Humanized monoclonal antibody targeting Claudin18.2 as well as preparation method and application thereof

A technology of monoclonal antibody and humanization, applied in chemical instruments and methods, antibodies, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can solve problems such as side effects, and achieve simple preparation steps , the effect of good ADCC and CDC activity

Active Publication Date: 2021-06-11
NANJING KAEDI BIOTHERAPEUTICS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, during the development of Claudin18.2 drugs, attention should be paid to the problem of cross-reaction and side effects with Claudin18.1

Method used

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  • Humanized monoclonal antibody targeting Claudin18.2 as well as preparation method and application thereof
  • Humanized monoclonal antibody targeting Claudin18.2 as well as preparation method and application thereof
  • Humanized monoclonal antibody targeting Claudin18.2 as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0134] Example 1: Antigen Preparation

[0135] According to the sequence information of Claudin18.2, the gene was synthesized after codon optimization, and subcloned into the adenovirus shuttle vector pShuttle-CMV-GFP. Recombination to obtain the recombinant adenovirus plasmid.

[0136] The recombinant adenovirus vector was purified, and then transfected into 293A cells using LVTransm transfection reagent to prepare adenovirus seeds. Based on the seed virus, large-scale amplification is carried out, the adenovirus is purified, and the titer is determined using Anti-Hexon antibody.

Embodiment 2

[0137] Example 2: Mouse Immunization

[0138] All mice were housed in a barrier system with sterile pelleted chow and autoclaved drinking water. Take 8 Balb / c mice (SPF grade) and mark them with ear tags, and follow the classic immunization process, using purified recombinant adenovirus, according to 1×10 7 pfu / mouse dosage, mice were immunized by intramuscular injection.

[0139] Table 1 Claudin18.2 recombinant adenovirus animal immunization scheme

[0140]

[0141]

Embodiment 3

[0142] Example 3: Detection of mouse serum titer after immunization

[0143] 1. Take the mouse out of the cage, use 75% medical alcohol cotton balls to disinfect the tail of the mouse, and use a blood collection needle to prick a small wound on the tail of the mouse;

[0144] 2. Use capillary glass blood collection tubes to collect blood drops;

[0145] 3. After collecting blood, use a dry sterile cotton ball to gently press the blood collection point to stop the bleeding, and return the mouse to the cage for observation;

[0146] 4. Place the centrifuge tube with collected blood samples in a 37°C incubator for 1 hour; then transfer the blood samples to 4°C overnight.

[0147] 5. Separate the serum from the blood clot, transfer it to a new sterile centrifuge tube, and centrifuge at 10,000 g for 10 min at 4°C;

[0148] 6. Dilute the serum at 1:1000, and perform FACS detection with CHO-Claudin18.2 cells and control cell CHO respectively.

[0149] The results of titer detectio...

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Abstract

The invention relates to a humanized monoclonal antibody targeting Claudin18.2 as well as a preparation method and application thereof. According to the present invention, the Claudin18.2 humanized monoclonal antibody obtained through the cell fusion and the hybridoma technology can be highly specifically combined with CHO-Claudin18.2 cells, but is almost not combined with CHO-Claudin18.1 cells, the preparation method of the Claudin18.2 humanized monoclonal antibody of the present invention has simple steps, and the obtained Claudin18.2 humanized monoclonal antibody has good ADCC and CDC activity on the expression of Claudin18.2 positive tumor cells. The humanized monoclonal antibody of Claudin18.2 disclosed by the invention is high in specificity and small in side effect, and has a very good prospect in application to treatment and / or prevention or diagnosis of Claudin18.2-related diseases such as tumors.

Description

technical field [0001] The invention belongs to the technical field of immunotherapy biomedicine, and relates to a humanized monoclonal antibody targeting Claudin18.2, its preparation method and application, especially in the treatment and / or prevention or diagnosis of diseases related to Claudin18.2 For example, tumor applications. Background technique [0002] Gastric cancer is one of the most common cancers worldwide, accounting for the fourth (male) and fifth (female) cause of cancer-related death in developed countries. Most gastric cancer patients are already in advanced stages after diagnosis. Early gastric cancer may be cured by gastrectomy, and the recurrence rate still reaches 50%. So far, advanced gastric cancer is incurable, and the median survival period after chemotherapy is 8-10 months. Multiple chemotherapy regimens are being investigated to improve response rates and tolerability, however 5-year survival remains elusive. [0003] Claudin-18 (CLDN18) is a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28A61K39/395A61P35/00G01N33/577G01N33/574G01N33/68
CPCC07K16/28A61P35/00G01N33/577G01N33/574G01N33/57492G01N33/6893C07K2317/565C07K2317/56C07K2317/24C07K2317/92C07K2317/94C07K2317/732C07K2317/734G01N2800/26G01N2333/7051
Inventor 代红久徐慧王梦瑶
Owner NANJING KAEDI BIOTHERAPEUTICS LTD
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