Monoclonal antibody binding to human CD4 and its preparation method and application

Abstract

The application relates to the technical field of biological medicine, and particularly discloses a monoclonal antibody combined with human CD4 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 CD4 polypeptide antigen. The antibody has high affinity and good specificity, and can effectively recognize CD4 positive T cells in human peripheral blood. The antibody is verified by ELISA and flow cytometry, and has high titer, low background and a signal-to-noise ratio superior to that of existing mouse-derived antibodies. Further, the amino acid sequences of the light chain and the heavy chain variable region of the antibody are obtained, so that controllability and repeatability of antibody expression are realized. The antibody can be widely applied to scenes such as immunodetection, HIV auxiliary diagnosis, T cell subgroup research and antibody engineering development, and has important scientific research and industrial values.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more particularly to a monoclonal antibody that binds to human CD4, its preparation method, and its applications, belonging to the fields of antibody engineering and immunoassay technology. This antibody can be widely used in flow cytometry detection, immune status assessment, research and diagnosis of infectious diseases and autoimmune diseases, and has significant scientific research and clinical application value. Background Technology

[0002] CD4 is a glycoprotein expressed on the surface of helper T cells. As a co-receptor in T cell activation, it participates in antigen recognition and immune response regulation. CD4-positive T cells (i.e., CD4+ T cells) play a crucial role in the body's immune system, and changes in their numbers are often used as an important indicator for assessing infectious diseases (such as HIV / AIDS) or autoimmune diseases. Clinically, CD4 antibodies are commonly used in vitro to detect CD4+ T cells in peripheral blood to assess a patient's immune status and disease progression.

[0003] Most existing CD4 antibodies are derived from murine hybridoma cells, which have limitations in affinity, specificity, and cross-reactivity. Furthermore, due to their animal origin, they pose potential safety risks in certain immunoassays or therapeutic applications. In addition, murine antibodies exhibit weak fluorescence signals and high background in flow cytometry and other detection methods, making it difficult to meet the increasingly demanding requirements for detection accuracy. Therefore, developing non-murine CD4 antibodies with more abundant sources, higher affinity, and superior application performance has become an important direction in current antibody engineering research.

[0004] Rabbit monoclonal antibodies, due to their unique B-cell immune response mechanism, can produce antibodies with high affinity and specificity for small molecules or weak antigens. Furthermore, rabbit antibodies exhibit greater structural diversity in their variant regions, resulting in a larger library capacity, which is beneficial for screening high-quality antibodies. Simultaneously, with the development of single-cell B-cell sorting and recombinant expression technologies, the reproducibility and purity of rabbit monoclonal antibody preparation have been significantly improved.

[0005] Therefore, developing a high-affinity rabbit-derived recombinant anti-human CD4 monoclonal antibody and achieving its recombinant expression through modern molecular cloning and expression systems can not only overcome the limitations of existing CD4 antibodies, but also promote the development of immune detection and disease research technologies, which has important practical significance and application prospects. Summary of the Invention

[0006] The purpose of this invention is to provide a high-affinity, highly specific, and reliably sourced anti-human CD4 rabbit recombinant monoclonal antibody, its preparation method, and its application, in order to solve the problems of insufficient affinity, high background signal, and safety issues caused by animal origin in existing murine antibodies, and to meet the urgent need for high-performance antibodies in immunoassay and scientific research applications.

[0007] To achieve the above objectives, the present invention employs the following technical solution: a monoclonal antibody that binds to human CD4, wherein the antibody comprises the following six complementarity-determining region (CDR) amino acid sequences:

[0008] Heavy chain CDR1 (HCDR1): SNSIS

[0009] Heavy chain CDR2 (HCDR2): YISYGGSAYYASWAKG

[0010] Heavy chain CDR3 (HCDR3): GVVGSTSGTNNL

[0011] Light chain CDR1 (LCDR1): QSSQSLYNNNQLS

[0012] Light chain CDR2 (LCDR2): SASTLES

[0013] Light chain CDR3 (LCDR3): LGDYKSDIDTFV

[0014] The antibody is obtained by isolating B cells from rabbits immunized with human CD4 antigen using monoclonal cloning technology and then recombinantly expressing them. The antibody can specifically bind to human CD4 protein and be used for the detection of CD4-positive T cells.

[0015] Furthermore, the amino acid sequence of the light chain variable region of this antibody is as follows:

[0016] AAVLTQTPASVSAAVGGSVTINCQSSQSLYNNNQLSWYQQKPGQPPKLLIYSASTLESGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCLGDYKSDIDTFVFGGGTEVVVK (SEQ ID NO: 1).

[0017] Furthermore, the amino acid sequence of the heavy chain variable region of this antibody is as follows:

[0018] QSVEESGGRLVTPGGSLTLTCTVSGIDLSSNSISWVRQAPGKGLEYIGYISYGGSAYYASWAKGRFTISRTSTTVDLRMTSLTTEDTATYFCARGVVGSTSGTNNLWGPGTLVTVSS (SEQ ID NO: 2).

[0019] The present invention discloses a method for preparing a monoclonal antibody that binds to human CD4, which is obtained through the following steps:

[0020] (1) The KLH-conjugated human CD4 antigen peptide was mixed with rabbit Quick Antibody™ immune adjuvant and then used to immunize rabbits. The rabbits were immunized three times in total for a period of eight weeks in the thigh muscles.

[0021] (2) After the immunization cycle, rabbit serum was collected and coated with human serum albumin HAS-coupled peptide for detection. The anti-CD4 antibody titer was detected by ELISA.

[0022] (3) After the qualified rabbits were treated with air injection, rabbit spleen lymphocytes were obtained and single B cells were sorted by biotin-labeled HAS-CD4 polypeptide.

[0023] (4) The obtained single B cells were cultured in vitro, and cell clones that produced high-titer anti-CD4 antibodies were screened.

[0024] (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;

[0025] (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.

[0026] Application of the antibody of the present invention, wherein the antibody is used for:

[0027] (1) Detection and auxiliary diagnosis of human immune status;

[0028] (2) CD4 count in the assessment of AIDS progression;

[0029] (3) Research on the function of immune cells in autoimmune diseases;

[0030] (4) Research on T cell differentiation, activation and signaling pathways;

[0031] (5) Used as an immunohistochemistry or magnetic bead sorting reagent in scientific research or clinical trials.

[0032] Application Validation

[0033] The rabbit recombinant monoclonal antibody obtained in this invention can specifically recognize CD4-positive T cells in human peripheral blood. It exhibits a clear fluorescence signal and good background control in flow cytometry staining experiments, superior to traditional mouse antibodies. Further plasmid sequencing yielded the complete variable region nucleic acid sequences of the antibody's light and heavy chains, as well as the translated amino acid sequences, possessing independent intellectual property rights.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] This invention achieves highly efficient immunization of New Zealand white rabbits by rationally designing highly antigenic polypeptide fragments of CD4 protein and using KLH conjugation technology combined with a rapid immunization adjuvant. It successfully obtains high-titer, high-affinity anti-CD4 antibodies, which have significant advantages such as short immunization cycle, high antibody titer and stability.

[0036] By using single B cell-specific screening and in vitro clonal culture, monoclonal B cells that secrete high-affinity antibodies can be accurately obtained, significantly improving screening efficiency and antibody quality, and ensuring excellent antibody specificity and affinity.

[0037] This invention utilizes rabbit antibody gene cloning and recombinant expression technology to achieve efficient recombinant production of anti-CD4 antibodies. The expression system is stable, the purification process is simple, the antibody purity is high, and the yield is stable (>30 mg / L), laying a solid foundation for large-scale industrial production.

[0038] The obtained 4D5 antibody not only has strong binding ability and high specificity, but can accurately identify CD4+ T cell subsets. It has strong fluorescence signal and low background, and the signal-to-noise ratio is significantly better than that of commercially available murine CD4 antibodies. It is suitable for flow cytometry and various immunoassay applications, improving detection sensitivity and accuracy.

[0039] The clarification of antibody gene sequences and the acquisition of independent intellectual property rights provide an important molecular basis for subsequent humanization of antibodies, development of dual antibodies, and design of antibody-drug conjugates (ADCs), and have broad potential for derivative development and application.

[0040] The antibody of this invention exhibits excellent stability and reproducibility. After multiple batch verifications and long-term storage tests, the antibody activity remains good, ensuring the reliability and reproducibility of results in clinical and scientific research applications.

[0041] Furthermore, the antibodies of this invention have a wide range of applications. In addition to conventional flow cytometry, they are also applicable to immunohistochemical staining, magnetic bead sorting, HIV auxiliary diagnosis, and immune pathway research, greatly enriching the application scenarios and value of CD4 antibodies.

[0042] In summary, the technical solution of this invention is systematic, simple to operate, and has significant effects. It not only provides an efficient route for the preparation of high-quality rabbit-derived anti-CD4 antibodies, but also provides high-performance, stable, and reliable key reagents for related immunodiagnostics and scientific research fields, and has important industrialization and application promotion value. Attached Figure Description

[0043] Figure 1 KLH-conjugated CD4 antigenic peptide sequences and their antigenicity prediction scores;

[0044] Figure 2 : ELISA detection image of anti-CD4 antibody titer in rabbit serum 8 weeks after immunization;

[0045] Figure 3 : Single B cell sorting data and ELISA bar chart of antibody titer in culture supernatant after sorting; (a) sorting data; (b) ELISA bar chart of antibody titer;

[0046] Figure 4 Flow cytometry results: 4D5 rabbit anti-CD4 antibody can recognize human peripheral blood CD4-positive cells, compared with commercial control antibody;

[0047] Figure 5 : Amino acid sequences of the light and heavy chain variable regions of the 4D5 antibody. Detailed Implementation

[0048] The technical solution of the present invention will be described in detail below with reference to specific embodiments. However, those skilled in the art should understand that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention.

[0049] I. Antigen Design and Immunization Procedures

[0050] like Figure 1 As shown, the antigenic peptide sequence (Sequence) and CD4 peptide antigen (GSFLTKGPSKLNDRA) were conjugated, and the antigenicity prediction score was calculated. High antigenicity region peptide fragments of the CD4 protein (such as GSFLTKGPSKLNDRA) were selected and conjugated with KLH (Keyhole Limpet Hemocyanin) to prepare an immunogenic antigen. The conjugated antigenic peptide was then thoroughly mixed with Bio-Long Quick Antibody™ rapid immunoadjuvant in a specific ratio to form an antigen-adjuvant mixture. This mixture was used to administer multiple injections into the thigh muscles of New Zealand white rabbits. After the initial immunization, booster immunizations were performed every two weeks for a total of three immunizations over an eight-week period. Rabbit serum was collected periodically after each immunization, and the anti-CD4 antibody titer was detected using ELISA to screen for immunized animals with high antibody titers.

[0051] Figure 2 The titer levels of the immunized rabbit serum under multiple dilution conditions were demonstrated, with a significant increase in OD452 value, indicating successful antibody production.

[0052] II. Screening and Culture of Single B Cells

[0053] After immunization, rabbits were euthanized by air injection to obtain their spleens. Splenic lymphocytes were obtained using mechanical grinding and filtration. Biotin-labeled human serum albumin (HSA) conjugated with CD4 antigen peptide was used as a probe for specific screening of single B cells by flow cytometry. The selected single B cells were distributed into 96-well plates and cultured in vitro for 7–10 days with a specific culture medium. The supernatant of each well was subjected to ELISA titer detection to screen out positive B cell clones that secrete high-affinity anti-CD4 antibodies.

[0054] like Figure 3 As shown, the antibody titers in the supernatant of positive clones were detected by ELISA. Among them, 1B9, 4D5, 4C7 and 4E8 had OD values ​​greater than 2.0, with 4D5 showing the best performance.

[0055] III. Cloning and Recombinant Expression of Antibody Genes

[0056] Total RNA was extracted from positive B cells and reverse transcribed to generate cDNA. PCR amplification was performed using specific primers targeting the variable regions (VH and VL) of rabbit antibodies to obtain heavy chain (HC) and light chain (LC) variable region gene fragments. These gene fragments were cloned into mammalian expression vectors such as pcDNA3.4. Transfected into HEK293F or CHO cells for transient or stable expression. Cell culture supernatant was collected and purified using protein A affinity chromatography to obtain a rabbit monoclonal recombinant antibody against human CD4. The binding ability, specificity, and signal intensity of the recombinant antibody were verified again using ELISA and flow cytometry.

[0057] IV. Antibody Application Validation

[0058] Peripheral blood samples were collected from healthy individuals using EDTA anticoagulant tubes and gently mixed. 100 μL of whole blood was added to flow cytometry tubes, with blank, single-color compensation, and sample groups set up. 5 μL of recombinant rabbit anti-CD4 antibody was added to each tube and incubated at 4°C in the dark for 20 min. 1–2 mL of hemolysin was added to lyse the red blood cells, and the supernatant was discarded after centrifugation. The cells were resuspended twice in PBS, and finally resuspended in PBS for cell detection. Flow cytometry analysis was performed, clearly observing the fluorescent labeling results of CD4+ T cell subsets with strong signal and low background. If immediate flow cytometry analysis was not possible, the samples could be fixed in 2% paraformaldehyde at 4°C for 30 min and stored for later use.

[0059] Figure 4 The results show that the 4D5 antibody obtained by this invention can effectively recognize the CD4+ T cell population, and its fluorescence signal is comparable to, or even clearer than, that of commercial mouse CD4 antibodies, with a lower background signal.

[0060] V. Obtaining Antibody Gene Sequences

[0061] Figure 5 The amino acid sequences of the heavy and light chain variable regions of the 4D5 antibody are provided. This invention further obtains the light and heavy chain variable region sequences of the 4D5 positive antibody clone through sequencing analysis.

[0062] 4D5 heavy chain variable region amino acid sequence:

[0063] QSVEESGGRLVTPGGSLTLTCTVSGIDLSSNSISWVRQAPGKGLEYIGYISYGGSAYYASWAKGRFTISRTSTTVDLRMTSLTTEDTATYFCARGVVGSTSGTNNLWGPGTLVTVSS

[0064] 4D5 light chain variable region amino acid sequence:

[0065] AAVLTQTPASVSAAVGGSVTINCQSSQSLYNNNQLSWYQQKPGQPPKLLIYSASTLESGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCLGDYKSDIDTFVFGGGTEVVVK

[0066] The antibody described above contains the following six complementarity-determining region (CDR) amino acid sequences:

[0067] Heavy chain CDR1 (HCDR1): SNSIS

[0068] Heavy chain CDR2 (HCDR2): YISYGGSAYYASWAKG

[0069] Heavy chain CDR3 (HCDR3): GVVGSTSGTNNL

[0070] Light chain CDR1 (LCDR1): QSSQSLYNNNQLS

[0071] Light chain CDR2 (LCDR2): SASTLES

[0072] Light chain CDR3 (LCDR3): LGDYKSDIDTFV

[0073] The antibody is obtained by isolating B cells from rabbits immunized with human CD4 antigen using monoclonal cloning technology and then recombinantly expressing them. The antibody can specifically bind to human CD4 protein and be used for the detection of CD4-positive T cells.

[0074] The above gene sequences have independent intellectual property rights and can be used for subsequent derivative applications such as antibody humanization and bispecific antibody development.

[0075] VI. ELISA Detection Method

[0076] To evaluate the titer of anti-CD4 antibodies in rabbit immune serum and culture supernatant, the following ELISA method was used:

[0077] 1. Coating antigen: Human serum albumin HAS-conjugated CD4 peptide was coated at a concentration of 2 μg / mL, 100 μL / well, onto the microplate and incubated overnight at 4°C.

[0078] 2. Cleaning: Use a plate washer to clean the ELISA plate 4-5 times;

[0079] 3. Blocking: Prepare 0.1% BSA, add 300 μL of blocking solution to each well, and block at room temperature for 2 hours;

[0080] 3. Cleaning: Use a plate washer to clean the ELISA plate 4-5 times;

[0081] 4. Add sample: Add 100 μL of sample to each well and incubate at 37℃ for 1 h;

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

[0083] 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.

[0084] 7. Cleaning: Use a plate washer to clean the ELISA plate 4-5 times;

[0085] 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;

[0086] 9. Stop color development: Add 100 μL of stop solution to each well;

[0087] 10. Reading: After turning on the microplate, read the OD452 value and save the data.

[0088] VII. Optimization of Flow Cytometry Staining Methods

[0089] To further verify the specificity and functionality of the antibody, the following optimized staining procedure was performed:

[0090] Sample processing:

[0091] 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)

[0092] Staining steps:

[0093] 1. Draw 100 μL of peripheral blood from the EDTA anticoagulant tube into the flow cytometer (as 1T).

[0094] 2. Set up blank tubes / monochromatographic 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 monochromatographic 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.

[0095] 3. Add 1-2 mL of 1*Schizolyl hemolysin / hemolysin to each tube (refer to the Schizolyl hemolysin instructions for specific timing and operation).

[0096] 4. After the red saturation is complete, centrifuge at 300g for 5 minutes at 4℃ and discard the supernatant.

[0097] 5. Add 1 mL of 1*PBS, resuspend, centrifuge at 300g for 5 min at 4℃, and discard the supernatant.

[0098] 6. Add 300-500 μL of 1*PBS to each tube to resuspend the cells.

[0099] 7. Flow cytometer was used to detect and analyze the data.

[0100] Note: If immediate analysis is not possible, stained cells can be fixed in 2% 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.

[0101] The results showed that the 4D5 rabbit anti-CD4 antibody could clearly identify the CD4+ T cell population, and the fluorescence intensity of positive cells was comparable to that of commercially available mouse antibodies, with lower background and higher signal-to-noise ratio.

[0102] VIII. Controlled Experiment and Repeatability Verification

[0103] To verify antibody stability and reproducibility, the following batch-to-batch repeatability tests were performed:

[0104] ELISA titer tests were performed on batches of 4D5 antibodies prepared at different times, and the OD values ​​were all >2.0 with a variation of less than 10%.

[0105] The flow cytometry morphology of the same batch of antibodies after staining by different operators was consistent, with a CV value of less than 5%.

[0106] The antibody maintained good recognition ability after being stored at 4°C for 3 months, without significant degradation or loss of function.

[0107] IX. Application Expansion

[0108] The obtained high-affinity rabbit-derived recombinant anti-human CD4 antibody can be used not only for routine flow cytometry but also in the following scenarios:

[0109] Immunohistochemical staining (IHC):

[0110] CD4+ cell distribution can be visualized in tissue sections by combining with horseradish peroxidase (HRP) or fluorescein-labeled secondary antibodies;

[0111] Magnetic bead sorting:

[0112] The antibody-conjugated magnetic beads of the present invention can be used for the magnetic separation of CD4+ cells, thereby improving the efficiency of cell subpopulation purification.

[0113] HIV / AIDS Auxiliary Diagnosis:

[0114] As a high-quality domestically produced CD4 antibody, it can be used for CD4 count and immune function assessment in HIV / AIDS patients;

[0115] Research on immune pathways:

[0116] When used in conjunction with TCR antibodies, it can be used for research on T cell activation, signal transduction, and subset differentiation.

[0117] Fundamentals of Antibody Humanization and Antibody Drug Development

[0118] Known gene sequences can be used to construct engineering development platforms for humanized, bispecific, or antibody-drug conjugates (ADCs).

[0119] This invention utilizes the rabbit immune system to generate high-affinity antibodies, enhancing the recognition ability of anti-CD4 antibodies; employs single B cell screening combined with recombinant expression technology to ensure the specificity, stability, and reproducibility of the antibodies; ensures a clear antibody source, avoiding animal-derived impurities and improving safety; obtains the antibody gene sequence, facilitating intellectual property protection and further modification; and can be widely applied in fields such as immune detection, flow cytometry, HIV surveillance, immunodeficiency research, and T cell subset function research.

Claims

1. A monoclonal antibody that binds to human CD4, characterized in that, The antibody contains six complementarity-determining regions (CDRs) as shown in the following amino acid sequence: Heavy chain CDR1 (HCDR1): SNSIS Heavy chain CDR2 (HCDR2): YISYGGSAYYASWAKG Heavy chain CDR3 (HCDR3): GVVGSTSGTNNL Light chain CDR1 (LCDR1): QSSQSLYNNNQLS Light chain CDR2 (LCDR2): SASTLES Light chain CDR3 (LCDR3): LGDYKSDIDTFV.

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: AAVLTQTPASVSAAVGGSVTINCQSSQSLYNNNQLSWYQQKPGQPPKLLIYSASTLESGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCLGDYKSDIDTFVFGGGTEVVVK (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: QSVEESGGRLVTPGGSLTLTCTVSGIDLSSNSISWVRQAPGKGLEYIGYISYGGSAYYASWAKGRFTISRTSTTVDLRMTSLTTEDTATYFCARGVVGSTSGTNNLWGPGTLVTVSS (SEQ ID NO: 2).

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