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Antibody combined with TIGIT antigen and preparation method and application of antibody

A technology of antibody and antigen, applied in the field of biotechnology-antibody

Active Publication Date: 2021-10-29
ACROIMMUNE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] At present, there is no TIGIT target-related drug approved for marketing in the world, but there are many pharmaceutical companies such as Genentech (Genentech), Bristol-Myers Squibb ( BMS), Merck & Co. (MSD), OncoMed, etc. launched the development of new anti-TIGIT antibody drugs and applied for relevant patent protection

Method used

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  • Antibody combined with TIGIT antigen and preparation method and application of antibody
  • Antibody combined with TIGIT antigen and preparation method and application of antibody
  • Antibody combined with TIGIT antigen and preparation method and application of antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Embodiment 1: the establishment and screening identification of the mouse hybridoma cell line that secretes anti-TIGIT antibody

[0083] 1) Alignment analysis of the amino acid sequence of human TIGIT protein and the amino acid sequence of mouse TIGIT protein

[0084] The amino acid sequence of the human TIGIT protein and the amino acid sequence of the mouse TIGIT protein are compared and analyzed as follows: figure 1 : wherein the amino acid sequence marked in italics of the box is the signal peptide (signalpeptide) for guiding the extracellular secretion and expression of the TIGIT protein, and the amino acid sequence marked in bold underlined in the box is the transmembrane domain of the TIGIT protein. like figure 1 Shown: the amino acid sequence homology between human TIGIT protein and mouse TIGIT protein is only about 60%; in the extracellular region directly involved in the recognition and binding of its ligand (CD155 or CD112), human TIGIT protein and mouse TIGI...

Embodiment 2

[0099] Example 2 Detection and analysis of the binding of mouse monoclonal antibody to TIGIT and other immune-related proteins by ELISA

[0100] The binding activities of purified mouse 33D2 monoclonal antibody, 188-2-D7 monoclonal antibody to TIGIT and other related proteins were compared and analyzed by ELISA method.

[0101] The basic steps of the ELISA method are as follows: Dilute the purified mouse 33D2 monoclonal antibody and 188-2-D7 monoclonal antibody to 1 μg / mL, and add them respectively to the pre-coated recombinant TIGIT-his protein or other immune-related gene-Fc fusion Protein (including CD28, B7-1, CTLA4, CD3, PD-1, PD1H (PD2), PD-L1, PD-L2 and other 12 kinds of antigens, the coating concentration of each antigen is 2μg / mL) in a 96-well plate After incubating at 37°C for 1h and washing, add HRP-labeled goat anti-mouse IgG (product of Jackson, USA) to each well of the 96-well plate, and after incubating at 37°C for 1h and washing, add OPD substrate to each well ...

Embodiment 3

[0106] Example 3 Flow cytometry (Flow-cytometry) detection and analysis of mouse antibody binding to CHO cells transfected and expressing human TIGIT gene (CHO / TIGIT)

[0107] In this example, the supernatant of the mouse-derived hybridoma cell line 33D2 was used as the primary antibody, and FITC fluorescently labeled goat anti-mouse IgG was used as the secondary antibody. CHO cell (CHO / TIGIT) conjugation of the human TIGIT gene. To this end, the CHO / TIGIT cells were immunized with positive serum containing mouse IgG (negative control sample, isotype control), mouse 33D2 supernatant and TIGIT antigen (positive control sample immu.sera, diluted 1:200); After incubating at 4°C for 30min and washing with PBS-1%BSA solution, add FITC-labeled goat anti-mouse IgG (product of Sigma); after incubating at 4°C for 30min and washing with PBS-1%BSA solution, the The sample was detected by Biosciences AccuriC6 flow cytometer (BD Company, USA).

[0108] Image 6 Schematic diagram of the ...

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Abstract

The invention discloses an antibody or a derivative thereof. The antibody is combined with a human TIGIT antigen with high affinity and is used for antagonizing and inhibiting the combination of TIGIT and a ligand such as CD155, wherein the antibody comprises amino acid sequences of antigen complementarity determining regions (CDR-L1, CDR-L2 and CDR3-L3) of a light chain variable region of the antibody, and amino acid sequences of antigen complementarity determining regions (CDR-H1, CDR-H2 and CDR-H3) of a heavy chain variable region of the antibody. The invention also discloses a humanized preparation process of the antibody and amino acid sequences of a heavy chain variable region and a light chain variable region of the humanized antibody. The antibody or the derivative thereof can be used as a pharmaceutical composition component or can be prepared into a proper pharmaceutical preparation, and the antibody or the derivative thereof can be independently taken or combined with other drugs such as anti-PD-1 monoclonal antibody and the like or treatment means for use, and can be used for treating diseases such as tumors and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology-antibody. The invention relates to an antibody for antagonizing and inhibiting the binding of T cell immunoglobulin and ITIM domain protein TIGIT (T-cell immunoreceptor with Ig and ITIM domains) to its ligand, its coding sequence, its preparation method and application. Background technique [0002] Cellular immunity mediated by T lymphocytes plays a key role in the process of identifying, monitoring, attacking and killing tumor cells. T lymphocytes conduct immune function by combining co-stimulatory molecules or co-inhibitory molecules with ligands expressed on tumor target cells or antigen-presenting cells (Antigenpresenting cells, APCs). Among them, co-inhibitory factors are also called immunosuppressive checkpoints (Inhibitory immune checkpoint molecules), mainly including CTLA-4 (cytotoxic T-lymphocyte antigen-4) and its ligands B7-1 (CD80), B7-2 (CD86); procedural Death receptor PD-1 (programm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13A61K39/395A61P35/00
CPCC07K16/2827C07K16/2818A61P35/00C07K2317/565C07K2317/56C07K2317/92C07K2317/76C07K2317/73C07K2317/24A61K2039/505A61K2039/507A61P31/00C07K16/2803Y02A50/30C07K16/28C12N15/85C12N2800/107
Inventor 周群敏孙薇薇陈蕞马晓晓范金玲胡红群
Owner ACROIMMUNE BIOTECH CO LTD
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