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A kind of antibody-conjugated drug against claudin 6 and its application

An antibody-conjugated drug, claudin technology, applied in the field of biomedicine, can solve problems such as high recurrence rate and side effects

Active Publication Date: 2020-12-08
GUANGZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods all exhibit high recurrence rates and cause serious side effects, such as transplant rejection
Even with successful resection, there is a 25% recurrence rate

Method used

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  • A kind of antibody-conjugated drug against claudin 6 and its application
  • A kind of antibody-conjugated drug against claudin 6 and its application
  • A kind of antibody-conjugated drug against claudin 6 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Antigen sequence and its preparation

[0047] Through the analysis and comparison of bioinformatics, a polypeptide sequence with the highest score and the least crossness with other tight junction structure-related proteins was screened out:

[0048] SEQ ID NO.1

[0049] LPMWKVTAFIGN

[0050] The polypeptide sequence corresponds to the 27th to 38th amino acids of the claudin 6 protein sequence.

[0051] In order to facilitate the access to the corresponding carrier KIH, a connection structure C was added at the end of the above sequence, and the following peptide synthesis of the polypeptide fragment with a purity of 90% was carried out. The sequence is:

[0052] LPMWKVTAFIGNC.

Embodiment 2

[0053] Example 2 Antibody Preparation

[0054] The synthesized polypeptide sequence was coupled with a carrier protein (such as KLH), and 3 BALB / c mice were boosted by standard procedures. The mouse with the best immune response was selected, and the splenocytes were fused with myeloma cells to prepare hybridoma cells.

[0055] The obtained hybridoma cells were screened by proliferation and ELISA to obtain high-titer clones; and 2 to 8 positive mother cells were subcloned by the limited dilution method, and finally a positive clone was determined for subtype identification. It was deposited in the China Center for Type Culture Collection on November 12, 2019, with the preservation number CCTCC NO.C2019282, address: China. Wuhan. Wuhan University, Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0056] After expanding the culture of the positive cloned cells, five mice were subjected to ascites production, and the corresponding monoclonal antibodies ...

Embodiment 3

[0057] Example 3 Preparation and Identification of Antibody-Drug Conjugates

[0058] 1. Main material

[0059] Antibody: the monoclonal antibody prepared in Example 2 (hereinafter referred to as Ab);

[0060] Connection structure: SMCC, purchased from AAT Bioquest (USA);

[0061] Drug: maytansine (hereinafter referred to as DM1).

[0062] 2. Preparation method

[0063] 1) Dissolve SMCC in DMSO and store at 4°C;

[0064] 2) Combine Ab and SMCC in conjugation buffer (50mm potassium phosphate, 50mm sodium chloride, 2mm EDTA,

[0065] pH 7.2) at a molar ratio of 1:10, stirred and reacted at 4°C for 4-6 hours;

[0066] 3) After the reaction, place the mixed solution in a dialysis bag (M=5000), and dialyze at 4°C for three days;

[0067] 4) Collect the dialysate and freeze-dry it in a vacuum freeze dryer;

[0068] 5) Quantitative analysis of protein samples with BCA;

[0069] 6) The modified antibody and DM1 molecules were mixed in PBS buffer (pH=7.0) at a molar ratio of 1:1...

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Abstract

The invention discloses an antibody-conjugated drug against claudin 6 and its application. The invention relates to an antigen sequence for preparing claudin 6 (CLDN6) antibody, its corresponding nucleotide sequence and a method for expressing the nucleoside Genetic engineering carrier, engineering bacteria or cell line of acid sequence; animal immunization through the antigenic polypeptide can produce highly specific claudin 6 antibody; further prepare hybridoma cells by immunizing animal cells to obtain corresponding monoclonal antibodies; Combining the anti-claudin 6 antibody with a linking structure and a drug can form an antibody-conjugated drug; the antibody-coupled drug can carry the drug to the tumor expressing positive claudin 6 according to the targeting effect of claudin 6 cells, so as to achieve the effect of specifically killing tumor cells, which is of great significance to the treatment of tumors.

Description

technical field [0001] The invention belongs to the field of biomedicine, relates to an antigen and its corresponding antibody application, in particular to an antibody-coupled drug against claudin 6 and its application. Background technique [0002] In the 20th century, primary liver cancer has risen to become the second cancer killer in my country, and more than 1 million patients die from liver cancer every year in the world, and the incidence of liver cancer in my country accounts for 40% of the world's total. The treatment of liver cancer is mainly based on surgery, but clinically 90% of liver cancer patients have lost the chance of surgery when they are diagnosed with liver cancer. Moreover, the recurrence rate within 5 years after hepatectomy is as high as 80%, and the long-term survival rate is still not high. Examples of other liver cancer treatments include tissue transplantation, systemic chemotherapy, radiation therapy, and electrocautery. However, these method...

Claims

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Application Information

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IPC IPC(8): C07K14/82C12N15/12C07K16/32C12N5/20A61K31/44A61K31/537A61K47/68A61P35/00
CPCC07K14/82C07K16/32A61K47/6803A61K31/44A61K31/537A61P35/00A61K31/455A61K47/6849C07K16/28A61K47/68033A61K39/39558A61K2039/505C07K2317/14
Inventor 刘铭孔繁恩马宁芳唐云强谢茂斌
Owner GUANGZHOU MEDICAL UNIV
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