Anti-human cd3 rabbit recombinant monoclonal antibody and preparation method and application thereof

Abstract

This invention relates to the field of biomedical technology, specifically disclosing a recombinant rabbit monoclonal antibody against human CD3, its preparation method, and its applications. This antibody, obtained by immunizing rabbits to elicit an anti-CD3 immune response, is derived through single-cell B-cell sorting, gene cloning, and a mammalian cell expression system, exhibiting high affinity and good specificity. The antibody can effectively recognize CD3-positive T cells in human peripheral blood and can be applied in flow cytometry, immunohistochemistry, and T cell function studies. Experimental results show that this antibody has advantages such as high titer, strong fluorescence signal, and low background, outperforming traditional murine anti-CD3 antibodies. Further, the amino acid sequences of the variable regions of the light and heavy chains of this antibody were obtained, enabling controllable and reproducible antibody expression. This antibody has significant scientific research value and promising industrial applications, suitable for various scenarios such as T cell marker detection, immune status assessment, and adjuvant research in immunotherapy.

Description

Technical Field

[0001] This invention discloses a recombinant monoclonal antibody against human CD3 rabbits, its preparation method and application, which relates to the field of biomedical technology. Background Technology

[0002] CD3 is a specific marker of T cells and is widely used in the typing and diagnosis of immune cells. For example, in the diagnosis of leukemia and lymphoma, CD3 expression can be used to differentiate between malignant tumors originating from T cells and those originating from B cells. Furthermore, CD3 detection can be used to assess the functional status of the immune system; for instance, changes in the number of CD3+ T lymphocytes can reflect the activity level of the immune system.

[0003] The role of CD3 antibodies in in vitro detection extends beyond the recognition and activation of immune cells; it also involves immune cell typing, diagnosis, and immunotherapy. These applications provide important tools for a deeper understanding of the immune system's function and the development of novel immunotherapies. CD3 antibodies can also be used in immunohistochemical detection to identify T cells and T-cell-derived malignancies in tissues. For example, CD3 expression can serve as an important diagnostic criterion in lymphoma and leukemia. Furthermore, CD3 antibodies can be used in in vitro activation experiments: they can be used to activate T cells in vitro. For instance, in studying the activation mechanisms and functions of T cells, anti-CD3 antibodies can induce T cell activation, thereby investigating their role in the immune response.

[0004] Most existing CD3 antibodies are produced from murine hybridomas, exhibiting animal origin and lower affinity compared to rabbit-derived antibodies. This technology utilizes rabbit-derived immunization and single-B cell screening, resulting in antibodies with higher affinity. The antibodies are expressed using recombinant methods, avoiding animal origin. Summary of the Invention

[0005] Based on the shortcomings of the prior art, this invention discloses a recombinant rabbit monoclonal antibody against human CD3, its preparation method and application, which can specifically recognize CD3-positive cells in human peripheral blood lymphocytes. In addition, by sequencing the constructed expression antibody light and heavy chain plasmids, the gene sequence and expressed amino acid sequence of the rabbit monoclonal antibody with intellectual property rights were obtained.

[0006] The technical solution of this invention is: a recombinant monoclonal antibody against human CD3 rabbits, which comprises the following 6 complementarity-determining region (CDR) amino acid sequences:

[0007] Heavy chain CDR1 (HCDR1): AYWMQ

[0008] Heavy chain CDR2 (HCDR2): VIYPGDGDARYTQKFQG

[0009] Heavy chain CDR3 (HCDR3): WFHHDYVMDY

[0010] Light chain CDR1 (LCDR1): SSQSVTVNNDLA

[0011] Light chain CDR2 (LCDR2): RASNLA

[0012] Light chain CDR3 (LCDR3): LGGYDDDASNA

[0013] The antibody is obtained by immunizing rabbits with human CD3 antigen to obtain B cells, which are then sorted by single B cells, cultured in vitro, cloned by gene and recombinantly expressed, and can specifically recognize human CD3 protein and be used for the detection of CD3 positive T cells.

[0014] Furthermore, the amino acid sequence of the light chain variable region of this antibody is as follows:

[0015] AAVLTQTPSAVSAAVGGTVTISCQSSQSVTVNNDLAWYQQRPGQPPKLLIYRASNLASRVPSRFSGSGSGTQFTLTISGVQCDDAATYYCLGGYDDDASNAFGGGTEVVVK (SEQ ID NO: 1).

[0016] Furthermore, the amino acid sequence of the heavy chain variable region of this antibody is as follows:

[0017] QCQVKLQQSGAELARPGGSVKLSCKASGYSFTAYWMQWLRQSPGQGLEWIGVIYPGDGDARYTQKFQGKATLTADKSSSTAYMQLSSLASEDSAVYYCARWFHHDYVMDYWGQGTTVTVSS (SEQ ID NO: 2).

[0018] The present invention discloses a method for preparing a recombinant monoclonal antibody against human CD3 rabbits, comprising the following steps:

[0019] Step 1: Immunize rabbits using KLH-conjugated CD3 antigen peptides and Bio-Lon Quick Antibody™ adjuvant. Mix the KLH-conjugated peptides and rabbit Quick Antibody™-8-week rapid adjuvant thoroughly and inject the antigen-adjuvant mixture into the thigh muscle of the rabbits. Repeat the immunization 3 times for a total of 8 weeks.

[0020] Step 2: After the immunization cycle is completed, coat the ELISA plate with human serum albumin HAS-coupled peptide for detection and detect the titer of anti-CD3 antibody in the blood by ELISA.

[0021] Step 3: After the qualified rabbits are treated with air injection, lymphocytes are obtained from the spleen of the immunized rabbits, and then specific single B cells are sorted using biotin-labeled HAS-CD3 antigen peptides.

[0022] Step 4: The positive rabbit B cells that recognize the antigen are cultured using rabbit single B cell culture technology. After 7-10 days of culture, the supernatant of the single B cells cultured in the 96-well plate is detected by ELISA, and human peripheral blood lymphocytes are detected by flow cytometry to confirm the acquisition of rabbit B cell clones that recognize human CD3.

[0023] Step 5: By extracting B cell RNA and reverse transcribing cDNA, the CDR region genes of the light and heavy chains in the B cell clones were amplified using specific primers and constructed into the pcDNA3.4 expression vector;

[0024] Step 6: After in vitro recombinant expression, a high concentration of recombinant rabbit monoclonal antibody was obtained and its titer was retested. The supernatant was then used for flow cytometry to verify its fluorescence effect.

[0025] Application of the antibody of the present invention, wherein the antibody is used for one of the following purposes:

[0026] (1) Detection and classification of CD3-positive T cells;

[0027] (2) Assessment of T cell function and immune activation status;

[0028] (3) Auxiliary diagnosis of T-cell-derived tumors such as lymphoma and leukemia;

[0029] (4) T cell markers in immunohistochemical staining analysis;

[0030] (5) T cell activation experiments and in vitro model construction in immunotherapy research.

[0031] The present invention has the following beneficial effects:

[0032] This invention overcomes the problems of insufficient affinity and strong cross-reactivity of traditional mouse antibodies by using monoclonal antibodies generated through rabbit immunization. The selected 1F2 clones exhibited strong fluorescence signals and extremely low background noise in flow cytometry, demonstrating excellent specific recognition capabilities and significantly improving the sensitivity and accuracy of T cell detection.

[0033] The clones have a clear origin, and the expression is controllable and reproducible: the obtained antibodies have a clear origin, and high-affinity B cells are precisely screened using single B cell screening technology. The antibodies are then prepared in a controllable manner through a recombinant expression system. The recombinant antibodies have high stability and strong batch-to-batch consistency, avoiding the defects of hybridoma antibodies, such as easy mutation and unsustainable expression.

[0034] The full-length amino acid sequences of the light and heavy chain variable regions of the recombinant antibody were obtained through sequencing, and six CDR regions were identified. This lays an important foundation for subsequent development such as humanization of the antibody, bispecific antibody construction, and ADC conjugation, and provides complete intellectual property protection capabilities.

[0035] This invention validated the antibody performance using ELISA and flow cytometry systems, demonstrating superior detection performance compared to commercially available murine anti-CD3 antibodies. It stably identified CD3-positive T cell subsets in human peripheral blood samples with good reproducibility and a high signal-to-noise ratio. This antibody is applicable not only to flow cytometry but also to various immunoassay and biomedical research fields, including immunohistochemistry, T cell activation studies, lymphoma / leukemia diagnosis, and the construction of immunotherapy models, exhibiting significant scientific and clinical value. The antibody gene was constructed on the pcDNA3.4 mammalian expression vector. High concentrations of recombinant rabbit monoclonal antibody were obtained after in vitro recombinant expression, and its titer was re-tested. The supernatant was then used for flow cytometry to verify its fluorescence effect. Attached Figure Description

[0036] Figure 1 KLH-conjugated antigen peptide sequence and CD3 peptide antigen DEDHLSLKEFSELEQSGYYV test.

[0037] Figure 2 The ELISA titer of rabbit serum was measured 8 weeks after immunization in this invention.

[0038] Figure 3 The images show the ELISA titer of the supernatant after B cell sorting in this invention; (a) shows the sorting data; and (b) shows the ELISA titer of the supernatant after sorting.

[0039] Figure 4 This is a diagram showing the positive 1F2 monoclonal strain selected by rabbit B cell culture in this invention, which can recognize CD3-positive cells in human peripheral blood, and compared with classic clone numbers.

[0040] Figure 5 This refers to the variable region of the light and heavy chains of the expressed amino acid sequence of the immunized monoclonal antibody strain 1F2 in this invention. Detailed Implementation

[0041] To further illustrate the technical solution of the present invention, the preparation process and verification method of the anti-human CD3 rabbit monoclonal antibody described in the present invention are explained in detail below with reference to embodiments. It should be understood that the following content is only a preferred embodiment and should not be construed as limiting the scope of protection of the present invention.

[0042] I. Antigen Design and Immunization Procedures

[0043] like Figure 1In this invention, the highly antigenic fragment (DEDHLSLKEFSELEQSGYYV) from the human CD3 molecule is selected as the immunogenicity. This sequence has an antigenicity prediction score of 12 (>3 points indicates a superior antigenic region), and Position is the range of values; it exhibits good immunogenicity. This polypeptide is coupled with KLH (Keyhole Limpet Hemocyanin) to enhance its immunostimulatory ability.

[0044] After thoroughly mixing the KLH-conjugated human CD3 antigen peptide with Bio-Long Quick Antibody™ rabbit rapid immunization adjuvant, multiple injections were administered into the thigh muscles of New Zealand white rabbits for a total of 3 injections over 8 weeks. A booster immunization was given every 2 weeks.

[0045] Rabbit serum was collected periodically after immunization, and the titer of anti-CD3 antibodies was detected using ELISA to determine the success of immunization. Results showed that at week 8 post-immunization, serum OD452 levels significantly increased, indicating successful induction of specific antibodies against human CD3 (see [link to study]). Figure 2 ).

[0046] II. Single B cell screening and in vitro culture

[0047] After the immunization cycle, rabbits were euthanized using air injection to obtain their spleens. Splenic lymphocytes were extracted using mechanical grinding and filtration. Biotin-labeled human serum albumin (HSA) conjugated with CD3 antigen peptides was used as a screening probe, combined with flow cytometry for specific screening of single B cells.

[0048] The selected single B cells were seeded into 96-well culture plates, and specific rabbit B cell culture medium was added. The cells were cultured in vitro for 7-10 days, and the supernatant was collected and its anti-CD3 antibody titer was detected by ELISA. Figure 3 The screening results were shown, with many clones having an OD452 value greater than 2.5, indicating that high-affinity rabbit B cell clones were successfully obtained.

[0049] III. Antibody Gene Cloning and Recombinant Expression

[0050] Total RNA was extracted from positive B cells obtained through screening, and cDNA was synthesized using reverse transcription. Specific primers targeting the variable regions of the rabbit light and heavy chains were designed, and PCR amplification was performed to obtain the complete antibody variable region gene fragment.

[0051] The CDR region genes of the light and heavy chains in the B cell clone were amplified using specific primers and constructed into the pcDNA3.4 expression vector. After in vitro recombinant expression, a high concentration of recombinant rabbit monoclonal antibody was obtained, and its titer was retested. The fluorescence effect was verified by flow cytometry using the supernatant.

[0052] The recombinant antibody was confirmed to have high and stable titer by a second ELISA test, and its recognition ability was then verified by flow cytometry.

[0053] IV. Antibody Function Verification

[0054] Peripheral blood samples were collected from healthy individuals using EDTA anticoagulant tubes and subjected to the following flow cytometry staining steps:

[0055] 1. Draw 100 μL of peripheral blood from the EDTA anticoagulant tube into the flow cytometer (as 1T).

[0056] 2. Set up blank tubes / monochromatographic compensation tubes / sample tubes (100 μL peripheral blood per tube). Do not add flow cytometry antibodies to the blank tubes. Add 5 μL of antibody to each monochromatographic compensation tube according to the label. Add 5 μL of antibody to each sample tube. Incubate at 4°C in the dark for 20 min. Note: If antibody titration has been performed previously, add the antibody according to the titration results.

[0057] 3. Add 1-2 mL of 1*Schizolyl hemolysin / hemolysin to each tube (refer to the Schizolyl hemolysin instructions for specific timing and operation).

[0058] 4. After the red saturation is complete, centrifuge at 300g for 5 minutes at 4℃ and discard the supernatant.

[0059] 5. Add 1 mL of 1*PBS, resuspend, centrifuge at 300g for 5 min at 4℃, and discard the supernatant.

[0060] 6. Add 300-500 μL of 1*PBS to each tube to resuspend the cells.

[0061] 7. Flow cytometer was used to detect and analyze the data.

[0062] Note: If immediate analysis is not possible, stained cells can be fixed in 2% paraformaldehyde (prepared with 1*PBS) at 4°C for 30 min, washed with 1*PBS (refer to step 5), and then resuspended in 300 μL of 1*PBS and stored at 4°C in the dark. Fixed cells should be analyzed as soon as possible.

[0063] Experimental results show that Figure 4 The positive 1F2 monoclonal strain selected through rabbit B cell culture in this invention can recognize CD3-positive cells in human peripheral blood, and is compared with classic clone numbers ( Figure 4 Where Count is the count and samplename is the sample name.

[0064] V. Antibody Sequence Information

[0065] like Figure 5The present invention further sequenced the light and heavy chains of the antibody expressing positive clone 1F2, and obtained its complete variable region amino acid sequence as follows:

[0066] 1F2 heavy chain variable region (SEQ ID NO:2):

[0067] QCQVKLQQSGAELARPGGSVKLSCKASGYSFTAYWMQWLRQSPGQGLEWIGVIYPGDGDARYTQKFQGKATLTADKSSSTAYMQLSSLASEDSAVYYCARWFHHDYVMDYWGQGTTVTVSS

[0068] 1F2 light chain variable region (SEQ ID NO:1):

[0069] AAVLTQTPSAVSAAVGGTVTISCQSSQSVTVNNDLAWYQQRPGQPPKLLIYRASNLASRVPSRFSGSGSGTQFTLTISGVQCDDAATYYCLGGYDDDASNAFGGGTEVVVK

[0070] This antibody contains the following 6 complementarity-determining region (CDR) amino acid sequences:

[0071] Heavy chain CDR1 (HCDR1): AYWMQ

[0072] Heavy chain CDR2 (HCDR2): VIYPGDGDARYTQKFQG

[0073] Heavy chain CDR3 (HCDR3): WFHHDYVMDY

[0074] Light chain CDR1 (LCDR1): SSQSVTVNNDLA

[0075] Light chain CDR2 (LCDR2): RASNLA

[0076] Light chain CDR3 (LCDR3): LGGYDDDASNA

[0077] These sequences are the core content of this invention and have been protected by intellectual property rights, facilitating subsequent engineering applications such as antibody humanization, bispecific antibody construction, and ADC drug development.

Claims

1. A recombinant monoclonal antibody against human CD3 in rabbits, characterized in that, This antibody contains six complementarity-determining regions (CDRs) as shown in the following amino acid sequence: Heavy chain CDR1 (HCDR1): AYWMQ Heavy chain CDR2 (HCDR2): VIYPGDGDARYTQKFQG Heavy chain CDR3 (HCDR3): WFHHDYVMDY Light chain CDR1 (LCDR1): SSQSVTVNNDLA Light chain CDR2 (LCDR2): RASNLA Light chain CDR3 (LCDR3): LGGYDDDASNA.

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

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

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