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Antibody capable of binding tigit or antigen-binding fragment thereof and use thereof

An antibody and application technology, applied in the field of antibodies to achieve the effect of inhibiting tumor growth

Active Publication Date: 2020-03-03
IMMUNOPHARMACEUTIC INST OF HEFEI RUIDA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using antibodies to block immunosuppressive receptors (including PD-1, CTLA4, etc.) Unresponsiveness to combination therapy, which may be due to the expression of multiple immunosuppressive receptors on the surface of T cells and NK cells

Method used

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  • Antibody capable of binding tigit or antigen-binding fragment thereof and use thereof
  • Antibody capable of binding tigit or antigen-binding fragment thereof and use thereof
  • Antibody capable of binding tigit or antigen-binding fragment thereof and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0158]Example 1: Preparation of Antibodies

[0159] Generate a mouse monoclonal antibody against human TIGIT, using purified recombinant TIGIT extracellular region Fc fusion protein (TIGIT-Fc) (recombinant TIGIT extracellular region Fc fusion protein, amino acid sequence shown in SEQ ID NO: 121) as an antigen , to immunize Balb / c mice (9 weeks old, purchased from Shanghai Leike, weighing about 20 g).

[0160] The immunized mice were immunized three times with purified antigen and complete Freund's adjuvant, and the immune response was detected after bloodletting from the tail vein. The serum was screened by ELISA and flow cytometry, and mice with anti-human TIGIT immunoglobulin were obtained. And for the mouse with the highest anti-TIGIT immunoglobulin, the splenocytes were taken out and fused with the mouse myeloma cell SP2 / 0 cell (ATCC number CRL-1581). The fused hybridoma cells were screened for antibodies to obtain mouse monoclonal antibodies 1H6, 2B11, 3A10, 4A5, 4A9, 4...

Embodiment 2

[0162] Example 2: Antibody binding ability screening

[0163] TIGIT Antibody ELISA Binding Experiment

[0164] ELISA experiments were used to examine the binding properties of TIGIT antibodies. The TIGIT extracellular region Fc fusion protein (TIGIT-Fc) was coated into a 96-well plate, and the signal intensity after the antibody was added was used to determine the binding characteristics of the antibody and TIGIT.

[0165] The TIGIT-Fc fusion protein (amino acid sequence shown in SEQ ID NO: 121) was diluted to 1 μg / ml with PBS buffer, added to a 96-well plate at a volume of 100 μl / well, and left overnight at 4°C. Aspirate the PBS buffer in the 96-well plate, wash the plate 6 times with PBST (pH7.2 PBS containing 0.1% Tween 20) buffer, add 200 μl / well PBS / 10% BSA, and incubate at 37°C for 2 hours to block. Remove the blocking solution, wash the plate 6 times with PBST, add 100 μl / well of the TIGIT antibody to be tested diluted to an appropriate concentration with PBST / 0.05% B...

Embodiment 3

[0170] Embodiment 3: In vitro binding affinity and kinetic experiments

[0171] The Biacore method is a recognized method for objectively detecting the mutual affinity and kinetics of proteins, and the TIGIT antibody of the present invention is analyzed by Biacore T200 to characterize the affinity and binding kinetics.

[0172] The TIGIT extracellular fragment Fc fusion protein (TIGIT-Fc) was covalently linked to the CM5(GE) chip by standard amino coupling method. A series of concentration gradients of TIGIT antibody diluted in PBS was then injected with each cycle and regenerated with 10 mM NaOH solution after injection. Track the antigen-antibody binding kinetics for 3 minutes and track the dissociation kinetics for 10 minutes, use GE's BIAevaluation software to analyze the data obtained with a 1:1 (Langmuir) binding model, and the ka (kon) and kd (koff) determined by this method and KD values ​​are shown in Table 3 below.

[0173] Table 3 TIGIT antibody affinity

[0174]...

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Abstract

The invention discloses an antibody or antigen binding fragment capable of being bonded with TIGIT and a use thereof. The invention relates to the field of antibodies, and discloses the antibody capable of being bonded with TIGIT and the use thereof. The antibody includes a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2 and a light chain CDR3. Theantibody has the following advantages: the antibody can block the interaction between TIGIT and CD155 with high affinity; the antibody can be bonded with TIGIT with high specificity, but cannot be bonded with other members of CD28 family (such as CD28, PD-1 and CTLA4); and the antibody can activate immune cells to kill tumor cells.

Description

technical field [0001] The present invention relates to the field of antibodies, in particular to an antibody capable of binding to TIGIT or an antigen-binding fragment thereof and its use. Background technique [0002] Tumor immunotherapy has been a hot spot in the field of tumor treatment for a long time. Using antibodies to block immunosuppressive receptors (including PD-1, CTLA4, etc.) Combination therapy did not respond, which may be caused by the expression of various immunosuppressive receptors on the surface of T cells and NK cells. Inhibitory receptors such as PD-1, CTLA4, and TIGIT may cooperate to inhibit the activation of immune cells including T cells and NK cells. Therefore, it is of great significance to develop therapeutic antibodies against immunosuppressive receptors such as TIGIT. [0003] TIGIT (T cell Ig and ITIM domain) molecule is an inhibitory receptor expressed on the surface of T cells and NK cells discovered in 2009. Inhibitory motif) intracellu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/30C12N15/13A61K39/395A61P35/00G01N33/574
CPCA61K2039/505A61P35/00C07K16/30C07K2317/24C07K2317/33C07K2317/56C07K2317/565C07K2317/76C07K2319/72G01N33/57484
Inventor 田志刚曹国帅王保如孙汭肖卫华魏海明王立
Owner IMMUNOPHARMACEUTIC INST OF HEFEI RUIDA CO LTD
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