Monoclonal antibody binding to human CD8 and its preparation method and application

Abstract

The application relates to the field of biological medicine, and particularly discloses a monoclonal antibody combined with human CD8 and a preparation method and application thereof. The antibody is obtained by combining single B cell sorting, gene cloning and mammalian cell expression technology after rabbit immunization of a CD8 polypeptide antigen, a New Zealand white rabbit is immunized by adopting a KLH coupled CD8 antigen polypeptide combined with a rapid immunoadjuvant, high-affinity antibody clones are obtained by single B cell sorting technology, and the antibody is prepared by recombination expression. The antibody is verified by ELISA and flow cytometry, has high titer and strong specificity, can effectively recognize human peripheral blood CD8+ T cells, and has a signal strength and a background signal-to-noise ratio which are significantly better than those of a traditional mouse-derived antibody. The application provides light and heavy chain variable region amino acid sequences of the antibody, realizes controllable preparation of the antibody and protection of intellectual property rights. The antibody can be widely applied to the fields of flow cytometry detection, immunohistochemistry, tumor immunotherapy monitoring and T cell function research, and has important scientific research and clinical application values.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a monoclonal antibody that binds to human CD8, its preparation method, and its application. Background Technology

[0002] CD8 molecules are important members of the immunoglobulin superfamily, primarily expressed on the surface of cytotoxic T cells (CTLs). As a co-receptor for MHC class I molecules, they participate in T cell antigen recognition and immune activation. CD8+ T cells play a central role in antiviral and antitumor immune responses, and their quantity and functional status are important indicators for assessing the body's immune capacity. Therefore, highly specific and high-affinity CD8 antibodies have broad application value in immunological research, clinical diagnosis, and immunotherapy monitoring.

[0003] Currently, most commercially available CD8 antibodies are derived from murine hybridoma technology. While they can meet basic detection needs, they still have the following limitations:

[0004] Insufficient affinity: Murine antibodies typically have low affinity for antigens, which may lead to insufficient detection sensitivity, especially in cells with low CD8 expression (such as certain tumor-infiltrating lymphocytes or macrophages) where the signal is weak.

[0005] Animal-derived risks: Mouse-derived antibodies may introduce foreign protein components, increasing the risk of immunogenicity and affecting the stability and safety of the test.

[0006] Large batch-to-batch variability: Antibodies produced by traditional hybridoma technology have poor batch-to-batch stability, which may affect the reproducibility of experiments.

[0007] Limited application scenarios: In detection methods such as flow cytometry, murine antibodies may result in high background signals due to non-specific binding, affecting the signal-to-noise ratio.

[0008] In recent years, rabbit monoclonal antibodies have demonstrated superior affinity and specificity due to the unique characteristics of the B-cell immune system. Furthermore, rabbit antibodies exhibit greater diversity in their CDR (complementarity-determining region) regions, facilitating the screening of superior antibody clones. In addition, rabbit monoclonal antibody preparation methods based on single B-cell sorting and recombinant expression technologies avoid animal-derived contamination, improve antibody stability and reproducibility, and are better suited to the high standards required for research and clinical testing. Summary of the Invention

[0009] In view of the shortcomings of the prior art, the present invention discloses a rabbit-derived recombinant anti-human CD8 monoclonal antibody with high affinity, high specificity and controllable source, which is of great significance for improving the accuracy of immune detection, promoting tumor immunotherapy research and infectious disease monitoring.

[0010] The technical solution of this invention is: a monoclonal antibody that binds to human CD8, wherein the antibody comprises the following 6 complementarity-determining region (CDR) amino acid sequences:

[0011] Heavy chain CDR1 (HCDR1): LSSGAMI;

[0012] Heavy chain CDR2 (HCDR2): FIGTAGWPTYASWAKG;

[0013] Heavy chain CDR3 (HCDR3): GVPGDASYSFNP;

[0014] Light chain CDR1 (LCDR1): QSSESVYKNNYLS;

[0015] Light chain CDR2 (LCDR2): STSTLAS;

[0016] Light chain CDR3 (LCDR3): QGAYDSSEWYFA;

[0017] The antibody is obtained by isolating B cells from rabbits immunized with human CD8 antigen using monoclonal technology and then recombinantly expressing them. The antibody can specifically bind to human CD8 protein and can be used to detect CD8-positive T cells.

[0018] Furthermore, the amino acid sequence of the light chain variable region of this antibody is as follows:

[0019]

[0020] Furthermore, the amino acid sequence of the heavy chain variable region of this antibody is as follows:

[0021]

[0022] The present invention discloses a method for preparing a monoclonal antibody binding to human CD8, which is obtained through the following steps:

[0023] (1) KLH-conjugated human CD8 antigen peptide was mixed with rabbit-specific Bio-Long Quick Antibody™ immune adjuvant and then used to immunize rabbits. The rabbits were immunized three times in the thigh muscles for a total of eight weeks.

[0024] (2) After the immunization cycle was completed, rabbit serum was collected and coated with human serum albumin HAS-coupled polypeptide for detection. The anti-CD8 antibody titer was detected by ELISA.

[0025] (3) After the qualified rabbits were treated with air injection, rabbit spleen lymphocytes were obtained and specific single B cells were sorted by biotin-labeled HAS-CD8 antigen peptide.

[0026] (4) The obtained single B cells were cultured in vitro, and cell clones that produced high-titer anti-CD8 antibodies were screened.

[0027] (5) By extracting B cell RNA and reverse transcribing cDNA, the CDR region genes of the light and heavy chains in the B cell clone were amplified using specific primers and constructed into the pcDNA3.4 expression vector;

[0028] (6) After in vitro recombinant expression, a high concentration of recombinant rabbit monoclonal antibody was obtained and its titer was retested. The supernatant was then used to verify its fluorescence effect by flow cytometry.

[0029] An application of an antibody according to the present invention, the antibody being used for:

[0030] (1) Detection and auxiliary diagnosis of human immune status;

[0031] (2) Evaluation of CD8-positive T cell function in viral infection or tumor immunotherapy;

[0032] (3) Study on the subtypes and activation status of immune cell subsets;

[0033] (4) Used as an immunohistochemistry or magnetic bead sorting reagent in scientific research or clinical trials.

[0034] Compared with existing technologies, the anti-human CD8 rabbit monoclonal antibody and its preparation method provided by this invention have the following significant advantages:

[0035] 1. High affinity and high specificity: This invention utilizes a rationally designed CD8 antigenic peptide (SQNKPKAAEGLDTQR, antigenicity prediction score of 13), combined with KLH conjugation and a rapid adjuvant (Quick Antibody™), to efficiently stimulate the rabbit immune system and obtain high-affinity antibodies (such as the 4C5 clone). Flow cytometry verification shows that this antibody can specifically recognize CD8+ T cells in human peripheral blood, exhibiting strong fluorescence signal, low background, and a signal-to-noise ratio significantly superior to traditional murine antibodies.

[0036] 2. No animal-derived risk: Antibodies are produced using recombinant expression technology, avoiding animal-derived components from hybridoma technology, thus improving the safety and stability of the antibodies and making them more suitable for clinical testing applications.

[0037] 3. Highly efficient and controllable preparation process: Utilizing single-cell B-cell sorting technology, high-affinity antibody clones (such as 4C5) are precisely screened and recombinantly expressed in mammalian cells (such as HEK293 or CHO), resulting in stable antibody yield (>30 mg / L) and high batch-to-batch consistency. Gene sequencing clearly identifies the variable region sequences of the antibody's light and heavy chains (SEQ ID NO: 1 and SEQ ID NO: 2), enabling antibody traceability and intellectual property protection.

[0038] 4. Broad Application Prospects: Applicable to various detection scenarios such as flow cytometry, immunohistochemistry (IHC), and magnetic bead sorting, meeting the needs of scientific research and clinical practice. It can be used in combination with other immune biomarkers (such as CD3, CD4, and IFN-γ) for T cell function research, tumor microenvironment analysis, and immunotherapy monitoring. It provides a molecular basis for subsequent antibody engineering (such as humanization and bispecific antibody development).

[0039] In summary, this invention not only solves the technical bottlenecks of existing CD8 antibodies, but also provides a more reliable tool for immune detection, tumor immunotherapy, and infectious disease research, and has significant scientific research and industrialization value. Attached Figure Description

[0040] Figure 1 KLH-conjugated CD8 antigenic polypeptide sequence (SQNKPKAAEGLDTQR) and its antigenicity prediction score (13 points).

[0041] Figure 2 Figure: ELISA results of serum anti-CD8 antibody titer in rabbits 8 weeks after immunization.

[0042] Figure 3 : Bar chart of ELISA titer of culture supernatant after sorting of single B cells. (a) is the ELISA titer of the sorted supernatant; (b) is the bar chart group.

[0043] Figure 4 The positive 4C5 monoclonal strain obtained by culturing rabbit B cells in this invention can recognize CD8 positive cells in human peripheral blood and is compared with the classic clone number.

[0044] Figure 5 : Amino acid sequences of the light and heavy chain variable regions of the 4C5 antibody, with CDR region positions marked. Detailed Implementation

[0045] I. Antigen Design and Immunization Procedures

[0046] 1. Antigen peptide design and KLH conjugation

[0047] like Figure 1 As shown, a highly antigenic region of the human CD8 protein was selected, and a polypeptide sequence SQNKPKAAEGLDTQR (antigenicity prediction score of 13, a score >3 indicates good antigenicity) was designed. This polypeptide was conjugated with KLH (keyhole hemocyanin) as an immunogen.

[0048] 2. Rabbit immunization program

[0049] Immunized animals: Healthy white rabbits (weighing 2.5-3.0 kg).

[0050] Immune adjuvant: Bio-Drone Quick Antibody TM -8-week rapid adjuvant.

[0051] Immunization methods:

[0052] First immunization: KLH-CD8 peptide (200 μg) was mixed with an adjuvant in equal volume and injected into the thigh muscle of rabbits at multiple sites.

[0053] Boost immunization: Vaccinate every 2 weeks for a total of 3 times, for a total of 8 weeks.

[0054] Serum titer detection: Rabbit serum was collected after each immunization, and the anti-CD8 antibody titer was detected using ELISA. Figure 2 As shown, the rabbit serum titer increased significantly after 8 weeks of immunization (OD value > 2.0), indicating that antibody production was successfully induced.

[0055] II. Single B-cell sorting and positive clone screening

[0056] 1. Splenic lymphocyte separation

[0057] Immunized rabbits were euthanized by air injection, and their spleens were aseptically harvested. After mechanical grinding, the spleens were passed through a 70μm cell sieve to obtain a single-cell suspension.

[0058] III. Preparation of Biotin-Labeled Antigen Probes

[0059] HSA (human serum albumin)-CD8 peptide (2 μg / mL) was conjugated with biotin at a 1:1 molar ratio to serve as a B cell sorting probe.

[0060] IV. Flow Cytometry Sorting of Single B Cells

[0061] Spleen cells were labeled with biotin-CD8 peptide and bound to streptavidin-fluorescent dye (e.g., PE). Antigen-specific B cells were then sorted by flow cytometry. Individual B cells were sorted into 96-well plates, and each well was added with B cell culture medium containing IL-6 (10 ng / mL) and CD40L (1 μg / mL).

[0062] V. ELISA detection of culture supernatant

[0063] After culturing for 7-10 days, collect the supernatant for ELISA testing. Figure 3 As shown, the OD values ​​of positive clones (such as 4C5 and 2C5) are all >2.5, with 4C5 having a higher titer (OD value ≈3.0).

[0064] VI. Antibody gene cloning and recombinant expression

[0065] 1. RNA extraction and cDNA synthesis

[0066] Total RNA was extracted from positive B cells (such as 4C5) and reverse transcribed into cDNA.

[0067] 2. PCR amplification of the variable regions of light and heavy chains

[0068] The heavy chain (VH) and light chain (VL) variable region genes were amplified using rabbit IgG-specific primers. The amplified products were purified after verification by gel electrophoresis.

[0069] 3. Vector construction and transfection

[0070] The VH and VL genes were cloned into the pcDNA3.4 expression vector, respectively. HEK293 cells were co-transfected (heavy chain:light chain plasmid ratio 1:1) and cultured at 37°C and 5% CO2 for 5–7 days.

[0071] 4. Antibody purification and identification

[0072] The culture supernatant was collected and purified by Protein A affinity chromatography. The purity was determined by SDS-PAGE (>95%), and the concentration was determined by the BCA method.

[0073] VII. Antibody Function Verification

[0074] ELISA steps:

[0075] 1. Coating antigen: Human serum albumin HAS-conjugated CD8 peptide was coated at a concentration of 2 μg / mL, 100 μL / well onto the microplate and incubated overnight at 4°C.

[0076] 2. Cleaning: Use a plate washer to clean the ELISA plate 4-5 times;

[0077] 3. Blocking: Prepare 0.1% BSA, add 300 μL of blocking solution to each well, and block at room temperature for 2 hours;

[0078] 3. Cleaning: Use a plate washer to clean the ELISA plate 4-5 times.

[0079] 4. Add sample: Add 100 μL of sample to each well and incubate at 37°C for 1 hour.

[0080] 5. Cleaning: Use a plate washer to clean the ELISA plate 4-5 times;

[0081] 6. Add secondary antibody: Prepare goat anti-rabbit HRP secondary antibody at a ratio of 1:8000. Add 100 μL of secondary antibody solution to each well, place on a shaker, adjust the speed to medium, and incubate in the dark for 40 min.

[0082] 7. Cleaning: Use a plate washer to clean the ELISA plate 4-5 times;

[0083] 8. Color development: Add 100 μL of TMB color development solution to each well, incubate in the dark, check the color development at any time, incubate for no more than 5 minutes, and stop the incubation immediately when the color turns dark blue;

[0084] 9. Stop color development: Add 100 μL of stop solution to each well;

[0085] 10. Reading: After turning on the microplate, read the value of 0D452 and save the data.

[0086] like Figure 4 As shown, the 4C5 antibody can clearly distinguish CD8+ cell populations in human peripheral blood (positive rate >20%), and its fluorescence signal intensity is superior to that of commercially available murine antibodies. Sample Name is the sample name; Subset Name is the cell subpopulation, Lymphocytes: the target cell population analyzed. Mean Fluorescence Intensity (MFI): x-axis (CD8-PE): signal intensity of the PE fluorescence channel labeled with CD8 antibody; y-axis (Count): cell number.

[0087] The specific experiment is as follows:

[0088] I. Sample Processing:

[0089] Sample processing: Peripheral blood, EDTA anticoagulant tube (purple cap) collection. When collecting peripheral blood, mix thoroughly to ensure even mixing with the anticoagulant (Note: Anticoagulant tube selection: Purple cap tube (EDTA anticoagulant tube) recommended)

[0090] II. Staining steps:

[0091] 1. Draw 100 μL of peripheral blood from the EDTA anticoagulant tube into the flow cytometer (as 1T).

[0092] 2. Set up blank tubes / single-color compensation tubes / sample tubes (100 μL peripheral blood per tube). Do not add flow cytometry antibodies to the blank tubes. Add 5 μL of antibody to each single-color compensation tube according to the label. Add 5 μL of antibody to each sample tube. Incubate at 4°C in the dark for 20 min. Note: If antibody titration has been performed previously, add the antibody according to the titration results.

[0093] 3. Add 1-2 mL of 1*Schizolyl hemolysin / hemolysin to each tube (refer to the Schizolyl hemolysin instructions for specific timing and operation).

[0094] 4. After the red cleavage is complete, centrifuge at 300g for 5 minutes at 4℃ and discard the supernatant.

[0095] 5. Add 1 mL of 1*PBS, resuspend, centrifuge at 300 g for 5 min at 4 °C, and discard the supernatant.

[0096] 6. Add 300-500 μL of 1*PBS to each tube to resuspend the cells.

[0097] 7. Flow cytometer was used to detect and analyze the data.

[0098] Note: If immediate analysis is not possible, stained cells can be fixed in 4% paraformaldehyde (prepared with 1*PBS) at 4°C for 30 min, washed with 1*PBS (refer to step 5), and then resuspended in 300 μL of 1*PBS and stored at 4°C in the dark. Fixed cells should be analyzed as soon as possible.

[0099] VIII. Antibody Sequence and Stability Testing

[0100] 1. Sequencing analysis

[0101] The light and heavy chain variable region sequences of the 4C5 clone (SEQ ID NO: 1 and SEQ ID NO: 2) were obtained by Sanger sequencing. Figure 5 As shown, the variable region of the heavy chain includes CDR1 (LSSGAMI), CDR2 (FIGTAGWPTYASWAKG) and CDR3 (GVPGDASYSFNP), while the variable region of the light chain includes CDR1 (QSSESVYKNNYLS), CDR2 (STSTLAS) and CDR3 (QGAYDSSEWYFA).

[0102] 2. Stability Verification

[0103] After storage at 4℃ for 3 months, the ELISA titer decreased by <10%, and there was no significant difference in the flow cytometry detection signal. It maintained >90% activity after repeated freeze-thaw cycles (5 times).

[0104] IX. Application Examples

[0105] 1. Immunohistochemistry (IHC)

[0106] After antigen retrieval, paraffin sections were treated with 4C5 antibody (1:200) and DAB staining was performed, which clearly marked CD8+ T cells in lymph nodes.

[0107] 2. Magnetic bead sorting

[0108] By conjugating antibodies to magnetic beads, high-purity (>95%) CD8+ T cells can be sorted from peripheral blood.

Claims

1. A monoclonal antibody that binds to human CD8, characterized in that, The antibody contains six complementarity-determining regions (CDRs) as shown in the following amino acid sequence: Heavy chain CDR1 (HCDR1): LSSGAMI; Heavy chain CDR2 (HCDR2): FIGTAGWPTYASWAKG; Heavy chain CDR3 (HCDR3): GVPGDASYSFNP; Light chain CDR1 (LCDR1): QSSESVYKNNYLS; Light chain CDR2 (LCDR2): STSTLAS; Light chain CDR3 (LCDR3): QGAYDSSEWYFA.

2. The antibody according to claim 1, characterized in that, The amino acid sequence of the light chain variable region of this antibody is as follows: MDTRAPTQLLGLLLLWLPGATFAVVLTQTPSPVSAAVGGTVTISCQSSESVYKNNYLSWFQQKPGQPPKLLIYSTSTLASGVPSRFKGSGSGTQFTLTISDLECDDAATYYCQGAYDSSEWYFAFGGGTEVVVK (SEQ ID NO: 1).

3. The antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of this antibody is as follows: METGLRWLLLVAVLKGVQCQSLEESGGRLVTPGTPLTLTCTVSGFVLSLSSGAMIWVRQPPEKGLEYIGFIGTAGWPTYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARGVPGDASYSFNPWGPGTLVTVSSG (SEQ ID NO: 2).

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