Anti-TIM-3 monoclonal antibody, and expression vector and application thereof

A technology of TIM-3 and monoclonal antibody, applied in the fields of cellular immunology and molecular biology

Active Publication Date: 2019-11-05
北京昭衍生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are currently no TIM-3 target d

Method used

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  • Anti-TIM-3 monoclonal antibody, and expression vector and application thereof
  • Anti-TIM-3 monoclonal antibody, and expression vector and application thereof
  • Anti-TIM-3 monoclonal antibody, and expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0103] Example 1 Construction of Anti-TIM-3 Antibody Expression Vector

[0104] Several antibodies with strong binding ability to TIM-3 antigen were obtained through the screening of phage antibody library in the early stage, and this application is one of the antibodies studied.

[0105] (1) Using the method of gene synthesis to synthesize the antibody heavy chain variable region sequence (the heavy chain variable region sequence of the antibody is shown in SEQ ID NO: 12), the light chain variable region sequence (the antibody light chain variable region sequence is shown in SEQ ID NO: 12) ID NO: 16), and use molecular cloning method to clone the fragment into the ZY-CDMO vector, the ZY-CDMO vector map is as follows figure 1 shown.

[0106] The specific operation is as follows: introduce an EcoRI restriction site at the 5' end of the light chain variable region by conventional PCR, introduce an XbaI restriction site at the 3' end, and insert it between EcoRI and XbaI of the ...

Embodiment 2

[0107] Example 2 Expression and purification of anti-TIM-3 antibody

[0108] Inoculate 30ml in a 125ml cell culture shaker flask with a density of 0.5×10 6 cells / ml of CHO cells, when the cell density grows to 2×10 6 cells / ml or more, the cell viability is more than 95%, and the cells are 1×10 7 The cells / ml density was inoculated in the electrotransfer buffer, 40 μg plasmid was added to the electric shock cup, and then 0.7ml of cell suspension was added, and finally the electrotransfer buffer was added to 0.8ml, and mixed gently to avoid bubbles. Electric shock once under the conditions of 300V, 900μF, place the electric shock cup in the ice box for 5min, and dilute it into 6ml of culture medium at 37°C CO 2 After recovering in the incubator for 48 hours, add 75 μM MSX to the seed medium to inoculate the 96-well plate and perform clone screening, and divide the monoclonal cells into the 96-well plate by the limited dilution method, 37 ° C, 5% CO 2 Cultivate in an incubator...

Embodiment 3

[0110] Example 3 Detection of affinity activity of anti-TIM-3 antibody to TIM-3 antigen

[0111] Use ELISA to detect affinity, the specific steps are as follows:

[0112] Coating: Take the ELISA plate and dilute the target protein human TIM-3 (Sino Biological, 10390-H08H) to 0.5 μg / ml with pH 9.6 coating solution, and coat 100 μl / well in a humid chamber overnight at 4°C.

[0113] Discard the liquid in the well, invert the plate and pat dry the residual liquid on a paper towel, add washing solution (0.1% PBST) 300 μl / well and wash 3 times.

[0114] Blocking: adding 300 μl / well of blocking solution (2% M-PBS) and standing at 37° C. for 1.0 h.

[0115] Discard the liquid in the well, invert the plate and pat dry the residual liquid on a paper towel, add washing solution (0.1% PBST) 300 μl / well and wash 3 times.

[0116] Add the primary antibody: Dilute the anti-TIM-3 antibody to 1mg / ml with DPBS, and then perform 10-fold dilutions successively, with a total of 7 concentration g...

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Abstract

The invention discloses an anti-TIM-3 monoclonal antibody, and an expression vector and an application thereof. The monoclonal antibody can specifically be combined with a TIM-3 protein, and generatessynergistic effect with the joint application of an anti-PD-1 antibody. The antibody can be used for treating tumor and has a wide application prospect in clinic.

Description

technical field [0001] The invention belongs to the fields of cell immunology and molecular biology, and relates to an anti-TIM-3 monoclonal antibody, an expression vector and applications thereof. Background technique [0002] Structurally, TIM-3 belongs to T cell immunoglobulin and mucin (T cell Immunoglobulin domain and Mucin domain protein, Tim) family members. Because the Tim family is widely involved in the immune regulation process of the body, its function is being paid more and more attention by people. TIM-3 is specifically expressed on the surface of activated Th1 and Th17 effector cells, but not on Th2 cells. It is now known that galactose-binding protein-9 (Galactin-9, Gal-9) is the natural ligand of TIM-3, which is widely expressed in the peripheral immune system, and Gal-9 and Tim-3 molecules on Th1 and Th17 cells Specific binding can trigger the latter's apoptosis, downregulate the immune response, and induce immune tolerance. At present, studies have conf...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13C12N5/10G01N33/577A61K39/395A61P35/00
CPCA61K2039/505A61P35/00C07K16/2803C07K2317/565C07K2317/92G01N33/577G01N2333/70503
Inventor 赵健郎小玲耿树生
Owner 北京昭衍生物技术有限公司
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