Full-human-derived anti-CD19 monoclonal antibody and production method thereof

A monoclonal antibody, fully human technology, used in biochemical equipment and methods, chemical instruments and methods, and botanical equipment and methods, etc., can solve the problems of reduced antibody efficacy and side reactions, and achieve good stability and safety. The effect of low sex and immunogenicity

Inactive Publication Date: 2017-09-22
SHANGHAI BIOMED UNION
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  • Summary
  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the mouse-derived components contained in the mouse-derived or humanized sequences may cause human anti-mouse anti

Method used

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  • Full-human-derived anti-CD19 monoclonal antibody and production method thereof
  • Full-human-derived anti-CD19 monoclonal antibody and production method thereof
  • Full-human-derived anti-CD19 monoclonal antibody and production method thereof

Examples

Experimental program
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Example Embodiment

[0026] Example 1: CD19 single chain antibody 2A3 sequence and vector construction

[0027] The CD19 single-chain antibody 2A3 sequence of the present invention is formed by VL and VH connected by a linker sequence, and the structure is VL-Linker-VH. The linker amino acid sequence is GGGGSGGGGSGGGGS.

[0028] The sequence of VL is characterized by containing three light chain complementarity determining regions CDR L1, CDR L2, and CDR L3 shown in Table 1. See the sequence table for the complete sequence of VL. This sequence is derived from human germline gene

[0029] The sequence of VH is characterized by containing three heavy chain complementarity determining regions VH CDR1, CDR2, CDR3 shown in Table 1. The complete sequence of VH is shown in the sequence listing. This sequence is derived from human germline gene

[0030] The complete 2A3 sequence is composed of VL, LINKER, and VH in sequence, as shown in the sequence table. It should be specially pointed out that although thi...

Example Embodiment

[0037] Example 2: Preparation of CD19 single chain antibody

[0038] Transfection of the recombinant plasmid pEF-2A3 into HEK293T cells: HEK293T cells were inoculated into T175 flasks one day before transfection, and transfection was carried out the next day when the cell density was about 80%. First, take 1.5ml of serum-free culture and add 60ug of plasmid; second, take 1.5ml of serum-free medium and add 120ul Lipofectamine2000 liposome transfection reagent; mix the plasmid and liposome solution evenly, let stand at room temperature for 15 minutes, then add to cell culture Base medium; after 6 hours, the medium was changed to fresh DMEM+10% FBS medium; after 72 hours, the culture supernatant was collected. The supernatant contains single-chain antibodies.

[0039] Purification of single-chain antibody 2A3: first add 500ul nickel filler suspension to the gravity column, add 10ml PBS balance system after the liquid dries; add the medium supernatant to the gravity column to allow it...

Example Embodiment

[0041] Example 3: CD19 antigen binding ability detection

[0042] ELISA was used to detect the binding ability of CD19 single-chain antibody 2A3 to CD19 antigen. First, use CD19 antigen to coat the ELISA plate, the antigen concentration is 2ug / ml, the coating solution is sodium carbonate buffer, and the coating is overnight at 4°C. Block the microplate and single-chain antibody 2A3 with 2% MPBS for 2 hours at room temperature. Add 100ul of blocked 2A3 to the blocked ELISA plate, add BSA to the control well as a control, and incubate at room temperature for 1 hour. Wash the plate 5 times with PBST and 2 times with PBS; add anti-HA-HRP antibody and incubate for 1 hour; wash the plate 5 times with PBST and wash the plate 2 times with PBS; add TMB color developing solution, 100 μl / well; room temperature Develop the color for 5-30 minutes, until the well shows a proper degree of blue, add 50ul concentrated sulfuric acid to stop the reaction, and immediately read the absorbance value...

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Abstract

The invention discloses a full-human-derived anti-CD19 monoclonal antibody. The full-human-derived anti-CD19 monoclonal antibody comprises a human-derived light-chain variable region and a human-derived heavy-chain variable region, and is characterized in that the light-chain variable region comprises CDR L1, CDR L2 and CDR L3 sequences shown in table 1, and the heavy-chain variable region comprises CDR H1, CDR H2 and CDR H3 sequences shown in table 1. The invention further provides a preparation method of the full-human-derived anti-CD19 monoclonal antibody. The anti-CD19 monoclonal antibody is a full-human-derived antibody, has lower immunogenicity, will not generate anti-mouse antibodies and can be widely applied to the medical field of immunization.

Description

technical field [0001] The invention belongs to the field of biological immunity, in particular, the invention relates to a fully human CD19 monoclonal antibody. Background technique [0002] CD19 is an important target of B-cell malignancies. CD19 is expressed at almost all stages of B-cell development, as well as in various B-cell tumors, but, importantly, CD19 is not expressed on pluripotent hematopoietic stem cells. These features make CD19 a good target for B cell tumors. Although targeting CD19 destroyed normal B cells, the defect was well tolerated by patients. The current monoclonal antibodies, chimeric antigen receptors or bispecific antibodies targeting CD19 all use murine or humanized sequences. However, the mouse-derived components contained in the mouse-derived or humanized sequences may cause human anti-mouse antibody (HAMA, human antimouse antibody) reactions, resulting in a decrease in antibody efficacy and even serious side effects. Therefore, there is a...

Claims

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Application Information

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IPC IPC(8): C07K16/30C07K16/28C12N15/13C12N5/10G01N33/68G01N33/577
CPCC07K16/2803C07K16/30C07K2317/24C07K2317/56C07K2317/565C07K2317/622
Inventor 郝瑞栋张大挺
Owner SHANGHAI BIOMED UNION
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