Antibody drug conjugate of CLDN 18.2-resistant antibody and preparation method and application of antibody drug conjugate

A technology of conjugates and drugs, which is applied in the field of biomedicine and can solve problems such as lack of

Pending Publication Date: 2020-05-08
L&L BIOPHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] In order to overcome the lack of drug conjugates of antibodies against human CLDN18.2 protein in the art

Method used

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  • Antibody drug conjugate of CLDN 18.2-resistant antibody and preparation method and application of antibody drug conjugate
  • Antibody drug conjugate of CLDN 18.2-resistant antibody and preparation method and application of antibody drug conjugate
  • Antibody drug conjugate of CLDN 18.2-resistant antibody and preparation method and application of antibody drug conjugate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0241] Example 1: Construction of high-expression cell lines of claudin 18.1, 18.2 (CLDN18.1, CLDN18.2)

[0242] The human CLDN18.1, human CLDN18.2, mouse CLDN18.1, and mouse CLDN18.2 high-expression cell lines used in the present invention are completed through the company's stable cell line construction platform. Specific steps are as follows:

[0243] On the first day of the experiment, 293T cells (Cat#GNHu17, a cell bank of the Type Culture Collection Committee of the Chinese Academy of Sciences) were seeded in two 6cm culture dishes, and the number of cells in each culture dish reached 7.5×10 5 indivual. On the second day, the plasmids (pGag-pol, pVSV-G, pBabe, etc. BioVector, Plasmid Vector Strain Cell Gene Collection Center) and the plasmid pBabe-CLDN18.2 or pBabe for cloning the human or mouse CLDN18.2 or CLDN18.1 gene will be wrapped. - Add OPTI-MEM (Thermofisher Scientific Cat#31985070) to 4 μg each of CLDN18.1 to make the final volume 200 μl, prepare 200 μl OPTI-M...

Embodiment 2

[0257] Example 2: Anti-CLDN18.2 antibody and CLDN18.2+ and CLDN18.1+ cell line binding (ELISA) experiment

[0258] After expanding the monoclonal cell line with high expression of human CLDN18.1, human CLDN18.2, mouse CLDN18.1 or mouse CLDN18.2 obtained in Example 1, press 5×10 4 96-well plate was spread per well, and the supernatant was removed after overnight incubation in a 37°C incubator, and fixed with 100 μl / well of immunostaining fixative (Shanghai Beyotime Biotechnology Co., Ltd. Cat#P0098) for half an hour at room temperature. After washing once with PBS (Yuanpei Biology, Cat#B320), block with 5% milk at 37°C for 2 hours, and wash 3 times with PBST. Add the sample to be tested (human or mouse antibody, Jackson Immuno Research). Incubate at 37°C for 1 hour, then wash 3 times with PBST. Add Anti-human or mouse HRP 1:2500 50μl / well and incubate at 37°C for 1 hour, then wash 3 times with PBST, develop color with TMB (Surmodic Cat#TTMB-1000-01), add 50μl / well 1M H 2 SO ...

Embodiment 3

[0259] Example 3: Cloning, expression and purification of recombinant proteins and antibodies

[0260] The cloning, expression and purification of the recombinant protein / antibody used in the present invention are carried out according to molecular cloning methods well known to those skilled in the art.

[0261] Specifically, the expression vector used in the present invention was purchased from Changsha Youbao Biotechnology Co., Ltd., and then introduced EcoRI enzyme cleavage site (GAATTC) by Shanghai Jianxin Biomedical Technology Co., Ltd. (Jinxin Biotechnology) to facilitate double enzyme digestion Or homologous recombination method to clone foreign genes. Gene synthesis is completed by companies such as Sangon Bioengineering (Shanghai) Co., Ltd. (Sangon Biotechnology). 293 cells and CHO-K were purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences.

[0262] The recombinant protein and antibody in the present invention are...

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Abstract

The present invention discloses an antibody drug conjugate of a CLDN 18.2-resistant antibody. The structure of the antibody drug conjugate (ADC) is shown as Ab-[(L2)n-L1-D]y in a formula I, wherein Dis a small-molecule cytotoxic drug, L1 and L2 are connected to the drug and the antibody separately, and n is 0 or 1; y represents the average number of D which is coupled to Ab, 0 < y <= 10, and Ab is an antibody which can be specifically bound to human CLDN 18.2, and includes a light-chain variable region (VL) and/or a heavy-chain variable region (VH); and the CLDN 18.2-resistant antibody correspondingly contains at least one specific complementary determining region (CDR) sequence or a mutant sequence of the specific complementary determining region (CDR) sequence in the light-chain variable region (VL) and/or a heavy-chain variable region (VH), and binding of the antibody to CLDN 18.2 is maintained or improved through the mutation. The invention also discloses a ADC-containing pharmaceutical composition and a preparation method and application of ADC. The antibody drug conjugate of the antibody has a large security window and low toxic and side effects, and provides more specific,more effective and better treatment option for tumor patients.

Description

technical field [0001] The present invention relates to the field of biomedicine, in particular to a drug conjugate of an anti-CLDN18.2 antibody and its preparation method and application. Background technique [0002] Cancer is a great threat to human health and one of the important causes of death in the field of disease. Cancer treatment has undergone various stages of development, such as surgery, chemotherapy, targeted drugs, tumor immunotherapy, and combination therapy, and has made great achievements in recent years. Among the numerous cancer patients, treatment options for cancer patients such as lung cancer, gastric cancer, pancreatic cancer, esophageal cancer, and ovarian cancer remain highly unmet. The treatment methods for these tumors include macromolecular targeted drugs, such as new monoclonal antibodies, and the combination of these monoclonal antibodies and existing tumor immunotherapy methods, including immune checkpoint inhibitors PD-1 and PD-L1 antibodie...

Claims

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Application Information

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IPC IPC(8): A61K47/68A61K31/537A61P35/00A61P35/02C07K16/18
CPCA61K31/537A61K47/6863A61P35/00A61P35/02C07K16/18C07K2317/51C07K2317/515C07K2317/56
Inventor 刘佳建
Owner L&L BIOPHARMA CO LTD
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