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Recombinant anti-CTLA4 monoclonal antibody, its production and use

A monoclonal antibody, heavy chain technology, applied in recombinant DNA technology, botanical equipment and methods, biochemical equipment and methods, etc., can solve problems such as systemic reactions, toxic and side effects, and achieve high sensitivity and specificity.

Active Publication Date: 2007-11-21
SHANGHAI NAT ENG RES CENT OF ANTIBODY MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In contrast, other immunosuppressants tend to indiscriminately kill all T cell clones, causing significant systemic reactions and toxic side effects

Method used

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  • Recombinant anti-CTLA4 monoclonal antibody, its production and use
  • Recombinant anti-CTLA4 monoclonal antibody, its production and use
  • Recombinant anti-CTLA4 monoclonal antibody, its production and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Screening the variable region of cytolytic T cell-associated antigen 4 antibody gene from the antibody library

[0042] 1) Construction of mouse antibody library

[0043] CTLA4-Fc fusion protein (R&D, 325-CT / CF) and Freund's adjuvant were used to immunize Balb / C mice. After four times of immunization, the mouse serum was diluted 1:500 and showed a strong positive reaction with CTLA4. Mouse spleen according to Marks et al. J. Mol. Biol., 222, 581-597. Hoogenboom and Winter, J. Mol. Biol., 227, 381-388. Haidaris CG et al. J Immunol Methods. 2001 Nov 1; 257( 1-2): 185-202, Griffiths, A.D. et al. EMBO J., 13, 3245-3260 (1994). The method described in Nissim, A. et al. EMBO J., 13, 692-698 (1994), Construction of murine antibody library.

[0044] 2) screening

[0045] Add 1 ml of the revived antibody library strain to 14 ml of fresh LB medium, and culture in a 50 ml Erlenmeyer flask at 37°C for 16 hours.

[0046] Centrifuge at a high speed of 12000rpm for 10 mi...

Embodiment 2

[0065] Example 2 Expression Intensity of Antibody Genes in CHO Cells

[0066]The 12 high-expression candidate clones obtained from the above screening were cultured in a 10 cm tissue culture dish, and the expression level of the antibody was determined by ELISA as described below. Coat goat anti-mouse IgG (Fc) on an ELISA plate, overnight at 4°C, block with 2% BSA at 37°C for 2 hours, add the culture supernatant to be tested and a standard (mouse IgG1), incubate at 37°C for 2 hours, add HRP-goat anti-mouse IgG (κ)) for binding reaction, incubated at 37°C for 1 hour, added TMB at 37°C for 10 minutes, and finally washed with H 2 SO 4 Termination reaction, test A 450 value. Table 1 below shows the expression levels of the 12 candidate clones obtained from the above screening.

[0067] Cell line number

[0068] As can be seen from Table 1 above, 3C4, 1A2 and 8H6 have very high expression levels.

Embodiment 3

[0069] Example 3 DNA sequencing of anti-CTLA4 monoclonal antibody gene

[0070] According to the pedigree, DNA sequencing was performed on the anti-CTLA4 antibody gene of the 8H6 cell line obtained above. The results are as follows: SEQ ID NO: 3 shows the 8H6 light chain variable region gene sequence (5' to 3') 336bp, and its deduced amino acid sequence is shown in SEQ ID NO: 1; SEQ ID NO: 4 shows the 8H6 heavy chain Variable region gene sequence ((5' to 3') 363 bp), its deduced amino acid sequence is shown in SEQ ID NO:2.

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Abstract

An anti-CTLA4 mono-clone antibody, its DNA molecule for encoding antibody, its carrier for expressing antibody and eukaryotic host cell converted by the expression carrier are disclosed. Light-chain variable zone has amino-acid sequence with SEQ ID NO:1, and heavy-chain variable zone has amino-acid sequence with SEQ ID NO:2. It can inspect CTLA4-Fc concentration in blood serum for examinee and patients with malignant diseases in hematopoietic system fast.

Description

technical field [0001] The invention relates to the technical field of antibody drugs. Specifically, the present invention relates to a new recombinant anti-cytolytic T cell-associated antigen 4 monoclonal antibody, its preparation method and application. Background technique [0002] The activation of T lymphocytes requires at least two signals. The first signal is generated by the combination of T cell antigen receptor (TCR) and antigen presenting cell (APC) surface MHC / antigen peptide complex. The binding of these two ligands requires Specific recognition is closely related to the specificity of immune response. The second signal is generated by the combination of other auxiliary molecules on the surface of T cells and the corresponding ligands on the surface of antigen-presenting cells. This signal is also called co-stimulatory signal, which is antigen-nonspecific. [0003] Molecules on the surface of T cells and antigen-presenting cells that participate in co-stimulat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N15/13C12N15/85G01N33/577C12P21/08
Inventor 郭亚军钱卫珠寇庚候盛
Owner SHANGHAI NAT ENG RES CENT OF ANTIBODY MEDICINE
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