in flow cytometry

[0078] (1) Cultured Molt-4 cells were used, washed with PBS, and then the cell density was adjusted to 1 M / mL.

[0079] (2) Divide the 1M / mL cells into 3 tubes, 1 tube for negative control experimental cells, 1 tube for isotype control experimental cells, and 1 tube for anti-CD8 antibody experimental cells.

[0080] (3) Cell fixation: Add 1 mL of 4% paraformaldehyde fixative to each of the three tubes of cells and fix the cells for 30 minutes. Then add 1 mL of 1% BSA in PBS and centrifuge at 1400 rpm at room temperature for 5 minutes.

[0081] (4) Add 1 mL of 1% BSA in PBS to each tube, resuspend the cells, and centrifuge at 1400 rpm at room temperature for 5 minutes.

[0082] (5) Cell membrane permeabilization: Add 1 mL of 0.1% Triton X-100 permeabilization reagent to each of the three tubes of cells and permeabilize the cells for 10 minutes. Then, add 1 mL of 1% BSA in PBS and centrifuge at 1400 rpm at room temperature for 5 minutes.

[0083] (6) Add 1 mL of 1% BSA in PBS to each tube, resuspend the cells, and centrifuge at 1400 rpm at room temperature for 5 minutes.

[0084] (7) Primary Antibody Incubation: Add 100 μL of 1% BSA in PBS to one tube of cells, 100 μL of 1 μg / mL isotype control antibody to another tube, and 100 μL of 1 μg / mL anti-CD8 rabbit monoclonal antibody to the final tube. Resuspend the cells and incubate at room temperature for 60 min. After incubation, add 1 mL of 1% BSA in PBS to each tube and centrifuge at 1400 rpm for 5 min at room temperature.

[0085] (8) Add 1 mL of 1% BSA in PBS to each tube, resuspend the cells, and centrifuge at 1400 rpm at room temperature for 5 minutes.

[0086] (9) Secondary antibody incubation: Add 100 μL of 1:2000 diluted goat Anti-Rabbit IgG H&L (Alexa Fluor 500) to each tube. 488), resuspend the cells and incubate at room temperature for 60 min. After incubation, add 1 mL of 1% BSA in PBS to each tube and centrifuge at 1400 rpm at room temperature for 5 min.

[0087] (10) Add 1 mL of 1% BSA in PBS to each tube, resuspend the cells, and centrifuge at 1400 rpm at room temperature for 5 minutes.

[0088] (11) Flow cytometry detection: Using the isotype control well as the standard, adjust the voltage for flow cytometry detection. The results are as follows Figure 4 shown.

[0089] The results showed that using an anti-CD8 rabbit monoclonal antibody in a flow cytometry experiment on the Molt-4 wild-type tumor cell line, the isotype control antibody did not recognize the CD8 protein on the Molt-4 wild-type cell line (left curve), while the anti-CD8 rabbit monoclonal antibody did recognize the CD8 protein on the Molt-4 wild-type cell line (right curve). This antibody can be used in flow cytometry applications.

[0090] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the present application.

Claims

1. An anti-CD8 antibody, characterized in that: It includes a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region CDR-H1 is shown in SEQ ID NO: 4, the amino acid sequence of CDR-H2 is shown in SEQ ID NO: 5, and the amino acid sequence of CDR-H3 is: GDL; the amino acid sequence of the light chain variable region CDR-L1 is shown in SEQ ID NO: 6, the amino acid sequence of CDR-L2 is shown in SEQ ID NO: 7, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO:

8.

2. The antibody according to claim 1, characterized in that: The antibody comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 2, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:

3.

3. The antibody according to claim 1 or 2, characterized in that: The antibody is a rabbit anti-human monoclonal antibody, and the rabbit immunogen comprises the amino acid sequence shown in SEQ ID NO:

1.

4. An expression vector, characterized in that: Comprising a nucleic acid encoding the antibody according to claim 1 or 2.

5. A host cell, characterized in that: Comprising the expression vector according to claim 4.

6. Use of the antibody according to any one of claims 1 to 3 in the preparation of an immunoassay tool for detecting and identifying CB8 protein.

7. The use according to claim 6, characterized in that: The immunoassay tool is a reagent, a test kit, a chip or a test paper.

8. The use according to claim 6, characterized in that: The immunoassay tool is used for at least one of immunohistochemistry, flow cytometry, and immunoblotting.

9. A kit for detecting and identifying CB8 protein, characterized in that: Comprising the antibody of claim 3.

10. A method for obtaining the antibody according to claim 1 or 2, characterized in that: include (1) culturing the host cell according to claim 5, and (2) Isolating and purifying the antibody from the cultured cells.

Images