Development of anti-human CD3 mouse and rabbit monoclonal antibodies and establishment of stable cell lines

Abstract

This invention provides an anti-human CD3 antibody, the amino acid sequence of which is selected from SEQ ID NO. 1-5 in the heavy chain CDR region and from SEQ ID NO. 6-9 in the light chain CDR region. This invention belongs to the field of antibodies. It provides the protein sequences of anti-human CD3 mouse and rabbit monoclonal antibodies. A stable CHO cell line was established for recombinant expression of this gene. The purified antibody specifically recognizes the human CD3 antigen, and the antibody yield is significantly higher than that produced by traditional hybridoma cell preparation. Furthermore, by replacing mouse IgG1 with rabbit IgG while retaining the variable region, a stable CHO cell line was established for recombinant expression of the rabbit monoclonal antibody. The purified antibody maintained its specific recognition of the human CD3 antigen.

Description

Technical Field

[0001] This invention belongs to the field of antibodies, specifically relating to the determination of protein sequences of anti-human CD3 mouse monoclonal antibody and rabbit monoclonal antibody and the establishment of stable cell lines. Background Technology

[0002] CD (Cluster of Differentiation): Also known as leukocyte differentiation antigens, these are cell surface markers that appear or disappear at different stages of normal differentiation and maturation, as well as during activation, in leukocytes of different lineages. They are a class of proteins or glycoproteins on the cell membrane. Physiologically, CD molecules have many uses, commonly serving as important receptors or ligands for cells. They not only act as surface markers for cell identification and isolation but also participate extensively in cell growth, maturation, differentiation, development, migration, and activation.

[0003] Under normal circumstances, different lymphocyte populations work synergistically to maintain the body's normal physiological immune function. When different subsets become abnormal in number or function, the body may experience immune dysregulation, leading to a series of pathological phenomena. These include autoimmune diseases (such as rheumatic diseases), immunodeficiency diseases (such as recurrent lung and intestinal infections), allergic diseases (such as urticaria and allergies), aplastic anemia, viral infections, and malignant tumors. Detection using anti-CD molecules can monitor these abnormalities, enabling timely and accurate diagnosis and treatment.

[0004] CD3 is a protein complex composed of five peptide chains (CD3γ, CD3δ, CD3ε, CD3ζ, and η chains), all of which are transmembrane proteins and are typically expressed on the surface of all mature T cells. Its primary function is as a co-receptor for T cells, promoting the activation of cytotoxic T cells (CD8+ T cells) and helper T cells (CD4+ T cells), and ultimately contributing to the formation of an adaptive immune response.

[0005] CD3 is the best marker for detecting mature T cells in peripheral blood. In flow cytometry, human CD3 is commonly used as a lineage-specific marker to distinguish T cells from other lymphocytes. The specificity of CD3 for T cells and its ability to bind at all stages of T cell development make it an ideal marker for detecting healthy T cells and T-cell cancers (such as leukemia and lymphoma). In immunohistochemical studies, CD3 is an immunohistochemical marker for T cells in tissue sections. Furthermore, CD3 monoclonal antibodies are widely used in immunosuppressive therapy during organ transplantation and bone marrow transplantation, as well as in immunomodulatory therapies aimed at eliminating T cells in severe autoimmune diseases. [1]The most famous of these is oxizumab, which was developed for the treatment of type 1 diabetes and autoimmune diseases.

[0006] CD3 monoclonal antibodies, as immunosuppressants, have advantages over conventional immunosuppressive drugs such as steroid hormones, including fewer side effects, lower dosages, and longer duration of action. Therefore, they have been widely used in clinical practice, especially in organ transplantation. Currently, the role of anti-CD3 monoclonal antibodies in the treatment of autoimmune diseases is receiving increasing attention, and preliminary progress has been made. [2] The uniqueness and numerous advantages of anti-CD3 monoclonal antibodies will make them a better means and method for treating organ transplants and autoimmune diseases.

[0007] When preparing antibodies using hybridomas, the instability of the hybridoma leads to the easy loss of antibody genes. Furthermore, hybridoma preparation technology does not identify the gene and protein sequences of the target antibody, hindering subsequent production, preparation, and research. Genetic engineering methods can effectively solve these problems. A necessary condition for antibody preparation via genetic engineering is the accurate determination of the antibody sequence. Therefore, identifying antibodies more suitable for CD3 detection is a more appropriate research direction at present.

[0008] [1] Bommireddy R, Doetschman T. TGF-β, T-ceII toIerance and anti-CD3therap[y J]. Trends Mol Med, 2004, 1(0 1):3-9.

[0009] [2] HeroId KC, Hagopian W, Auger JA, et al. Anti-CD3 monocIonaIantibody in new-onset type 1 diabetes meIIitu[s J]. N Engl Med, 2002, 34(622): 1692-1698. Summary of the Invention

[0010] To address the aforementioned issues, this invention discovered four novel mouse and rabbit monoclonal antibodies against human CD3. By designing gene and protein sequences, stable CHO cell lines expressing these genes were established for recombinant expression. Verification showed that the expressed and purified antibodies specifically recognize the human CD3 antigen, with antibody yields significantly higher than those produced using traditional hybridoma cells. Furthermore, by replacing mouse IgG1 with rabbit IgG while retaining the variable region, stable CHO cell lines expressing rabbit monoclonal antibodies were established for recombinant expression. Verification showed that the expressed and purified antibodies maintained their specific recognition of the human CD3 antigen.

[0011] the term:

[0012] In this invention, CDRs (complementarity-determining regions) are called complementarity-determining regions or complementarity-determining clusters. They are located in the hypervariable region of immunoglobulins, which are antigen-binding sites of antibodies and are structurally complementary to the antigenic determinants. They generally include CDR1, CDR2, and CDR3.

[0013] In this invention, the variable region refers to the region of the immunoglobulin light chain and heavy chain near the N-terminus where the amino acid sequence changes significantly.

[0014] In this invention, the heavy chain refers to two longer, relatively larger identical heavy chains (H chains) in the antibody; the light chain refers to two shorter, relatively smaller identical light chains (L chains) in the antibody.

[0015] In this invention, similarity refers to the consistency of the amino acid sequence of homologous proteins and the proportion of substituted amino acids.

[0016] In this invention, monoclonal antibody refers to a highly homogeneous antibody produced by a single B cell clone that targets only a specific antigenic epitope.

[0017] In this invention, murine antibodies refer to antibodies secreted by murine hybrid fusion cells obtained by fusing B cells from immunized mice with myeloma cells.

[0018] On the one hand, the present invention provides an anti-human CD3 antibody.

[0019] The anti-human CD3 antibody comprises a heavy chain and a light chain, wherein the heavy chain includes a heavy chain variable region, and the light chain includes a light chain variable region; the heavy chain variable region includes HCDR1, HCDR2, and HCDR3, and the light chain variable region includes LCDR1, LCDR2, and LCDR3, wherein:

[0020] (1) HCDR1 is the amino acid sequence shown in SEQ ID NO.1;

[0021] (2) HCDR2 is the amino acid sequence shown in SEQ ID NO.2 or the amino acid sequence shown in SEQ ID NO.3;

[0022] (3) HCDR3 is the amino acid sequence shown in SEQ ID NO.4 or the amino acid sequence shown in SEQ ID NO.5;

[0023] (4) LCDR1 is the amino acid sequence shown in SEQ ID NO.6;

[0024] (5) LCDR2 is the amino acid sequence shown in SEQ ID NO.7;

[0025] (6) LCDR3 is the amino acid sequence shown in SEQ ID NO.8 or the amino acid sequence shown in SEQ ID NO.9.

[0026] Specifically, the amino acid sequence of the variable region of the heavy chain of the anti-human CD3 antibody is SEQ ID NO.10 or a sequence having more than 80% sequence similarity to SEQ ID NO.10; or the amino acid sequence of the variable region of the heavy chain of the anti-human CD3 antibody is SEQ ID NO.11 or a sequence having more than 80% sequence similarity to SEQ ID NO.11; the amino acid sequence of the variable region of the light chain of the anti-human CD3 antibody is selected from SEQ ID NO.12 or a sequence having more than 80% sequence similarity to SEQ ID NO.12; or the amino acid sequence of the variable region of the light chain of the anti-human CD3 antibody is selected from SEQ ID NO.13 or a sequence having more than 80% sequence similarity to SEQ ID NO.13.

[0027] Preferably, the amino acid sequence of the heavy chain variable region of the anti-human CD3 antibody is SEQ ID NO.10 or SEQ ID NO.11; and the amino acid sequence of the light chain variable region is SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, or SEQ ID NO.15.

[0028] Specifically, the heavy chain amino acid sequence of the anti-human CD3 antibody is SEQ ID NO.16, or a sequence having more than 65% sequence similarity to SEQ ID NO.16; or the heavy chain amino acid sequence of the anti-human CD3 antibody is SEQ ID NO.17, or a sequence having more than 65% sequence similarity to SEQ ID NO.17; or the heavy chain amino acid sequence of the anti-human CD3 antibody is SEQ ID NO.18, or a sequence having more than 65% sequence similarity to SEQ ID NO.18; or the heavy chain amino acid sequence of the anti-human CD3 antibody is SEQ ID NO.19, or a sequence having more than 65% sequence similarity to SEQ ID NO.19; the light chain amino acid sequence of the anti-human CD3 antibody is SEQ ID NO.20, or a sequence having more than 65% sequence similarity to SEQ ID NO.20; or the light chain amino acid sequence of the anti-human CD3 antibody is SEQ ID NO.21, or a sequence having more than 65% sequence similarity to SEQ ID NO.21; or the light chain amino acid sequence of the anti-human CD3 antibody is SEQ ID NO.22, or a sequence having more than 65% sequence similarity to SEQ ID NO.16. NO.22 is a sequence with more than 65% sequence similarity; or the light chain amino acid sequence of the anti-human CD3 antibody is SEQ ID NO.23, or a sequence with more than 65% sequence similarity to SEQ ID NO.23.

[0029] Preferably, the heavy chain amino acid sequence of the anti-human CD3 antibody is selected from SEQ ID NO.16-19, and the light chain amino acid sequence is selected from SEQ ID NO.20-23.

[0030] Specifically, the anti-human CD3 antibody is a monoclonal antibody.

[0031] Preferably, the anti-human CD3 antibody is a murine-derived antibody.

[0032] On the other hand, the present invention provides a nucleotide sequence expressing the aforementioned anti-human CD3 antibody.

[0033] The nucleotide sequence can be a sequence optimized by codon degeneracy, and can be used to encode the aforementioned anti-CD3 antibody.

[0034] Preferably, the nucleotide sequences corresponding to the heavy chain SEQ ID NO.16-19 are shown in SEQ ID NO.24-27, and the nucleotide sequences corresponding to the light chain SEQ ID NO.20-23 are shown in SEQ ID NO.28-31.

[0035] In another aspect, the present invention provides an expression vector comprising the aforementioned nucleotide sequence.

[0036] The expression vector can be a plasmid, bacteriophage, or virus.

[0037] In another aspect, the present invention provides cells expressing the aforementioned anti-human CD3 antibody or nucleotide sequence or expression vector.

[0038] The cells described above can efficiently express anti-human CD3 antibodies.

[0039] Specifically, the cells may be HEK293 or CHO.

[0040] In another aspect, the present invention provides a method for preparing the aforementioned anti-human CD3 antibody, wherein the method includes culturing the aforementioned cells.

[0041] The method may also include an antibody purification step.

[0042] The antibody purification steps can be precipitation, broad-spectrum affinity purification, antigen-specific purification, or ion exchange.

[0043] In another aspect, the present invention provides a method for fluorescently labeling the aforementioned anti-human CD3 antibody, wherein the method includes labeling the anti-human CD3 antibody using macromolecular dyes or small molecule dyes.

[0044] Preferably, the macromolecular dye is APC, and the small molecule dye is FITC.

[0045] In another aspect, the present invention provides the application of the aforementioned anti-human CD3 antibody, nucleotide sequence, expression vector, or cell in CD3 detection, wherein the application is not for disease diagnosis or treatment.

[0046] The application described is achieved through antibody-antigen binding.

[0047] The application could be the detection of CD3 in T lymphocytes.

[0048] In another aspect, the present invention provides the application of the aforementioned anti-human CD3 antibody, nucleotide sequence, expression vector, or cell in the preparation of a CD3 detection kit.

[0049] Specifically, the kit also includes other reagents for CD3 detection, such as buffer solutions and sample processing agents.

[0050] Specifically, the kit may also include molecules for fluorescently labeled antibodies.

[0051] More specifically, the molecule can be a macromolecular dye or a small molecule dye.

[0052] Preferably, the macromolecular dye is APC, and the small molecule dye is FITC.

[0053] In another aspect, the present invention provides a CD3 detection method, wherein the detection method uses the aforementioned anti-human CD3 antibody for detection.

[0054] Specifically, the detection method may also include a sample pretreatment step.

[0055] The preprocessing step involves sample collection and processing.

[0056] Preferably, the detection method is not a disease diagnosis or treatment method.

[0057] In another aspect, the present invention provides a CD3 detection kit, wherein the kit includes the aforementioned anti-CD3 antibody, nucleotide sequence, expression vector, or cells.

[0058] Specifically, the kit also includes other reagents for CD3 detection, such as buffer solutions and sample processing agents.

[0059] Specifically, the kit may also include molecules for fluorescently labeled antibodies.

[0060] More specifically, the molecule can be a macromolecular dye or a small molecule dye.

[0061] Preferably, the macromolecular dye is APC, and the small molecule dye is FITC.

[0062] The technical effects achieved by this invention are as follows: This invention provides the protein sequences of four anti-human CD3 mouse and rabbit monoclonal antibodies. A stable CHO cell line was established for recombinant expression of these genes. The purified antibodies expressed from these antibodies specifically recognize the human CD3 antigen, and the antibody yield is significantly higher than that produced using traditional hybridoma cells. Furthermore, by replacing the mouse IgG1 of this antibody with rabbit IgG while retaining the variable region, a stable CHO cell line was established for recombinant expression of the rabbit monoclonal antibody. The purified antibodies expressed from this rabbit monoclonal antibody maintain their specific recognition of the human CD3 antigen. Attached Figure Description

[0063] Figure 1 SDS gel electrophoresis images of anti-human CD3 antibodies; where 1 is purchased Biolegend mouse anti-human CD3 [UCHT1]; 2 is self-made mouse anti-human CD3 [ZXCloneHCD3-06]; 3 is self-made rabbit anti-human CD3 [ZXCloneHCD3-08]; 4 is purchased Biolegend mouse anti-human CD3 [SK7]; 5 is self-made mouse anti-human CD3 [ZXCloneHCD3-05]; 6 is self-made rabbit anti-human CD3 [ZXCloneHCD3-07].

[0064] Figure 2Flow cytometry results for Biolegend mouse anti-human CD3[SK7]-FITC purchased from outside sources.

[0065] Figure 3 Flow cytometry results for the self-made mouse anti-human CD3 [ZXCloneHCD3-05]-FITC.

[0066] Figure 4 The flow cytometry result of the self-made rabbit anti-human CD3 [ZXCloneHCD3-07]-FITC.

[0067] Figure 5 Flow cytometry data of purchased Biolegend mouse anti-human CD3[UCHT1]-FITC.

[0068] Figure 6 Flow cytometry results for the self-made mouse anti-human CD3 [ZXCloneHCD3-06]-FITC.

[0069] Figure 7 Flow cytometry results for the self-made rabbit anti-human CD3 [ZXCloneHCD3-08]-FITC.

[0070] Figure 8 Flow cytometry results for Biolegend mouse anti-human CD3[SK7]-APC purchased externally.

[0071] Figure 9 Flow cytometry analysis of the self-made mouse anti-human CD3 [ZXCloneHCD3-05]-APC.

[0072] Figure 10 Flow cytometry analysis of self-made rabbit anti-human CD3 [ZXCloneHCD3-07]-APC.

[0073] Figure 11 Flow cytometry results for Biolegend mouse anti-human CD3[UCHT1]-APC purchased from outside the company.

[0074] Figure 12 Flow cytometry analysis of the self-made mouse anti-human CD3 [ZXCloneHCD3-06]-APC.

[0075] Figure 13 Flow cytometry analysis of self-made rabbit anti-human CD3 [ZXCloneHCD3-08]-APC.

[0076] Figure 14 The image shows the potency of Biolegend mouse anti-human CD3 [SK7] purchased from outside sources.

[0077] Figure 15 The potency test chart of the self-made mouse anti-human CD3 [ZXCloneHCD3-05].

[0078] Figure 16 The potency test chart of the self-made rabbit anti-human CD3 [ZXCloneHCD3-07].

[0079] Figure 17 The image shows the potency of Biolegend mouse anti-human CD3 [UCHT1] purchased from outside sources.

[0080] Figure 18 The potency test chart of the self-made mouse anti-human CD3 [ZXCloneHCD3-06].

[0081] Figure 19 The potency test chart of the self-made rabbit anti-human CD3 [ZXCloneHCD3-08].

[0082] Figure 20 This is a specificity diagram of the purchased Biolegend mouse anti-human CD3[SK7] antibody.

[0083] Figure 21 The specificity diagram of the self-made mouse anti-human CD3 [ZXCloneHCD3-05] antibody.

[0084] Figure 22 The specificity diagram of the self-made rabbit anti-human CD3 [ZXCloneHCD3-07] antibody.

[0085] Figure 23 This is a specificity diagram of the positive control CD3[SK7] antibody.

[0086] Figure 24 This is a specificity diagram of the purchased Biolegend mouse anti-human CD3[UCHT1] antibody.

[0087] Figure 25 The specificity diagram of the self-made mouse anti-human CD3 [ZXCloneHCD3-06] antibody.

[0088] Figure 26 The specificity diagram of the self-made rabbit anti-human CD3 [ZXCloneHCD3-08] antibody.

[0089] Figure 27 This is a specificity diagram of the positive control CD3[UCHT1] antibody. Detailed Implementation

[0090] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0091] Example 1: Preparation and Detection of Antibodies

[0092] Four types of antibodies were prepared:

[0093] Antibody 1: Self-made mouse anti-human CD3 [ZXCloneHCD3-05];

[0094] Antibody 2: Self-made rabbit anti-human CD3 [ZXCloneHCD3-07];

[0095] Antibody 3: Self-made mouse anti-human CD3 [ZXCloneHCD3-06];

[0096] Antibody 4: Self-made rabbit anti-human CD3 [ZXCloneHCD3-08].

[0097] The heavy chain amino acid sequence of the self-made mouse anti-human CD3 [ZXCloneHCD3-05] is SEQ ID NO.16, and the light chain amino acid sequence is SEQ ID NO.20; the heavy chain amino acid sequence of the self-made rabbit anti-human CD3 [ZXCloneHCD3-07] is SEQ ID NO.17, and the light chain amino acid sequence is SEQ ID NO.21; the heavy chain amino acid sequence of the self-made mouse anti-human CD3 [ZXCloneHCD3-06] is SEQ ID NO.18, and the light chain amino acid sequence is SEQ ID NO.22; the heavy chain amino acid sequence of the self-made rabbit anti-human CD3 [ZXCloneHCD3-08] is SEQ ID NO.19, and the light chain amino acid sequence is SEQ ID NO.23. Nucleotide sequences for expressing the corresponding antibodies were designed using conventional methods. The nucleotide sequences corresponding to the heavy chain SEQ ID NO.16-19 are shown in SEQ ID NO.24-27, and the nucleotide sequences corresponding to the light chain SEQ ID NO.20-23 are shown in SEQ ID NO.28-31. After cloning the nucleotide sequence into the CHO stable cell line, antibody stable expression was performed, and the constructed vector was further introduced into CHO cells.

[0098] Transfection and screening:

[0099] (1) CHO cells were cultured in CHO CD medium at a concentration of 2 × 10⁻⁶. 5 Inoculate cells / mL with 4 mM glutamine. Build up cell density to 4 × 10⁶ cells / mL. 6 Cells were collected at a density of 1 × 10⁶ cells / mL with a viability greater than 95%. Cells were collected by centrifugation, resuspended in electroporation buffer, and adjusted to a density of 1 × 10⁶ cells / mL. 7 cells / mL.

[0100] (2) Take a sterile EP tube, add 0.8 mL of cell suspension and 5 μg of plasmid, mix gently, incubate for 15 min, and then place on ice for 5 min. Set the electroporator parameters as follows: voltage 280 V, pulse count 3, pulse duration 5 ms, pulse interval 0.784 s, and electroporation cuvette inner diameter 4 mm. Add 0.5 mL of the cell and plasmid mixture to each electroporation cuvette, place it in the electroporator, and start the electroporation program. After completion, quickly aspirate the cells from the electroporation cuvette and transfer them to a 125 mL Erlenmeyer flask containing 20 mL of CHO CD medium. Repeat the above steps to electroporate the second electroporation cuvette. Gently mix the cells in the shake flask, then tighten the cap and incubate in a shaker at 37 °C.

[0101] (3) After 48 hours, transfer the cells to centrifuge tubes and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant and resuspend the cells in 20 mL of CHO CD medium containing 2, 5, and 20 μM MSX. Calculate cell density and viability. The cell density should be around 1.2 × 10⁻⁶. 6 Approximately [number] cells / mL.

[0102] (4) Transfer the cells to a new shake flask. Starting from day 7, observe cell density and viability every two days. Cell density started at 1.2 × 10⁻⁶ cells / day. 6 The cell density decreases from 10 to 15 days, reaching its lowest point between days 5 and 7. The cell density then begins to increase. When the cell density reaches 1 × 10⁻⁶, the density gradually decreases. 6 After reaching a cell density of 3 × 10⁶ cells / mL, the cells were passaged to a cell density of 3 × 10⁶ cells / mL using CD OPM medium containing 50 μM MSX. 5 cells / mL, continue culturing.

[0103] (5) When the cell density increases to 4×10 6 After reaching a cell / mL level, cells were expanded or passaged using CD OPM medium containing 50 μM MSX, with a minimum seeding density of 3 × 10⁻⁶ cells / mL. 5 When the number of cells / mL reaches a certain level, preparations for cryopreservation or large-scale culture can begin.

[0104] (6) Antibody collection method: Antibody purification was performed using protein A affinity chromatography.

[0105] Purity testing:

[0106] (1) Sample processing: Take 10 μg of each of the following: purchased Biolegend mouse anti-human CD3 [SK7], purchased Biolegend mouse anti-human CD3 [UCHT1], self-made mouse anti-human CD3 [ZXCloneHCD3-05], self-made rabbit anti-human CD3 [ZXCloneHCD3-07], self-made mouse anti-human CD3 [ZXCloneHCD3-06] and self-made rabbit anti-human CD3 [ZXCloneHCD3-08] (sample: loading buffer volume ratio = 4:1), incubate at 100℃ for 30 min, centrifuge at 10000 rpm at room temperature for 1 min, and then set aside for use.

[0107] (2) Sample loading: Take out the pre-cast gel and install it in the electrophoresis tank, inject the electrophoresis buffer, turn the comb, and load the sample. Protein Marker 10 μL, sample loading 5 μg.

[0108] (3) Electrophoresis: Voltage 80V, electrophoresis time 90min. Electrophoresis can be stopped when the bromophenol blue indicator band reaches the bottom.

[0109] (4) Staining: Take out the gel and place it in a suitable Coomassie Brilliant Blue staining solution for 30 min, decolorize with decolorizing solution for 1 h, repeat 5 times, and then take a picture.

[0110] The results showed that the four antibodies prepared in Example 1 had intact heavy and light chains, clear bands, and a purity of over 90%. (See attached figures.) Figure 1 .

[0111] Example 2 Antibody labeling

[0112] 1. FITC (fluorescein isothiocyanate) labeling

[0113] (1) Take 0.2 mg of each of the self-made mouse anti-human CD3 [ZXCloneHCD3-05], self-made rabbit anti-human CD3 [ZXCloneHCD3-07], self-made mouse anti-human CD3 [ZXCloneHCD3-06], and self-made rabbit anti-human CD3 [ZXCloneHCD3-08] into an ultrafiltration tube. Wash the antibodies three times with PBS with a final concentration of 1 mM EDTA. Centrifuge the ultrafiltration tubes at 4000 g for 2 min inverted position, collect the concentrated antibodies, and dilute them to 10 mg / mL with PBS with a final concentration of 1 mM EDTA.

[0114] (2) Take small molecule dye FITC (according to n) （摩尔质量） Antibody: n （摩尔质量） Add FITC (1:15 with dye) to the monoclonal antibody reaction solution and mix well.

[0115] (3) React at room temperature of 25℃ for 45 min in the dark on a shaker.

[0116] (4) Transfer the reaction solution to an ultrafiltration centrifuge tube, wash the antibody 5 times with PBS with a final concentration of 1 mM EDTA. Centrifuge the ultrafiltration tube inverted at 4000g for 2 min, collect the concentrated antibody, and add 368 μL of PBS with a final concentration of 1 mM EDTA to make the final antibody concentration 0.5 mg / mL.

[0117] The labeled antibodies were named as self-made mouse anti-human CD3 [ZXCloneHCD3-05]-FITC, self-made rabbit anti-human CD3 [ZXCloneHCD3-07]-FITC, self-made mouse anti-human CD3 [ZXCloneHCD3-06]-FITC, and self-made rabbit anti-human CD3 [ZXCloneHCD3-08]-FITC.

[0118] 2. APC tag

[0119] According to the method described in the patent "Method and Kit for Labeling Fluorescent Proteins and / or Coupled Proteins with Monoclonal Antibodies (CN202010972671.X)," self-made mouse anti-human CD3 [ZXCloneHCD3-05], self-made rabbit anti-human CD3 [ZXCloneHCD3-07], self-made mouse anti-human CD3 [ZXCloneHCD3-06], and self-made rabbit anti-human CD3 [ZXCloneHCD3-08] were labeled respectively. The labeled antibodies were named self-made mouse anti-human CD3 [ZXCloneHCD3-05]-APC, self-made rabbit anti-human CD3 [ZXCloneHCD3-07]-APC, self-made mouse anti-human CD3 [ZXCloneHCD3-06]-APC, and self-made rabbit anti-human CD3 [ZXCloneHCD3-08]-APC.

[0120] 3. Detection of the effect of labeled fluorescent antibodies

[0121] (1) Add 100 μL of cell quality control material (manufacturer: Beckman Coulter, catalog number: 6607077) to a 1.5 mL centrifuge tube.

[0122] (2) Take 1 μL of each of the following fluorescently labeled antibodies: self-made mouse anti-human CD3 [ZXCloneHCD3-05]-FITC, self-made rabbit anti-human CD3 [ZXCloneHCD3-07]-FITC, self-made mouse anti-human CD3 [ZXCloneHCD3-06]-FITC, self-made rabbit anti-human CD3 [ZXCloneHCD3-08]-FITC, self-made mouse anti-human CD3 [ZXCloneHCD3-05]-APC, self-made rabbit anti-human CD3 [ZXCloneHCD3-07]-APC, self-made mouse anti-human CD3 [ZXCloneHCD3-06]-APC, and self-made rabbit anti-human CD3 [ZXCloneHCD3-08]-FITC. Add 1 μL of the control fluorescent antibody, Biolegend mouse anti-human CD3[SK7]-FITC (manufacturer: Biolegend, catalog number: 981002), Biolegend mouse anti-human CD3[UCHT1]-FITC (manufacturer: Biolegend, catalog number: 300405), Biolegend mouse anti-human CD3[SK7]-APC (manufacturer: Biolegend, catalog number: 981012), and Biolegend mouse anti-human CD3[UCHT1]-APC (manufacturer: Biolegend, catalog number: 300411), to the sample respectively.

[0123] (3) All the above samples were reacted in the dark for 15 minutes.

[0124] (4) Add 1 mL of hemolysin and continue the reaction for 15 minutes. The hemolysin used is the hemolysin for flow cytometers from Zhengxi Bio (Zhengxi Bio® (ZXBio®) hemolysin: Zhejiang Medical Device Registration No. 20200133).

[0125] (5) Centrifuge at 4000 rpm for 2 min, discard the supernatant, and add 500 μL PBS to resuspend the sample to be tested.

[0126] (6) Flow cytometer (Kerman DxFlex flow cytometer) detects the proportion of positive cells in the sample, the clustering of the chromatogram, and other indicators, thereby determining whether the fluorescent dyes FITC and APC can be labeled and whether the labeled fluorescent antibody is correctly used to detect the target antigen.

[0127] FITC and APC successfully labeled self-made mouse anti-human CD3 [ZXCloneHCD3-05], self-made rabbit anti-human CD3 [ZXCloneHCD3-07], self-made mouse anti-human CD3 [ZXCloneHCD3-06], and self-made rabbit anti-human CD3 [ZXCloneHCD3-08], with proportions similar to those of purchased Biolegend mouse anti-human CD3 [SK7]-FITC and purchased Biolegend mouse anti-human CD3 [UCHT1]-APC. (See [link to documentation]). Figures 2-13 .

[0128] Example 3 Antibody titer

[0129] The antibody used in this embodiment is:

[0130] Antibody 1: Self-made mouse anti-human CD3 [ZXCloneHCD3-05];

[0131] Antibody 2: Self-made rabbit anti-human CD3 [ZXCloneHCD3-07];

[0132] Antibody 3: Self-made mouse anti-human CD3 [ZXCloneHCD3-06];

[0133] Antibody 4: Self-made rabbit anti-human CD3 [ZXCloneHCD3-08];

[0134] Antibody 5: Biolegend mouse anti-human CD3 [SK7] purchased externally (manufacturer: Biolegend, catalog number: 344802);

[0135] Antibody 6: Biolegend mouse anti-human CD3 [UCHT1] purchased externally (manufacturer: Biolegend, catalog number: 300402).

[0136] First, antibodies 1-6 of known concentration were diluted to 1 mg / mL. Then, they were further diluted with PBS at ratios of 1:100, 1:1000, 1:3000, 1:6000, 1:12000, 1:24000, 1:48000, 1:60000, and 1:96000. Antibodies 1-4 served as the experimental group, and antibodies 5-6 served as the control group.

[0137] The experimental group consisted of four groups, and the control group consisted of two groups. For each group, 100 μL of healthy human blood (company reference) was added, along with 1 μL of commercially available PE goat anti-mouse IgG flow cytometry antibody (manufacturer: Biolegend, catalog number: 405307). After incubation in the dark for 15 minutes, 1 mL of hemolysin (Zhengxi flow cytometry hemolysin (Zhengxi Bio® ZXBio®) hemolysin: Zhejiang Medical Device Registration No. 20200133) was added to each of the six experimental groups, and the reaction was continued for 15 minutes. The mixture was then centrifuged at 4000 rpm for 2 minutes, and the supernatant was discarded.

[0138] The experimental group was treated with 100 μL of proportionally diluted antibody 1, antibody 2, antibody 3, and antibody 4, respectively, while the control group was treated with proportionally diluted antibody 5 and antibody 6, respectively. After mixing, the mixture was incubated at room temperature in the dark for 30 min. The sample was then resuspended in 900 μL of PBS and centrifuged at 4000 rpm for 2 min. The supernatant was discarded, and the sample was resuspended in 500 μL of PBS for flow cytometry analysis.

[0139] The affinities of the six antibodies are as follows:

[0140] Antibody 1: Self-made mouse anti-human CD3 [ZXCloneHCD3-05], with an affinity (kd) of 829.6 (1 mg / mL).

[0141] Antibody 2: Self-made rabbit anti-human CD3 [ZXCloneHCD3-07], with an affinity (kd) of 795.3 (1 mg / mL).

[0142] Antibody 3: Self-made mouse anti-human CD3 [ZXCloneHCD3-06], with an affinity (kd) of 908.9 (1 mg / mL).

[0143] Antibody 4: Self-made rabbit anti-human CD3 [ZXCloneHCD3-08], with an affinity (kd) of 362.6 (1 mg / mL).

[0144] Antibody 5: Biolegend mouse anti-human CD3 [SK7] purchased externally, with an affinity (kd) of 867.0 (1 mg / mL).

[0145] Antibody 6: Biolegend mouse anti-human CD3 [UCHT1] purchased externally, with an affinity (kd) of 903.8 (1 mg / ml).

[0146] The results showed that the self-made CD3 mouse anti-human antibody had similar affinity to the commercially available Biolegend mouse anti-human antibody; the self-made CD3 rabbit anti-human antibody had stronger affinity than the commercially available Biolegend mouse anti-human antibody.

[0147] Example 4 Antibody Specificity

[0148] The antibody used in this embodiment is:

[0149] Pure antibiotics prepared in Example 1:

[0150] Antibody 1: Self-made mouse anti-human CD3 [ZXCloneHCD3-05];

[0151] Antibody 2: Self-made rabbit anti-human CD3 [ZXCloneHCD3-07];

[0152] Antibody 3: Self-made mouse anti-human CD3 [ZXCloneHCD3-06];

[0153] Antibody 4: Self-made rabbit anti-human CD3 [ZXCloneHCD3-08].

[0154] Commercially available, outsourced pure antibiotics:

[0155] Antibody 5: Biolegend mouse anti-human CD3 [SK7] purchased externally (manufacturer: Biolegend, catalog number: 344802);

[0156] Antibody 6: Biolegend mouse anti-human CD3 [UCHT1] purchased externally (manufacturer: Biolegend, catalog number: 300402).

[0157] Commercially available fluorescent antibodies used for detection:

[0158] Fluorescent antibody 7: Biolegend mouse anti-human CD3 [SK7]-FITC (manufacturer: Biolegend, catalog number: 981002);

[0159] Fluorescent antibody 8: Purchased Biolegend mouse anti-human CD3[UCHT1]-FITC (Manufacturer: Biolegend, Catalog No.: 300405)

[0160] Commercially available fluorescent antibodies for co-staining:

[0161] Fluorescent antibody 9: CD19-APC (Manufacturer: Longyang Zhengxi Biotechnology Co., Ltd., Product No.: ZXDU061-APC-0.5).

[0162] Antibody-specific detection methods:

[0163] Take eight 1.5 mL centrifuge tubes and add 100 μL of cell quality control material (manufacturer: Beckman Coulter, catalog number: 6607077) and 1 mL of hemolysin to each tube. Incubate for 15 minutes. The hemolysin used is ZXBio® hemolysin for flow cytometry (Zhejiang Medical Device Registration No. 20200133). Centrifuge at 4000 rpm for 2 minutes, discard the supernatant, and resuspend the cells in 100 μL of PBS.

[0164] The experiment was divided into a pure antibody test group and a positive control group.

[0165] Pure Antibody Assay: Using the cell suspension prepared as described above, take six centrifuge tubes and add 1 μL of antibody 1-antibody 6 to each tube to block the CD3 antigen reaction site. After incubation in the dark for 15 minutes, resuspend the samples in 500 μL of PBS. Centrifuge at 4000 rpm for 2 minutes, discard the supernatant, and resuspend the samples in 100 μL of PBS. Add 1 μL of fluorescent antibody 7 and 1 μL of fluorescent antibody 9 to samples 1, 2, and 5 after blocking for co-staining; add 1 μL of fluorescent antibody 8 and 1 μL of fluorescent antibody 9 to samples 3, 4, and 6 for co-staining. Mix well and incubate at room temperature in the dark for 15 minutes. After incubation, add 900 μL of PBS and vortex to mix. Centrifuge at 4000 rpm for 2 minutes. Discard the supernatant, add 500 μL of PBS, mix well, and then analyze on a flow cytometer.

[0166] Positive control group: Cell suspension prepared as described above was divided into two centrifuge tubes. One tube contained 1 μL of fluorescent antibody 7 and 1 μL of fluorescent antibody 9 for co-staining; the other tube contained 1 μL of fluorescent antibody 8 and 1 μL of fluorescent antibody 9 for co-staining. After mixing, the mixture was incubated at room temperature in the dark for 15 minutes. After incubation, 900 μL of PBS was added, and the mixture was vortexed. The mixture was centrifuged at 4000 rpm for 2 minutes. The supernatant was discarded, and 500 μL of PBS was added. The mixture was then analyzed using flow cytometry.

[0167] Flow cytometry (Agilent NovoCyte flow cytometer) is used to detect indicators such as the proportion of positive cells in the sample and the clustering of cell patterns, thereby determining whether antibodies 1-6 can specifically recognize CD3 target antigens.

[0168] Depend on Figure 20-27 It was found that the positive control group showed positive signals for both CD3 and CD19; the pure antibody test group showed no positive signal for CD3 but positive signal for CD19. Based on this analysis, the CD3 antibody prepared from CHO / HEK293 can effectively block the CD3 antigen reaction site on the cell surface and can specifically recognize the CD3 target antigen.

Claims

1. An anti-human CD3 antibody, characterized in that, It includes a heavy chain and a light chain, wherein the heavy chain includes a heavy chain variable region, and the light chain includes a light chain variable region; the heavy chain variable region includes HCDR1, HCDR2, and HCDR3, and the light chain variable region includes LCDR1, LCDR2, and LCDR3, wherein: The amino acid sequence of HCDR1 is shown in SEQ ID NO.1, the amino acid sequence of HCDR2 is shown in SEQ ID NO.2, the amino acid sequence of HCDR3 is shown in SEQ ID NO.4, the amino acid sequence of LCDR1 is shown in SEQ ID NO.6, the amino acid sequence of LCDR2 is shown in SEQ ID NO.7, and the amino acid sequence of LCDR3 is shown in SEQ ID NO.

8.

2. The anti-human CD3 antibody according to claim 1, characterized in that, The amino acid sequence of the variable region of the heavy chain of the anti-human CD3 antibody is SEQ ID NO.10 or a sequence having more than 80% sequence similarity to SEQ ID NO.10; the amino acid sequence of the variable region of the light chain of the anti-human CD3 antibody is SEQ ID NO.12 or a sequence having more than 80% sequence similarity to SEQ ID NO.12; or the amino acid sequence of the variable region of the light chain of the anti-human CD3 antibody is SEQ ID NO.13 or a sequence having more than 80% sequence similarity to SEQ ID NO.

13.

3. The anti-human CD3 antibody according to claim 1, characterized in that, The heavy chain amino acid sequence of the anti-human CD3 antibody is SEQ ID NO.16, or a sequence having more than 65% sequence similarity to SEQ ID NO.16; or the heavy chain amino acid sequence of the anti-human CD3 antibody is SEQ ID NO.17, or a sequence having more than 65% sequence similarity to SEQ ID NO.17; the light chain amino acid sequence of the anti-human CD3 antibody is SEQ ID NO.20, or a sequence having more than 65% sequence similarity to SEQ ID NO.20; or the light chain amino acid sequence of the anti-human CD3 antibody is SEQ ID NO.21, or a sequence having more than 65% sequence similarity to SEQ ID NO.

21.

4. The anti-human CD3 antibody according to any one of claims 1-3, characterized in that, It is a monoclonal antibody.

5. The anti-human CD3 antibody according to claim 4, characterized in that, These are murine or rabbit-derived antibodies.

6. A nucleic acid expressing the anti-human CD3 antibody according to any one of claims 1-5.

7. The nucleic acid according to claim 6, characterized in that, The nucleotide sequences corresponding to the heavy chain SEQ ID NO.16-17 are shown in SEQ ID NO.24-25, and the nucleotide sequences corresponding to the light chain SEQ ID NO.20-21 are shown in SEQ ID NO.28-29.

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

9. Cells comprising the anti-human CD3 antibody according to any one of claims 1-5, the nucleic acid according to any one of claims 6-7, or the expression vector according to claim 7.

10. A CD3 detection method, characterized in that, The detection is performed using the anti-human CD3 antibody according to any one of claims 1-5, and the method described is a non-disease diagnosis or treatment method.

11. A CD3 detection kit, characterized in that, It includes the anti-human CD3 antibody according to any one of claims 1-5, the nucleic acid according to any one of claims 6-7, the expression vector according to claim 8, or the cell according to claim 9.

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