Light chain variable region and heavy chain variable region of rabbit anti-canine CD3E monoclonal antibody and application of light chain variable region and heavy chain variable region

Abstract

The invention relates to the technical field of immunology and biology, in particular to a light chain variable region and a heavy chain variable region of a rabbit anti-canine CD3E monoclonal antibody and application of the light chain variable region and the heavy chain variable region. The rabbit anticanine CD3E monoclonal antibody is successfully developed, and the antibody can be applied to experiments such as ELISA (enzyme-linked immunosorbent assay), flow cytometry, immunohistochemistry, stimulation of canine T cell activation and the like. Meanwhile, after being transfected to the surface of a tumor cell, the T cell can be promoted to kill the tumor. The development of the antibody provides a basic tool for detection and subsequent tumor immunotherapy. Meanwhile, the problems of low affinity, poor antigen specificity and the like of a traditional antibody are solved, the performance of the antibody is improved, and the application scene of the antibody is expanded.

Description

Technical Field

[0001] The present invention relates to the fields of immunology and biotechnology, and in particular to a light chain variable region and a heavy chain variable region of a rabbit anti-canine CD3E monoclonal antibody and applications thereof. Background Art

[0002] As important companion animals, dogs are capable of spontaneously developing tumors and are five times more likely to develop cancer than humans. Dogs can spontaneously develop a variety of tumor types, including but not limited to mammary cancer, B-cell lymphoma, osteosarcoma, and melanoma. Currently, treatment options for canine tumors are limited, and identification of lymphomas is challenging. Therefore, the development of canine CD3E monoclonal antibodies has become a key tool for studying canine T cell biology and pathology. However, antibodies targeting canine CD3 are currently scarce both domestically and internationally. According to research, the only commercially available canine CD3E antibody is a mouse anti-canine CD3E (CA17.2A12), which is mouse-derived. Mouse-derived antibodies are obtained through hybridomas, which require a long acquisition cycle and have low efficiency in fusion between spleen cells and myeloma cells. Only a small fraction of the total B cell population completes fusion, while the vast majority are lost. Therefore, they are not suitable for comprehensive screening of large antibody libraries. Furthermore, mouse-derived antibodies often have weak antigen recognition, low affinity, and low specificity and stability.

[0003] Currently, Blinatumomab targets CD3 and CD19 and is primarily used to treat acute B-lymphocytic leukemia; Teclistamab targets CD3 and BCMA and is primarily used to treat relapsed / refractory multiple myeloma. However, there is a lack of therapeutic immune antibodies for dogs. Therefore, the development of canine CD3 antibodies can be used for subsequent identification, diagnosis, and immune antibody preparation. Summary of the Invention

[0004] The purpose of the present invention is to provide a light chain variable region and a heavy chain variable region of a rabbit anti-canine CD3E monoclonal antibody and applications thereof, so as to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a light chain variable region and a heavy chain variable region of a rabbit anti-canine CD3E monoclonal antibody, wherein the heavy chain variable region comprises CDR-H1, CDR-H2 and CDR-H3; the light chain variable region comprises CDR-L1, CDR-L2 and CDR-L3;

[0007] The amino acid sequence of the CDR-H1 is shown in any one of SEQ ID NO.1 to SEQ ID NO.5; the amino acid sequence of the CDR-H2 is shown in any one of SEQ ID NO.6 to SEQ ID NO.10; the amino acid sequence of the CDR-H3 is shown in any one of SEQ ID NO.11 to SEQ ID NO.15; the amino acid sequence of the CDR-L1 is shown in any one of SEQ ID NO.16 to SEQ ID NO.20; the amino acid sequence of the CDR-L2 is shown in any one of QAS, SAS, RTS and RAS; the amino acid sequence of the CDR-L3 is shown in any one of SEQ ID NO.21 to SEQ ID NO.25.

[0008] Preferably, the amino acid sequence of the CDR-H1 is shown in SEQ ID NO.1; the amino acid sequence of the CDR-H2 is shown in SEQ ID NO.6; the amino acid sequence of the CDR-H3 is shown in SEQ ID NO.11; the amino acid sequence of the CDR-L1 is shown in SEQ ID NO.16; the amino acid sequence of the CDR-L2 is shown in QAS; and the amino acid sequence of the CDR-L3 is shown in SEQ ID NO.21.

[0009] Alternatively, the amino acid sequence of the CDR-H1 is shown in SEQ ID NO.2; the amino acid sequence of the CDR-H2 is shown in SEQ ID NO.7; the amino acid sequence of the CDR-H3 is shown in SEQ ID NO.12; the amino acid sequence of the CDR-L1 is shown in SEQ ID NO.17; the amino acid sequence of the CDR-L2 is shown in SAS; and the amino acid sequence of the CDR-L3 is shown in SEQ ID NO.22;

[0010] Alternatively, the amino acid sequence of the CDR-H1 is shown as SEQ ID NO.3; the amino acid sequence of the CDR-H2 is shown as SEQ ID NO.9; the amino acid sequence of the CDR-H3 is shown as SEQ ID NO.12; the amino acid sequence of the CDR-L1 is shown as SEQ ID NO.16; the amino acid sequence of the CDR-L2 is shown as QAS; and the amino acid sequence of the CDR-L3 is shown as SEQ ID NO.25.

[0011] The present invention provides a rabbit anti-canine CD3E monoclonal antibody, wherein the heavy chain variable region of the rabbit anti-canine CD3E monoclonal antibody comprises CDR-H1, CDR-H2 and CDR-H3; the light chain variable region of the rabbit anti-canine CD3E monoclonal antibody comprises CDR-L1, CDR-L2 and CDR-L3;

[0012] The amino acid sequence of the CDR-H1 is shown in any one of SEQ ID NO.1 to SEQ ID NO.5; the amino acid sequence of the CDR-H2 is shown in any one of SEQ ID NO.6 to SEQ ID NO.10; the amino acid sequence of the CDR-H3 is shown in any one of SEQ ID NO.11 to SEQ ID NO.15; the amino acid sequence of the CDR-L1 is shown in any one of SEQ ID NO.16 to SEQ ID NO.20; the amino acid sequence of the CDR-L2 is shown in any one of QAS, SAS, RTS and RAS; the amino acid sequence of the CDR-L3 is shown in any one of SEQ ID NO.21 to SEQ ID NO.25.

[0013] Preferably, the amino acid sequence of the CDR-H1 is shown in SEQ ID NO.1; the amino acid sequence of the CDR-H2 is shown in SEQ ID NO.6; the amino acid sequence of the CDR-H3 is shown in SEQ ID NO.11; the amino acid sequence of the CDR-L1 is shown in SEQ ID NO.16; the amino acid sequence of the CDR-L2 is shown in QAS; and the amino acid sequence of the CDR-L3 is shown in SEQ ID NO.21.

[0014] Alternatively, the amino acid sequence of the CDR-H1 is shown in SEQ ID NO.2; the amino acid sequence of the CDR-H2 is shown in SEQ ID NO.7; the amino acid sequence of the CDR-H3 is shown in SEQ ID NO.12; the amino acid sequence of the CDR-L1 is shown in SEQ ID NO.17; the amino acid sequence of the CDR-L2 is shown in SAS; and the amino acid sequence of the CDR-L3 is shown in SEQ ID NO.22;

[0015] Alternatively, the amino acid sequence of the CDR-H1 is shown as SEQ ID NO.3; the amino acid sequence of the CDR-H2 is shown as SEQ ID NO.9; the amino acid sequence of the CDR-H3 is shown as SEQ ID NO.12; the amino acid sequence of the CDR-L1 is shown as SEQ ID NO.16; the amino acid sequence of the CDR-L2 is shown as QAS; and the amino acid sequence of the CDR-L3 is shown as SEQ ID NO.25.

[0016] The present invention provides the use of the above-mentioned light chain variable region and heavy chain variable region or the above-mentioned rabbit anti-canine CD3E monoclonal antibody in preparing a product for detecting canine CD3E protein.

[0017] Preferably, the product is a reagent, a kit or a chip;

[0018] The kit includes an ELISA kit, an immunohistochemistry kit or a flow cytometry kit.

[0019] The present invention provides a product for detecting canine CD3E protein, which comprises the above-mentioned light chain variable region and heavy chain variable region or the above-mentioned rabbit anti-canine CD3E monoclonal antibody.

[0020] Further preferably, the product is a reagent, a kit or a chip.

[0021] Further preferably, the reagents include ELISA reagents, immunohistochemistry reagents, T cell separation reagents or flow cytometry reagents.

[0022] Further preferably, the kit comprises an ELISA kit, an immunohistochemistry kit, a T cell separation kit or a flow cytometry kit.

[0023] The present invention provides the use of the light chain variable region and heavy chain variable region or the rabbit anti-canine CD3E monoclonal antibody in preparing a product for diagnosing tumors or a drug for treating tumors.

[0024] Further preferably, the tumor includes leukemia, lymphoma and / or breast cancer.

[0025] Further preferably, the tumor is canine leukemia, canine lymphoma and / or canine mammary cancer.

[0026] The present invention provides a drug for treating tumors, which comprises the above-mentioned light chain variable region and heavy chain variable region or the above-mentioned rabbit anti-canine CD3E monoclonal antibody.

[0027] Further preferably, the tumor includes leukemia, lymphoma and / or breast cancer.

[0028] Further preferably, the tumor is canine leukemia, canine lymphoma and / or canine mammary cancer.

[0029] As an additional solution, the present invention provides a product for diagnosing tumors, which comprises the above-mentioned light chain variable region and heavy chain variable region or the above-mentioned rabbit anti-canine CD3E monoclonal antibody.

[0030] Further preferably, the product is a reagent, a kit or a chip.

[0031] Further preferably, the reagents include ELISA reagents, immunohistochemistry reagents, T cell separation reagents or flow cytometry reagents.

[0032] Further preferably, the kit comprises an ELISA kit, an immunohistochemistry kit, a T cell separation kit or a flow cytometry kit.

[0033] The present invention provides use of the above-mentioned light chain variable region and heavy chain variable region or the above-mentioned rabbit anti-canine CD3E monoclonal antibody in preparing a product for diagnosing T cell-related diseases.

[0034] The present invention provides the use of the above-mentioned light chain variable region and heavy chain variable region or the above-mentioned rabbit anti-canine CD3E monoclonal antibody in preparing a product for improving the killing ability of T cells.

[0035] The present invention discloses the following technical effects:

[0036] The present invention has successfully developed a rabbit anti-canine CD3E monoclonal antibody, which can be used in experiments such as ELISA, flow cytometry, immunohistochemistry, and stimulation of canine T cell activation. Furthermore, when transfected onto the surface of tumor cells, it can promote T cell killing of tumors. The development of this antibody provides a basic tool for detection and subsequent tumor immunotherapy. The present invention also has the following advantages:

[0037] 1. Addressing the limitations of traditional antibodies: Traditional mouse-derived monoclonal antibodies have problems such as low affinity and poor antigen specificity, which limit their application in diagnosis and treatment. The rabbit-derived antibodies provided by the present invention can address this problem.

[0038] 2. Improve antibody performance: The somatic hypermutation and gene conversion mechanism of rabbit monoclonal antibodies enable their affinity to reach the picomolar level (mouse monoclonal antibodies are mostly nanomolar), and they can recognize more conformational epitopes, making them suitable for the recognition of complex antigens (canine CD3E transmembrane complex).

[0039] 3. Expanded application scenarios: Dogs are important experimental animals. Genomic analysis shows that the similarity between the human and dog genomes is much greater than that between the mouse and human genomes. Rabbit anti-canine CD3E monoclonal antibodies can fill the gap in cross-species research tools. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0041] Figure 1 The results of Western blot verification are shown in Figure 1. 1 is a marker, and 2 is a canine CD3E-Fc antigen.

[0042] Figure 2 The antibody titer test results;

[0043] Figure 3 It is the specific test result;

[0044] Figure 4 The results of flow cytometry verification are shown in Figure 2; (A) and (B) are the results of flow cytometry verification of uncultured PBMCs; (C) and (D) are the results of flow cytometry verification of canine T cells;

[0045] Figure 5 The results of immunohistochemistry verification are shown. A is a negative sample under a 40x eyepiece; B is a negative sample under an 80x eyepiece; C is a positive sample under a 40x eyepiece; and D is a positive sample under an 80x eyepiece. The arrow in B points to negative unstained cells; the arrow in D points to a positive successfully stained area.

[0046] Figure 6 The data are statistical results of canine IFN-γ release; canine T cell refers to canine T cells, and canine Tcel+HORCF-CD3.1 refers to canine T cells combined with canine CD3E monoclonal antibody.

[0047] Figure 7 The figures are cell killing results; A is the killing result after co-culture of wild-type CMT-U27 cells with T cells; B is the killing result after co-culture of CMT-U27 cells successfully transfected with canine CD3E monoclonal antibody with T cells. DETAILED DESCRIPTION

[0048] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0049] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0050] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0051] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0052] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0053] Example 1 Preparation of Canine CD3E-Fc Antigen

[0054] The CD3E-Fc plasmid was obtained by using the canine CD3 protein extracellular domain (XP_025321127.1) provided by NCBI as the exogenous gene and pcDNA3.1 as the vector.

[0055] One day before transfection, HEK293 cells were seeded at a density of 1×10 6 cells / mL, and culture in an incubator at 37°C and 5% CO2. On the day of transfection, the cell density was controlled at 1-1.5×10 6 cells / mL. Add the CD3E-Fc plasmid and transfection reagent to the transfection buffer, mix thoroughly, and incubate at 37°C to obtain a DNA-transfection reagent mixture. Add the DNA-transfection reagent mixture to the cells to be transfected and incubate in an incubator at 37°C, 5% CO2.

[0056] About 4-6 days after the above cell transfection, the cell culture was removed and centrifuged to collect the supernatant and cells. The centrifuged cells were dissolved in a buffer (50mM Tris, 300mM NaCl, 8M Urea, 20mM Imidazole, pH 8.0) and purified using a nickel column to obtain canine CD3E-Fc antigen. The purified canine CD3E-Fc antigen was verified by Western blot, and the results were as follows: Figure 1 The results showed that canine CD3E-Fc antigen (CD3E protein or canine CD3E-Fc antigen) was successfully expressed and purified and located in the correct position.

[0057] Example 2 Preparation of monoclonal antibodies

[0058] (1) After emulsifying the eukaryotic expressed canine CD3E-Fc antigen with equal volumes of Freund's complete adjuvant, the vaccine was subcutaneously injected into New Zealand white rabbits at multiple points on the back. The immunization dose was 0.5 mg / rabbit. After 14 days, a booster immunization was performed once, for a total of 2 booster immunizations. Except for the first immunization, Freund's incomplete adjuvant was used for subsequent immunizations, once every 7 days, for a total of 4 immunizations. Blood was collected from the ear vein 7 days after the last immunization. The serum was collected after centrifugation and the antibody titer was determined. The results are as follows Figure 2 The results showed that the serum titer after immunization of rabbits was higher than 512K and could be used for subsequent B cell sorting.

[0059] (2) Single B cell isolation

[0060] After the antibody titer reaches 512K or above, preliminary screening is performed using B cell surface markers. Rabbit blood is collected and prepared into a single-cell suspension. Single B cells are then sorted using flow cytometry. The sorting process uses memory B cell markers and antigen dual staining. If a single B cell (MBC) displays IgG antibodies that specifically bind to the antigen on its surface, the cell will have two fluorescent markers. The target MBCs can be sorted individually from the cell suspension using flow cytometry and placed in a 96-well plate.

[0061] Example 3 High-throughput expression, cell supernatant detection and antibody variable region sequencing

[0062] At room temperature, the B cells isolated in Example 2 were lysed and a single-cell antibody gene amplification system was used to obtain the complete light chain sequence and heavy chain Fab region fragment. The amplified heavy chain target gene was ligated into a pcDNA3.4 vector encoding a rabbit IgG Fc fragment, and the antibody light chain gene was ligated into a blank pcDNA3.4 vector to construct a dual plasmid. The dual plasmid was then transiently expressed in HEK293 cells to express the CD3E monoclonal antibody. After 5 days, the supernatant was collected for specificity detection.

[0063] The specific steps of specific detection are:

[0064] Antigen Coating: Dilute canine CD3-Fc protein (prepared in Example 1), canine CD3-his protein, human Fc protein, and feline CD3 protein to 2 μg / mL in PBS buffer. Add 100 μL per well to a 96-well ELISA plate and incubate overnight at 4°C. Wash the coated plate twice with a plate washer and tap dry. Add 200 μL of blocking solution (0.1% BSA) per well and incubate in a 37°C incubator for 2 h. Then, wash the blocked plate twice with a plate washer and tap dry.

[0065] (2) Incubation of primary antibody: dilute the antibody with PBS buffer, rabbit pre-immune blood as negative control, dilute at a ratio of 1:1000, add 100 μL of the diluted antibody to the corresponding wells, incubate at 37°C for 2 h, and after incubation, wash the plate three times in a plate washer and dry.

[0066] (3) Incubation with secondary antibody: Add 100 μL of HRP-labeled goat anti-rabbit secondary antibody (abclonal, AS002) to the corresponding wells, incubate at 37°C for 30 min, wash the plate in a plate washer 5 times, and then dry.

[0067] (4) Color development: TMB color development solution (solarbio, PR1200) was added to a 96-well plate, 100 μL per well, and incubated at 37°C for 15 min.

[0068] (5) Stop and read: Add stop solution to the 96-well plate, 50 μL per well, and then place it in the microplate reader for reading. The detection wavelength is set to 450 nm and the test results are read. Figure 3 The results showed that the canine CD3E monoclonal antibody produced could bind to canine CD3 protein, but not to feline CD3E protein and human Fc protein, indicating that the canine CD3E monoclonal antibody produced had high specificity.

[0069] After detection, the variable regions of the corresponding canine CD3E monoclonal antibodies were sequenced. The detection results are shown in Table 1.

[0070] Table 1 Variable region sequences of canine CD3E monoclonal antibodies

[0071]

[0072]

[0073] Example 2 Flow cytometry verification

[0074] Materials: Canine T cells cultured in our laboratory and uncultured human peripheral blood mononuclear cells (PBMCs).

[0075] Methods: The generated canine CD3E monoclonal antibody (CDR-H1 is shown in SEQ ID NO.3; CDR-H2 is shown in SEQ ID NO.9; CDR-H3 is shown in SEQ ID NO.12; CDR-L1 is shown in SEQ ID NO.16; CDR-L2 is shown in QAS; CDR-L3 is shown in SEQ ID NO.25) was used as the primary antibody, and goat anti-rabbit AlexaFluor 488 antibody was used as the secondary antibody for staining.

[0076] Canine T cells and uncultured canine peripheral blood mononuclear cells were stained with CD3E monoclonal antibody (as primary antibody) and goat anti-rabbit Alexa Fluor 488 antibody (Abclonal, catalog number AS053) (as secondary antibody), respectively. After incubation at 4°C for 20 minutes, the cells were washed and resuspended in buffer. The cell suspension was then filtered through a cell strainer, collected and kept at low temperature for detection using a flow cytometer. The results are shown in Figure 2. Figure 4 The results showed that the positive rate of uncultured PBMC after staining with CD3E monoclonal antibody was 81.1%; the positive rate of laboratory-cultured canine T cells after staining with CD3E monoclonal antibody was 97.2%.

[0077] Implementation Case 3: Immunohistochemistry Validation

[0078] Canine spleen tissue sections were processed for immunohistochemistry using an immunohistochemistry kit according to the protocol (abcam, ab64264). The specific steps were as follows: After deparaffinization and rehydration, the canine spleen tissue was microwaved at medium power for 20 minutes using citrate buffer (pH 6.0). Epitope retrieval was performed, and endogenous peroxidase activity was quenched with 3% H₂O₂ for 30 minutes at room temperature. Nonspecific protein-protein interactions were blocked with a protein-free blocking serum reagent for 30 minutes at room temperature. A canine CD3E monoclonal antibody (primary antibody, described in Example 2) was diluted 1:3000 in blocking buffer and incubated at 4°C for 12 hours. A biotinylated goat anti-polyvalent IgG secondary antibody was then added and incubated at room temperature for 15 minutes, followed by a streptavidin-peroxidase conjugate for 10 minutes at room temperature. Immunoreactivity was visualized using diaminobenzidine (DAB) substrate. The sections were counterstained with hematoxylin, dehydrated through a series of different ethanol concentrations, processed in xylene, and mounted with a synthetic resin mounting medium. Figure 5 The results showed that canine spleen tissue was clearly positive after staining with CD3 monoclonal antibody. This antibody can be used to diagnose T cell-related diseases.

[0079] Example 4 Canine CD3 monoclonal antibody stimulates T cells

[0080] The cultured T cells were plated onto a 96-well cell culture plate with 100,000 cells per well. Canine CD3E monoclonal antibody (canine CD3E monoclonal antibody in Example 2) was added at a final concentration of 10 ng / mL. After culturing for 48 h, the cell supernatant was taken to detect the release of canine IFN-γ (interferon-γ). The results were as follows: Figure 6The results showed that after T cells were stimulated with CD3E antibodies, IFN-γ release doubled, indicating that T cells were effectively activated through the TCR signaling pathway and initiated a Th1 immune response. This result not only demonstrates the reliability of the experimental system, but also provides key data for research on infection immunity, tumor treatment, and the mechanisms of autoimmune diseases.

[0081] Example 5 Antibodies expressed on tumor cell membranes can promote T cells to kill tumors

[0082] The sequences of the generated canine CD3E monoclonal antibody (canine CD3E monoclonal antibody or rabbit anti-canine CD3E antibody) (CDR-H1 shown in SEQ ID NO. 3; CDR-H2 shown in SEQ ID NO. 9; CDR-H3 shown in SEQ ID NO. 12; CDR-L1 shown in SEQ ID NO. 16; CDR-L2 shown in QAS; and CDR-L3 shown in SEQ ID NO. 25) were designed and synthesized using the pcDNA3.1 vector to synthesize an anti-CD3E membrane protein plasmid. 293T cells were plated onto 6 cm cell culture dishes. When cells reached 70%-80% confluence, a plasmid mixture containing psPAX2 and pMD2.G plasmids (mass ratio of psPAX2 to pMD2.G was 2:1) was mixed with the anti-CD3E membrane protein plasmid at a ratio of 3:1. The resulting mixture was then mixed with Lipofectamine reagent (DNA:reagent, 1:3) and incubated at room temperature for 15 minutes. The transfection complex was added to the cells and cultured in serum-free DMEM for 6 hours, then replaced with complete culture medium (containing 10% FBS). The viral supernatant was harvested 48 and 72 hours after transfection, combined, and the viral liquid was obtained by centrifugation (3500rpm, 10 minutes) and 0.45μm filtration. CMT-U27 cells (canine breast cancer cell line, wild-type CMT-U27 cells) were incubated with 10mL of viral supernatant supplemented with polybrene (8μg / mL), centrifuged at 1100×g for 1 hour, and then inoculated into 24-well plates. After 48h, 2μg / mL of puromycin was used for screening. After successful screening, the successfully transfected U27 cells were co-cultured with canine PBMCs in different ratios, and then the crystal violet test was used to detect cell killing. The results are shown in Figure 2. Figure 7 The results showed that after co-culture of CMT-U27 cells (U27 cells) with canine PBMCs, it was found that T cells had no obvious killing effect on CMT-U27 cells ( Figure 7 A in the figure), while CMT-U27 cells (anti-CD3-U27 cells) successfully transfected with canine CD3E monoclonal antibody showed a significant killing effect after co-culture with T cells, even though the number of PBMCs was far lower than that of CMT-U27 cells ( Figure 7 B) in.

[0083] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A light chain variable region and a heavy chain variable region of a rabbit anti-canine CD3E monoclonal antibody, characterized in that: The heavy chain variable region includes CDR-H1, CDR-H2 and CDR-H3; the light chain variable region includes CDR-L1, CDR-L2 and CDR-L3; The amino acid sequence of the CDR-H1 is shown in any one of SEQ ID NO.1 to SEQ ID NO.5; the amino acid sequence of the CDR-H2 is shown in any one of SEQ ID NO.6 to SEQ ID NO.10; the amino acid sequence of the CDR-H3 is shown in any one of SEQ ID NO.11 to SEQ ID NO.15; the amino acid sequence of the CDR-L1 is shown in any one of SEQ ID NO.16 to SEQ ID NO.20; the amino acid sequence of the CDR-L2 is shown in any one of QAS, SAS, RTS and RAS; the amino acid sequence of the CDR-L3 is shown in any one of SEQ ID NO.21 to SEQ ID NO.

25.

2. The light chain variable region and heavy chain variable region according to claim 1, characterized in that: The amino acid sequence of the CDR-H1 is shown in SEQ ID NO.1; the amino acid sequence of the CDR-H2 is shown in SEQ ID NO.6; the amino acid sequence of the CDR-H3 is shown in SEQ ID NO.11; the amino acid sequence of the CDR-L1 is shown in SEQ ID NO.16; the amino acid sequence of the CDR-L2 is shown in QAS; and the amino acid sequence of the CDR-L3 is shown in SEQ ID NO.

21. Alternatively, the amino acid sequence of the CDR-H1 is shown in SEQ ID NO.2; the amino acid sequence of the CDR-H2 is shown in SEQ ID NO.7; the amino acid sequence of the CDR-H3 is shown in SEQ ID NO.12; the amino acid sequence of the CDR-L1 is shown in SEQ ID NO.17; the amino acid sequence of the CDR-L2 is shown in SAS; and the amino acid sequence of the CDR-L3 is shown in SEQ ID NO.22; Alternatively, the amino acid sequence of the CDR-H1 is shown as SEQ ID NO.3; the amino acid sequence of the CDR-H2 is shown as SEQ ID NO.9; the amino acid sequence of the CDR-H3 is shown as SEQ ID NO.12; the amino acid sequence of the CDR-L1 is shown as SEQ ID NO.16; the amino acid sequence of the CDR-L2 is shown as QAS; and the amino acid sequence of the CDR-L3 is shown as SEQ ID NO.

25.

3. A rabbit anti-canine CD3E monoclonal antibody, characterized in that The heavy chain variable region of the rabbit anti-canine CD3E monoclonal antibody includes CDR-H1, CDR-H2 and CDR-H3; the light chain variable region of the rabbit anti-canine CD3E monoclonal antibody includes CDR-L1, CDR-L2 and CDR-L3; The amino acid sequence of the CDR-H1 is shown in any one of SEQ ID NO.1 to SEQ ID NO.5; the amino acid sequence of the CDR-H2 is shown in any one of SEQ ID NO.6 to SEQ ID NO.10; the amino acid sequence of the CDR-H3 is shown in any one of SEQ ID NO.11 to SEQ ID NO.15; the amino acid sequence of the CDR-L1 is shown in any one of SEQ ID NO.16 to SEQ ID NO.20; the amino acid sequence of the CDR-L2 is shown in any one of QAS, SAS, RTS and RAS; the amino acid sequence of the CDR-L3 is shown in any one of SEQ ID NO.21 to SEQ ID NO.

25.

4. The rabbit anti-canine CD3E monoclonal antibody according to claim 3, characterized in that The amino acid sequence of the CDR-H1 is shown in SEQ ID NO.1; the amino acid sequence of the CDR-H2 is shown in SEQ ID NO.6; the amino acid sequence of the CDR-H3 is shown in SEQ ID NO.11; the amino acid sequence of the CDR-L1 is shown in SEQ ID NO.16; the amino acid sequence of the CDR-L2 is shown in QAS; and the amino acid sequence of the CDR-L3 is shown in SEQ ID NO.

21. Alternatively, the amino acid sequence of the CDR-H1 is shown in SEQ ID NO.2; the amino acid sequence of the CDR-H2 is shown in SEQ ID NO.7; the amino acid sequence of the CDR-H3 is shown in SEQ ID NO.12; the amino acid sequence of the CDR-L1 is shown in SEQ ID NO.17; the amino acid sequence of the CDR-L2 is shown in SAS; and the amino acid sequence of the CDR-L3 is shown in SEQ ID NO.22; Alternatively, the amino acid sequence of the CDR-H1 is shown as SEQ ID NO.3; the amino acid sequence of the CDR-H2 is shown as SEQ ID NO.9; the amino acid sequence of the CDR-H3 is shown as SEQ ID NO.12; the amino acid sequence of the CDR-L1 is shown as SEQ ID NO.16; the amino acid sequence of the CDR-L2 is shown as QAS; and the amino acid sequence of the CDR-L3 is shown as SEQ ID NO.

25.

5. Use of the light chain variable region and heavy chain variable region according to claim 1 or 2 or the rabbit anti-canine CD3E monoclonal antibody according to claim 3 or 4 in the preparation of a product for detecting canine CD3E protein.

6. A product for detecting canine CD3E protein, characterized in that: The product comprises the light chain variable region and heavy chain variable region of claim 1 or 2 or the rabbit anti-canine CD3E monoclonal antibody of claim 3 or 4.

7. Use of the light chain variable region and heavy chain variable region according to claim 1 or 2 or the rabbit anti-canine CD3E monoclonal antibody according to claim 3 or 4 in the preparation of a product for diagnosing tumors or a drug for treating tumors.

8. A drug for treating tumors, characterized in that: The drug comprises the light chain variable region and the heavy chain variable region according to claim 1 or 2 or the rabbit anti-canine CD3E monoclonal antibody according to claim 3 or 4.

9. Use of the light chain variable region and heavy chain variable region according to claim 1 or 2 or the rabbit anti-canine CD3E monoclonal antibody according to claim 3 or 4 in the preparation of a product for diagnosing T cell-related diseases.

10. Use of the light chain variable region and heavy chain variable region according to claim 1 or 2 or the rabbit anti-canine CD3E monoclonal antibody according to claim 3 or 4 in the preparation of a product for improving T cell killing ability.

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