Anti-TRAILR2 antibody-toxin-conjugate and pharmaceutical application thereof in anti-tumor therapy

An antibody drug and conjugate technology, which can be used in anti-tumor drugs, drug combinations, medical preparations with inactive ingredients, etc., and can solve the problems of poor patient compliance, high cost, and high toxicity of chemical drugs.

Active Publication Date: 2018-08-28
YANTAI OBIOADC BIOMEDICAL TECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the cost of combined medication is high, especially when used in combination with chemical drugs, the problem of high toxicity of chemical drugs has not been fundamentally solved, the patient's compliance is poor, and the toxic and side effects on patients are still very large

Method used

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  • Anti-TRAILR2 antibody-toxin-conjugate and pharmaceutical application thereof in anti-tumor therapy
  • Anti-TRAILR2 antibody-toxin-conjugate and pharmaceutical application thereof in anti-tumor therapy
  • Anti-TRAILR2 antibody-toxin-conjugate and pharmaceutical application thereof in anti-tumor therapy

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0198] Antibody preparation

[0199] The sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by conventional techniques, such as PCR amplification or genomic library screening. In addition, the coding sequences for the light and heavy chains can be fused together to form single-chain antibodies.

[0200] Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

[0201]In addition, related sequences can also be synthesized by artificial synthesis, especially when the fragment length is relatively short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.

[0202] At present, the DNA sequence encoding the antibody of the p...

Embodiment 1

[0269] Example 1 In vitro antitumor activity of Zapadcine-3

[0270] Various lymphocytic leukemia cell lines (Jurkat E6-1, Reh), lung cancer cell line (MSTO-211H), glioma cell line (A172), pancreatic cancer Mia PaCa-2 and human normal cells with high expression of TRAILR2 were used Lines or peripheral blood cells, such as human normal peripheral blood mononuclear cells (PBMC), human normal colonic epithelial cells (NCM-460), human normal colonic tissue cells (CCD-18Co) and human normal lung epithelial cells (BEAS-2B), To evaluate the cytotoxic effect of Zapadcine-3 on various tumor cells and normal cells. The specific research process is as follows: use trypsin (0.25%, V / V) to digest adherent cultured cells (such as MSTO-211H, etc.), detach the cells and / or directly collect suspension cultured cells (Jurkat E6-1, Reh) , resuspended in 100 μL complete medium. 5,000 adherent cells or 16,000 suspension cells were inoculated in 96-well plates for culture at 37°C overnight. Then...

Embodiment 2

[0277] Example 2 Detection of the affinity between Zapadcine-3a and TRAILR2 by ELISA

[0278] ELISA was used to evaluate the binding of Zapadcine-3a to humanized recombinant protein TRAILR2. The specific process was as follows: 2 μg / ml humanized recombinant protein TRAILR2 was coated with 1×PBS buffer (pH 7.4) at a volume of 100 μl / well 96-well plate, placed overnight at 4°C. The supernatant was discarded, and the plate was washed 3 times with PBST (PH 7.4PBS containing 0.05% Tweeen20) buffer, 5 min each time, 240 μl / well of PBS containing 5% skimmed milk powder was added, incubated at 37°C for 3 h, and blocked. Discard the blocking solution, wash the plate 3 times with 300 μl / well PBST, and add 50 μl / well of the antibody to be tested (primary antibody) or ADC gradiently diluted with PBS containing 1% skimmed milk powder or BSA, the concentration is from Start with 2-fold serial dilution at 4 μg / ml, a total of 12 concentrations, 3 replicate wells, and incubate at room tempera...

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Abstract

The invention provides an broad-spectrum, efficient and anti-tumor anti-TRAILR2 antibody-toxin-conjugate (ADC, called after Zapadcine-3(a,b,c,d,e). Toxin with cytotoxic effect is connected with an anti-TRAILR2 humanized monoclonal antibody through covalent bonds by using disulfide bond bridge connection or conventional coupling technology and chemical linkers, so that the anti-TRAILR2 humanized antibody-toxin-conjugate is formed. The ADC has the specificity of TRAILR2 positive tumors, after the combination with TRAILR2, the positive tumors can be endocytosed and enter into lysosomes of tumor cells, proteases inside the lysosomes are degraded to release free micromolecule urotoxins, so that various TRAILR2 positive tumor cells are killed specifically, the tumor growth is suppressed, the tumor cells are even completely eliminated, and the tumor is cured.

Description

technical field [0001] The present invention relates to the technical fields of biochemistry, immunochemistry, organic chemistry and medicinal chemistry, in particular, the present invention relates to an antibody-toxin-conjugate (ADC, named Zapadcine-3) and its drug use for treating tumors. Background technique [0002] According to the "Global Cancer Report 2014" published by WHO, the global cancer cases are increasing rapidly, from 14 million in 2012 to 19 million in 2025 and 24 million in 2035. The global cancer patients and deaths are disturbing Nearly half of the new cancer cases occurred in Asia, most of which were in China, and China ranked first in the number of new cancer cases. In 2012, the three most common cancers in the world were lung cancer (1.8 million), breast cancer (1.7 million), and colorectal cancer (1.4 million). The top three cancers with the highest mortality rates were lung cancer, liver cancer, and gastric cancer. Leukemia is one of the top ten hi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/68A61K38/07A61P35/00
CPCA61K38/07
Inventor 郑德先张书永潘讴东郑超朱婉夏清梅
Owner YANTAI OBIOADC BIOMEDICAL TECH LTD
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