Fully humanized Anti-human CD4 antibody and use thereof

Abstract

Provided is a fully humanized anti-human CD4 antibody, comprising a heavy chain and a light chain, the amino acid sequence of the variable region of the heavy chain being a sequence shown in SEQ ID NO.2, and the amino acid sequence of the variable region of the light chain being a sequence as shown in SEQ ID No.4. The light chain and the heavy chain of the fully humanized anti-human CD4 antibody are all derived from humans, so that said antibody has low immunogenicity and high safety, and antibody-coupled magnetic beads formed by coupling the fully humanized anti-human CD4 antibody and magnetic beads have low immunogenicity, high safety and good sorting effect.

Description

A fully human anti-human CD4 antibody and its application Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a fully human anti-human CD4 antibody and its applications. Background Technology

[0002] CD4 T cells are important immune cells. They play several key roles in the immune system: 1. Helping the immune response: assisting other immune cells in recognizing and responding to pathogens; 2. Regulating the immune response: maintaining the balance and stability of the immune system; 3. Participating in adaptive immunity: generating long-term immune memory against specific pathogens.

[0003] Adoptive cell transfer therapy (ACT) involves collecting the patient's own immune cells (cytotoxic T cells), culturing and modifying them in vitro to enhance their targeted killing function, and then reinfusing them into the patient to eliminate tumor cells. Chimeric antigen receptor T cells (CAR-T) are a revolutionary cancer immunotherapy. CARs are artificially synthesized receptors that can redirect lymphocytes, most commonly T cells, to recognize and kill target cells expressing specific antigens. In recent years, rapidly developing CAR-T cell therapy clinical trials have actively explored its potential applications.

[0004] Meanwhile, the production of CAR-T cells involves multiple steps and requires quality control testing throughout the process. Simply put, firstly, blood is removed from the patient / healthy individual using leukocyte separation, leukocytes are separated, and the remaining blood is returned to the circulatory system. Next, the leukocytes are sorted into CD4 T cells and CD8 T cells to enrich T cells. The enriched T cells are then activated and genetically engineered to express specific CARs, further expanding the CAR-T cell count to an appropriate level. Finally, the cells are reinfused into the patient. During reinfusion, in addition to the T cells, the magnetic beads used for sorting and the antibodies conjugated to the beads are also reinfused into the body. Therefore, the development of magnetic beads with antibodies exhibiting lower immunogenicity and better safety is of great significance. However, current technology lacks a magnetic bead-conjugated fully human anti-human CD4 antibody with lower immunogenicity and better safety.

[0005] The above background information is provided only to assist in understanding the inventive concept and technical solution of this invention. It does not necessarily belong to the prior art of this application. In the absence of clear evidence that the above information was disclosed before the filing date of this application, the above background information should not be used to evaluate the novelty and inventiveness of this application. Summary of the Invention

[0006] In view of this, in order to provide antibody-conjugated magnetic beads with low immunogenicity and good safety, the present invention aims to provide a sequence of a fully human anti-human CD4 antibody, a fully human antibody, and antibody-conjugated magnetic beads.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] This invention provides a fully human anti-human CD4 antibody, comprising a heavy chain and a light chain, wherein the amino acid sequence of the variable region of the heavy chain is the sequence shown in SEQ ID NO.2, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID NO.4.

[0009] The present invention provides a nucleic acid encoding a fully human anti-human CD4 antibody as described above.

[0010] According to some preferred embodiments of the present invention, the nucleotide sequence encoding the antibody heavy chain variable region is the sequence shown in SEQ ID NO.1, and the nucleotide sequence encoding the antibody light chain variable region is the sequence shown in SEQ ID NO.3.

[0011] This invention provides a fully human anti-human CD4 antibody, comprising a heavy chain and a light chain, wherein the nucleotide sequence of the variable region of the heavy chain is the sequence shown in SEQ ID NO.1, and the nucleotide sequence of the variable region of the light chain is the sequence shown in SEQ ID NO.3.

[0012] The present invention provides a nucleic acid encoding a fully human anti-human CD4 antibody as described above.

[0013] The present invention also provides a recombinant expression vector containing the above-mentioned nucleic acid.

[0014] According to some preferred embodiments of the present invention, an expression vector for a heavy chain antibody is included, wherein the amino acid sequence corresponding to the nucleotide sequence of the expression cassette is the sequence shown in SEQ ID NO.7.

[0015] Preferably, the amino acid sequence corresponding to the nucleotide sequence of the expression vector of the heavy chain antibody is obtained by constructing the amino acid sequence of the variable region of the heavy chain of the fully human anti-human CD4 antibody shown in SEQ ID NO.2 and the amino acid sequence of human IgG1-LALA shown in SEQ ID NO.5 into the pcDNA3.4 vector.

[0016] According to some preferred embodiments of the present invention, an expression vector for a light chain antibody is included, wherein the amino acid sequence corresponding to the nucleotide sequence of the expression cassette is the sequence shown in SEQ ID NO. 8.

[0017] Preferably, the amino acid sequence corresponding to the nucleotide sequence of the expression vector is obtained by constructing the pcDNA3.4 vector with the amino acid sequence of the variable region of the light chain of the fully human anti-human CD4 antibody shown in SEQ ID NO.4 and the amino acid sequence of the human Kappa sequence in SEQ ID NO.6.

[0018] The present invention also provides a cell transformed using the above-described recombinant expression vector.

[0019] The present invention also provides the use of the antibody, nucleic acid, recombinant expression vector and cells described above in the preparation of drugs or related kits for diseases such as AIDS, rheumatoid arthritis, insulin-dependent diabetes mellitus, and systemic lupus erythematosus.

[0020] The present invention also provides a pharmaceutical composition comprising the antibody described above, and / or the nucleic acid described above, and / or the recombinant expression vector described above, in combination with one or more pharmaceutically acceptable excipients.

[0021] The present invention also provides a magnetic bead coupled with the fully human anti-human CD4 antibody as described above, and the use of the coupled magnetic bead in CD4T cell sorting.

[0022] A method for preparing magnetic beads as described above includes the following steps:

[0023] FeCl3·6H2O and FeCl2·4H2O were dissolved in deionized water, NH4OH was added, magnetic separation was performed, and the mixture was washed with deionized water and dried to obtain nano-magnetic beads.

[0024] A carboxyglucan aqueous solution and magnetic nanobeads were mixed and heated to react. The magnetic beads were then screened using magnetic separation to obtain carboxyglucan-coated magnetic beads.

[0025] Using buffer as the washing solution, the carboxydextran-coated magnetic beads were washed with magnetic separation, and EDC and NH4S were added to activate the carboxyl groups. The supernatant was removed by magnetic separation, and the beads were resuspended with buffer. The fully human anti-human CD4 antibody as described above was added, and the reaction was incubated. The beads were then separated with magnetic separation, blocked with blocking solution, washed with preservation solution, and stored in preservation solution.

[0026] A method for sorting CD4T cells includes the following steps:

[0027] Peripheral blood mononuclear cells were extracted, resuspended, and magnetic beads as described above were added. The mixture was mixed, incubated in the dark, and the cells were washed with buffer solution. After centrifugation, the supernatant was discarded, and the cells were resuspended with buffer solution to obtain a cell suspension.

[0028] Place the sorting column on a magnetic rack and rinse it. Add the cell suspension to the sorting column placed in the magnetic field. The cells flowing out of the sorting column at this time are non-target cells. Add PBS to wash away the remaining cells. When the droplets at the bottom of the sorting column stop dripping, add the washing solution to the sorting column and repeat. At the same time, collect the non-target cells flowing out. When all the liquid has flowed out, remove the sorting column from the magnetic rack and place it in a new collection tube. Add PBS to the sorting column, insert the stopcock into the sorting column and push it to the bottom of the sorting column. The liquid collected at this time contains the target cells.

[0029] The proportion of CD4T cells was detected by flow cytometry of the collected target cells and non-target cells.

[0030] The present invention provides an amino acid sequence of a fully human anti-human CD4 antibody that specifically binds to human CD4, comprising an amino acid sequence of a variable region of a heavy chain and an amino acid sequence of a variable region of a light chain; the amino acid sequence of the variable region of the heavy chain comprises the sequence shown in SEQ ID NO.2; the amino acid sequence of the variable region of the light chain comprises the sequence shown in SEQ ID NO.4.

[0031] The present invention provides a nucleotide sequence of a fully human anti-human CD4 antibody that specifically binds to human CD4, comprising a nucleotide sequence encoding a variable region of the heavy chain and a nucleotide sequence encoding a variable region of the light chain; the nucleotide sequence encoding the variable region of the heavy chain comprises the sequence shown in SEQ ID NO.1, and the nucleotide sequence encoding the variable region of the light chain comprises the sequence shown in SEQ ID NO.3.

[0032] Due to the adoption of the above technical solutions, the advantages of the present invention compared with the prior art are as follows: the fully human anti-human CD4 antibody of the present invention has both light and heavy chains derived from humans, thus having lower immunogenicity and better safety; at the same time, the antibody-conjugated magnetic beads formed by the fully human anti-human CD4 antibody and magnetic beads in this application have low immunogenicity, good safety and good sorting effect. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 is a schematic diagram of the results of flow cytometry identification of CD4 monoclonal antibodies in Example 1 of the present invention;

[0035] Figure 2 is an SDS-PAGE non-reducing gel electrophoresis image of the fully human CD4 antibody in Example 2 of the present invention.

[0036] Figure 3 shows the affinity curve of the fully human anti-human CD4 antibody and human CD4 in Example 2 of the present invention.

[0037] Figure 4 shows the FACS identification results of the fully human anti-human CD4 antibody binding analysis in Example 2 of the present invention: negative control;

[0038] Figure 5 shows the FACS identification results of the fully human anti-human CD4 antibody binding analysis in Example 2 of the present invention: positive control;

[0039] Figure 6 shows the FACS identification results of the fully human anti-human CD4 antibody binding analysis in Example 2 of the present invention: CD4 antibody (sample to be tested).

[0040] Figure 7 shows the FACS identification results of the fully human anti-human CD4 antibody after conjugation with magnetic beads in Example 3 of the present invention: before sorting;

[0041] Figure 8 shows the FACS identification results of the fully human anti-human CD4 antibody after conjugation with magnetic beads in Example 3 of the present invention: after sorting;

[0042] Figure 9 shows the FACS evaluation results of existing commercially available products: after sorting. Detailed Implementation

[0043] To enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0044] Mouse monoclonal antibodies are commonly used in disease diagnosis and treatment. However, in actual clinical applications, heterologous monoclonal antibodies can induce human anti-mouse antibody (HAMA) reactions due to their high immunogenicity, leading to a short half-life of the mouse monoclonal antibody drug, poorer therapeutic effect, and increased risk of safety issues. To overcome this difficulty, this application uses yeast display technology to screen a human natural antibody library, prepares and obtains fully human antibodies, whose amino acid sequences are 100% derived from humans, thus exhibiting advantages such as lower immunogenicity and better safety.

[0045] Example 1: Screening of fully human anti-human CD4 antibodies

[0046] 1.1 First-round magnetic bead enrichment in the library

[0047] (1) Take 100 OD of yeast library strain (human antibody scfv yeast display library) from the -80℃ freezer and add it to 100 mL of SD-Trp deficient medium. Incubate at 30℃ and 220 rpm for 3-4 h.

[0048] (2) After culturing for 3-4 hours, the absorbance at OD600 was measured. The defective culture medium was removed by centrifugation, and the cells were collected. The cells were added to 200 mL of SG-CAA medium and cultured at 20 °C and 220 rpm for 24 hours. Yeast cells with an OD of 500 were collected by centrifugation.

[0049] (3) The yeast cells were resuspended in 40 mL PBS + 1% BSA.

[0050] (4) Centrifuge and discard the supernatant. Resuspend the yeast in 5 mL PBS + 1% BSA, add 100 nM CD4-biotin, and incubate at room temperature with shaking for 1 h.

[0051] (5) After incubating for 1 hour, centrifuge at 3000 rpm for 3 minutes and discard the supernatant.

[0052] (6) Add 40 mL PBS + 1% BSA and centrifuge at 3000 rpm for 3 min.

[0053] (7) Repeat step (6) twice.

[0054] (8) Add 5 mL PBS + 1% BSA to resuspend the cells, then add 40 μL SA-beads and mix thoroughly.

[0055] (9) Incubate for 20 minutes.

[0056] (10) Place the magnetic column on the magnetic rack, pass the incubated cells through the column, and then wash the column three times with 5 mL PBS + 1% BSA.

[0057] (11) Remove the magnetic column from the magnetic rack and use 5 mL of defective culture medium for cells.

[0058] (12) The eluted cells were cultured overnight at 30°C and 220 rpm.

[0059] (13) After overnight incubation, the medium was replaced with SG-CAA medium and cultured at 20°C and 220 rpm for 24 h before flow cytometry analysis was performed.

[0060] 1.2 Second Round FACS Enrichment of the Library

[0061] (1) Take 10 OD of bacterial culture into a 50 mL centrifuge tube, centrifuge and discard the supernatant, wash 3 times with 1 mL PBSA (3000 rpm, 3 min), and then reselect the bacterial culture with 200 μL PBSA.

[0062] (2) Dilute the anti-FLAG antibody solution with PBSA at a ratio of 1:1000, and prepare a 50 nm CD4-Biotin solution using the anti-FLAG antibody solution.

[0063] (3) Add 1 mL of 50 nm CD4-Biotin solution, then add 20 μL of the bacterial solution treated in step (1), and incubate at room temperature for 1 h.

[0064] (4) After incubation, centrifuge at 3000 rpm for 3 min and discard the supernatant. Wash the unbound CD4 with 40 mL PBSA, mix by blowing and aspiration, and wash 3 times at 3000 rpm for 1 min.

[0065] (5) Prepare a mixture of SA-PE and goat anti-human-647 with PBSA at a ratio of 1:1000, add 1 mL to the wells of the plate after (4) treatment, and incubate at room temperature for 1 h.

[0066] (6) After incubation, centrifuge at 3000 rpm for 3 min and discard the supernatant. Wash the unbound fluorescein with PBSA, 200 μL / well, mix by blowing and aspiration, and wash 3 times at 3000 rpm for 3 min.

[0067] The bacterial culture was resuspended in 4 mL of PBSA and sorted by flow cytometry. Cell populations that were positive for both PE and 647 were sorted by flow cytometry.

[0068] 1.3 Cloning FACS Identification

[0069] (1) The enriched yeast cells were spread on SD-Trp plates and cultured in a 30°C incubator until single clones grew.

[0070] (2) Select clones and inoculate them in SD-Trp medium and culture overnight at 30°C with shaking.

[0071] (3) The bacterial cells cultured overnight were transferred to SG-CAA medium and cultured at 20°C for 48 hours.

[0072] (4) Perform FACS testing according to step 1.2 of Example 1.

[0073] (5) The results of flow cytometry identification of CD4 monoclonal clones are shown in Figure 1. The left figure is the negative control, and the right figure is the CD4 yeast monoclonal clones that are positive for both PE and 647 after screening.

[0074] 1.4 Yeast Cloning and Sequencing

[0075] (1) Yeast cloning: Plasmids were extracted using a yeast plasmid extraction kit.

[0076] (2) Plasmid transformation of Top10 Escherichia coli competent cells.

[0077] (3) Select clones for sequencing to obtain fully human anti-human CD4 antibody sequences. The nucleotide sequence of the heavy chain variable region of the fully human anti-human CD4 antibody is shown in SEQ ID NO.1; the amino acid sequence of the heavy chain variable region of the fully human anti-human CD4 antibody is shown in SEQ ID NO.2; the nucleotide sequence of the light chain variable region of the fully human anti-human CD4 antibody is shown in SEQ ID NO.3; and the amino acid sequence of the light chain variable region of the fully human anti-human CD4 antibody is shown in SEQ ID NO.4.

[0078] Example 2: Expression and Validation of Fully Human Anti-Human CD4 Antibody

[0079] 2.1 Expression and purification of fully human anti-human CD4 antibody

[0080] (1) The amino acid sequence of the variable region of the heavy chain of the fully human anti-human CD4 antibody shown in SEQ ID NO.2 and the amino acid sequence of human IgG1-LALA shown in SEQ ID NO.5 were constructed into the pcDNA3.4 vector to obtain the amino acid sequence shown in SEQ ID NO.7. The amino acid sequence was transformed to obtain the nucleotide sequence of the expression cassette corresponding to the expression vector of the heavy chain antibody.

[0081] (2) The amino acid sequence of the light chain variable region of the fully human anti-human CD4 antibody shown in SEQ ID NO.4 and the human Kappa amino acid sequence shown in SEQ ID NO.6 were constructed into the pcDNA3.4 vector to obtain the amino acid sequence shown in SEQ ID NO.8. The amino acid sequence was transformed to obtain the nucleotide sequence of the expression cassette corresponding to the expression vector of the light chain antibody.

[0082] (3) The light chain plasmid and heavy chain plasmid were transformed into 293 cells at a ratio of 1:1 and then the antibody was purified.

[0083] 2.2 SDS-PAGE of fully human anti-human CD4 antibody

[0084] (1) Add 4 μL of loading buffer to 16 μL of sample and denature at 100 °C for 5-10 min.

[0085] (2) Load 10 μL of sample, run SDS-PAGE electrophoresis at 150V for 45 min.

[0086] (3) After electrophoresis, turn off the power, take out the gel, put it into a large petri dish for staining, use rapid staining solution, room temperature, 10 min, discard the staining solution, and decolorize with distilled water overnight.

[0087] (4) The purified fully human CD4 antibody was obtained, and the results are shown in Figure 2, indicating the molecular weight of the fully human anti-human CD4 antibody. Band M is the marker; band 1 is the purified fully human anti-human CD4 antibody. In Figure 2, the markers from top to bottom are: 270kDa, 175kDa, 130kDa, 95kDa, 65kDa, 50kDa, 35kDa, 30kDa, 15kDa, and 5kDa.

[0088] 2.3 Fully Human Anti-Human CD4 Antibody Binding Analysis - ELISA

[0089] The binding correlation with CD4 was detected by dilution method.

[0090] (1) Coating: Dilute the antigen protein with coating solution to 1 μg / mL, 100 μL / well, and incubate at 37℃ for 1 h;

[0091] (2) Washing: Wash three times with 200 μL / well of PBS containing 0.05% Tween 20, and shake off the liquid in the plate;

[0092] (3) Blocking: Use 200 μL / well, 2.5% BSA blocking solution, and block at 37℃ for 1 h;

[0093] (4) Washing: Wash three times with 200 μL / well of PBS containing 0.05% Tween 20, and shake off the liquid in the plate;

[0094] (5) The purified antibody produced by 293 cells was diluted in a gradient using a dilution method, and 100 μL of each gradient was added to the wells coated with the antigen. The negative control was protein extracted from 293 cells without the transformed plasmid and incubated at 37°C for 1 h.

[0095] (6) Washing: Wash three times with 200 μL / well of PBS containing 0.05% Tween 20, and shake off the liquid in the plate;

[0096] (7) Add anti-Human-Fc(HRP) at 100 μL / well to the wells of the previously incubated samples and incubate at 37°C for 1 h;

[0097] (8) Washing: Wash three times with 200 μL / well of PBS containing 0.05% Tween 20, and shake off the liquid in the plate;

[0098] (9) Color development: Add 100 μL of TMB to each well and incubate at 37°C for 10 min in the dark.

[0099] (10) Reading: Add 50 μL of 1M HCl to each well to terminate the colorimetric reaction and read the photometric value at OD450nm.

[0100] (11) Data analysis (CD4 antibody affinity curve) As shown in Figure 3, the fully human anti-human CD4 antibody binds strongly to human CD4 and has high affinity.

[0101] 2.4 Fully Human Anti-Human CD4 Antibody Binding Analysis - FACS

[0102] (1) Take 2.00E+5 Sup-T1 cells per well into a 96-well plate, centrifuge at 1000xg for 5 min, remove the supernatant, and wash three times with 1xPBS;

[0103] (2) Centrifuge the supernatant at 5000xg for 10-20 min and retain the supernatant;

[0104] (3) Take 100 μL of transiently transformed supernatant to resuspend Sup-T1 cells as the test sample, take 100 μL of commercial CD4 antibody (2 μg / mL) to resuspend Sup-T1 cells as the positive control, and take 100 μL of protein extracted from 293 cells without transformation plasmid to resuspend Sup-T1 cells as the negative control. Incubate at room temperature for 1 h at 400 rpm.

[0105] (4) After incubation, centrifuge 1000xg of sample for 3min, discard the supernatant, and wash three times with 0.01% PBSA;

[0106] (5) Add 100 μL / well of goat anti-human-647 (1:1000 dilution) and incubate at room temperature for 1 h at 400 rpm;

[0107] (6) After incubation, centrifuge 1000xg of sample for 3min, discard the supernatant, and wash three times with 0.01% PBSA;

[0108] (7) Finally, resuspend in 300 μL of 0.01% PBSA and perform flow cytometry identification.

[0109] (8) Data analysis (CD4 antibody binding analysis - FACS identification results) are shown in Figure 4: negative control, Figure 5: positive control, and Figure 6: sample to be tested. This shows that the fully human anti-human CD4 antibody has a strong binding to human CD4.

[0110] Example 3: Verification of the conjugation and sorting effect of fully human anti-human CD4 antibody with magnetic beads.

[0111] 3.1 Preparation of magnetic beads

[0112] (1) 2.2 g FeCl3·6H2O and 0.8 g FeCl2·4H2O were dissolved in 40 mL of deionized water in a 2:1 ratio, and the solutions were sealed in a three-necked flask under vigorous stirring and an inert nitrogen atmosphere. 5 mL of NH4OH was added dropwise to the reaction vessel, and the reaction was carried out at 85 °C for 1 hour. After magnetic separation, the solutions were washed three times with deionized water and dried to obtain magnetic beads.

[0113] (2) Coating magnetic beads with carboxyglucan: Carboxyglucan aqueous solution and magnetic beads were added to a flask at a mass ratio of 1:5 or 1:10, stirred and heated at 50°C for 4 hours, and magnetic beads coated with carboxyglucan were obtained by magnetic separation and screening.

[0114] (3) Conjugation of fully human anti-human CD4 antibody: The carboxydextrose-coated magnetic beads were magnetically washed using 10 mM pH 5.0 MES buffer as the washing solution. 10 μL of EDC (10 mg / mL) and 20 μL of NHS (10 mg / mL) were added, and the carboxyl groups were activated at 37°C for 0.5 h. The supernatant was removed by magnetic separation, and the mixture was resuspended in 25 mM pH 5.0 MES buffer to 1 mL. 60 μg of fully human anti-human CD4 antibody was added, and the mixture was incubated at 37°C in a rotary incubator for 6 h. After magnetic separation, blocking buffer (PBS solution containing 2% BSA) was added, and the mixture was washed with preservation buffer (PBS solution containing 1% Tween 20) and stored in the preservation buffer.

[0115] 3.2 Verification of sorting effect

[0116] (1) Take 30 mL of peripheral blood and extract PBMCs using Ficoll, then count the cells. Take 1.00E+07 PBMCs, add 90 μL of PBS per tube to resuspend the cells, add magnetic beads and mix well, then incubate at 4°C in the dark for 20 min. Add 1 mL of buffer to wash the cells, centrifuge at 300×g for 5 min, discard the supernatant and resuspend in 500 μL of buffer.

[0117] (2) Place the Miltenyi MS sorting column on a magnetic rack and rinse the column with 1 mL of PBS. Add the cell suspension to the sorting column placed in the magnetic field. At this point, the cells flowing out of the sorting column are non-target cells. Add 750 μL of PBS to wash away the remaining cells. When the droplets at the bottom of the sorting column stop dripping, add the washing solution to the sorting column. Repeat twice, collecting the non-target cells flowing out at the same time. When all the liquid has flowed out, remove the sorting column from the magnetic rack and place it in a new 1.5 mL EP tube. Add 0.5 mL of PBS to the sorting column, quickly insert the stopcock into the column, and push it firmly to the bottom of the sorting column. At this point, the collected liquid contains the target cells.

[0118] (3) The collected target cells and non-target cells were analyzed by flow cytometry to determine the proportion of CD4+ cells. The sorting results are shown in Figure 7: before sorting, Figure 8: after sorting, and Figure 9: after sorting (existing commercially available products). The results show that the fully human anti-human CD4 antibody and the self-made magnetic beads conjugated in this application have better sorting performance.

[0119] Fully human antibodies, with both their light and heavy chains derived from humans, exhibit the lowest rejection rates and the best safety profile, representing a significant trend in therapeutic antibody development. This application utilizes yeast display to obtain a fully human antibody with even lower immunogenicity and improved safety. This antibody is obtained by screening a yeast display library using human CD4 antigen, resulting in a shorter development cycle compared to hybridoma methods. Furthermore, being derived from a human antibody library, the obtained antibody sequence is fully human, offering advantages such as lower immunogenicity and better safety. Additionally, coupling the fully human anti-human CD4 antibody with magnetic beads yields excellent sorting performance. As a component of immunoassay reagents, it can be applied in immunoassay and immunodiagnosis, playing a crucial role in the diagnosis and treatment of CD4-related diseases.

[0120] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

[0121] Table of sequence numbers and specific sequence correspondences for this invention:

Claims

1. A fully human anti-human CD4 antibody, characterized in that, It includes a heavy chain and a light chain, wherein the amino acid sequence of the variable region of the heavy chain includes the sequence shown in SEQ ID NO.2, and the amino acid sequence of the variable region of the light chain includes the sequence shown in SEQ ID NO.

4.

2. A nucleic acid encoding the fully human anti-human CD4 antibody as described in claim 1.

3. The nucleic acid according to claim 2, characterized in that, The nucleotide sequence encoding the antibody heavy chain variable region includes the sequence shown in SEQ ID NO.1, and the nucleotide sequence encoding the antibody light chain variable region includes the sequence shown in SEQ ID NO.

3.

4. A fully human anti-human CD4 antibody, characterized in that, It includes a heavy chain and a light chain, wherein the nucleotide sequence of the variable region of the heavy chain is the sequence shown in SEQ ID NO.1, and the nucleotide sequence of the variable region of the light chain is the sequence shown in SEQ ID NO.

3.

5. A nucleic acid encoding the fully human anti-human CD4 antibody as described in claim 4.

6. A recombinant expression vector comprising the nucleic acid according to claim 2, claim 3 or claim 5.

7. The recombinant expression vector according to claim 6, characterized in that, The expression vector includes a heavy chain antibody, wherein the amino acid sequence corresponding to the nucleotide sequence of the expression cassette is the sequence shown in SEQ ID NO.

7.

8. The recombinant expression vector according to claim 7, characterized in that, The amino acid sequence corresponding to the nucleotide sequence of the expression vector of the heavy chain antibody is obtained by constructing the amino acid sequence of the variable region of the heavy chain of the fully human anti-human CD4 antibody shown in SEQ ID NO.2 and the amino acid sequence of human IgG1-LALA shown in SEQ ID NO.5 into the pcDNA3.4 vector.

9. The recombinant expression vector according to claim 8, characterized in that, The expression vector includes a light chain antibody, wherein the amino acid sequence corresponding to the nucleotide sequence of the expression cassette is the sequence shown in SEQ ID NO.

8.

10. The recombinant expression vector according to claim 9, characterized in that, The amino acid sequence corresponding to the nucleotide sequence of the expression vector of the light chain antibody is obtained by constructing the amino acid sequence of the variable region of the light chain of the fully human anti-human CD4 antibody shown in SEQ ID NO.4 and the amino acid sequence of human Kappa in SEQ ID NO.6 into the pcDNA3.4 vector.

11. A cell transformed with the recombinant expression vector according to any one of claims 6-10.

12. The use of the antibody of claim 1 or 4, the nucleic acid of claim 2, 3 or 5, the recombinant expression vector of any one of claims 6-10, and the cell of claim 11 in the preparation of a medicament or kit for a related disease.

13. A pharmaceutical composition comprising the antibody of claim 1 or 4, and / or the nucleic acid of claim 2, 3 or 5, and / or the recombinant expression vector of any one of claims 6-10, in combination with one or more pharmaceutically acceptable excipients.

14. A magnetic bead, characterized in that, The magnetic beads are coupled with the fully human anti-human CD4 antibody as described in claim 1 or 4.

15. A method for preparing magnetic beads as described in claim 14, characterized in that, Includes the following steps: FeCl3·6H2O and FeCl2·4H2O were dissolved in deionized water, NH4OH was added, magnetic separation was performed, and the mixture was washed with deionized water and dried to obtain nano-magnetic beads. A carboxyglucan aqueous solution and magnetic nanobeads were mixed and heated to react. The magnetic beads were then screened using magnetic separation to obtain carboxyglucan-coated magnetic beads. Using buffer as the washing solution, the carboxy-dextran-coated magnetic beads were washed with magnetic separation, and EDC and NHS were added to activate the carboxyl groups; the supernatant was removed by magnetic separation, and the beads were resuspended in buffer solution. Add the fully human anti-human CD4 antibody as described in claim 1 or 4, and culture the reaction; use magnetic separation, add blocking solution, wash with preservation solution, and store in preservation solution.

16. The application of the magnetic beads as described in claim 14 in sorting CD4 T cells.

17. A method for sorting CD4 T cells, characterized in that, Includes the following steps: Peripheral blood mononuclear cells were extracted, resuspended, and magnetic beads as described in claim 14 were added. The mixture was stirred, incubated in the dark, and the cells were washed with buffer solution. After centrifugation, the supernatant was discarded, and the cells were resuspended with buffer solution to obtain a cell suspension. Place the sorting column on a magnetic rack and rinse it. Add the cell suspension to the sorting column placed in the magnetic field. The cells flowing out of the sorting column at this time are non-target cells. Add PBS to wash away the remaining cells. When the droplets at the bottom of the sorting column stop dripping, add the washing solution to the sorting column and repeat. At the same time, collect the non-target cells flowing out. When all the liquid has flowed out, remove the sorting column from the magnetic rack and place it in a new collection tube. Add PBS to the sorting column, insert the stopcock into the sorting column and push it to the bottom of the sorting column. The liquid collected at this time contains the target cells. The proportion of CD4 T cells was detected by flow cytometry of the collected target cells and non-target cells.

18. The amino acid sequence of a fully human anti-human CD4 antibody that specifically binds to human CD4, characterized in that, The amino acid sequence includes the variable region of the heavy chain and the variable region of the light chain; the amino acid sequence of the variable region of the heavy chain includes the sequence shown in SEQ ID NO.2; the amino acid sequence of the variable region of the light chain includes the sequence shown in SEQ ID NO.

4.

19. A nucleotide sequence of a fully human anti-human CD4 antibody that specifically binds to human CD4, characterized in that, It includes nucleotide sequences encoding a variable region of the heavy chain and nucleotide sequences encoding a variable region of the light chain; the nucleotide sequence encoding the variable region of the heavy chain includes the sequence shown in SEQ ID NO.1, and the nucleotide sequence encoding the variable region of the light chain includes the sequence shown in SEQ ID NO.3.

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