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Antibody-drug conjugate targeting gpc3 and its preparation method and use

A technology of drug conjugates and antibody conjugates, which is applied in the field of medicine, can solve the problems of poor chemical stability of coupling reagents, long synthetic routes of coupling reagents, and messy electrophoretic images of antibody conjugates, etc.

Active Publication Date: 2021-05-04
SHANGHAI INST OF MATERIA MEDICA CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the methods in the prior art have long synthesis routes of coupling reagents, poor chemical stability of coupling reagents, and relatively messy electrophoresis of antibody conjugates, suggesting that there may be side reactions in the coupling process. Solve problems such as sulfhydryl exchange (reverse Michael addition reaction) during in vivo circulation

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  • Antibody-drug conjugate targeting gpc3 and its preparation method and use
  • Antibody-drug conjugate targeting gpc3 and its preparation method and use
  • Antibody-drug conjugate targeting gpc3 and its preparation method and use

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preparation example Construction

[0209] Antibody preparation

[0210] 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.

[0211] 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.

[0212] 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.

[0213] At present, the DNA sequence encoding the antibody of the ...

Embodiment 1

[0389] Synthesis and preparation of the compound shown in embodiment 1, formula Ic

[0390] The substituted maleamide linker fragment molecule represented by formula Ia listed in the first aspect of the present invention can be synthesized by the method in Scheme 1, and intermediate A is obtained by reacting n glycol with tert-butyl bromoacetate, and then combined with Aromatic nucleophilic substitution of substituted nitrofluorobenzenes affords intermediate B. In addition, intermediate B can also be obtained by reacting p-tosylate-protected intermediate F with substituted nitrofluorophenols. The nitro group in intermediate B is reduced to amino to obtain intermediate C, which is then reacted with 2,3-dibromomaleic anhydride to obtain intermediate D, and then subjected to substitution reaction with arylthiophenol to obtain the linker fragment Molecule E. Molecules of series F are obtained by condensation with dipeptide / tripeptide-PAB cytotoxic drug linkers. The reaction sch...

Embodiment 2

[0435] Example 2, Preparation and identification of anti-GPC3 antibody

[0436] The GPC3 antibody of the present invention is prepared by the following method: use a phage display antibody library to screen against GPC3 to obtain a human anti-GPC3 antibody, and sequence and identify the antibody with good affinity. The sequence information is as follows:

[0437] Table 2 Human anti-GPC3 antibody GPC3-6 heavy chain variable region CDR and light chain variable region CDR

[0438]

[0439] Table 3 Sequences of human anti-GPC3 antibody GPC3-6 heavy chain and light chain framework regions (FR 1-4)

[0440]

[0441]

[0442] Note: In the above table, aa represents the amino acid sequence, and nt represents the nucleotide sequence.

[0443] SEQ ID NO: 30GPC3-6 heavy chain variable region (VH) amino acid sequence 120aa

[0444] QVHLVQSGAEVQKPGSSVKVSCKASGGTFSSYGINWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTA VYYCARGSGLLPRIGGYWGKGTMVTVSS

[0445] SEQ ID NO:29GPC3...

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Abstract

The present invention describes an antibody-drug conjugate against Glypican protein 3 (GPC3), which uses a novel double-substituted maleimide linker to combine strong cytotoxic active substances and biomacromolecules coupling. This type of linker can selectively act on the disulfide chain at the same time, thereby greatly improving the material uniformity of the conjugate. The conjugate prepared by the linker of the present invention has high inhibitory activity on cell lines expressing GPC3 antigen. In addition, the present invention also provides the preparation method and application of the above-mentioned conjugate.

Description

technical field [0001] The present invention relates to the field of medicine, more specifically, to an antibody-drug conjugate targeting glypican protein 3 (GPC3) and its preparation method and application. Background technique [0002] Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third most common cause of cancer death in the world. According to the American Cancer Society, hepatocellular carcinoma (HCC) accounts for approximately 75 percent of liver cancer cases, and symptoms are often not present until advanced stages of liver cancer. Currently, surgery is the most effective treatment for HCC. However, the tumor recurrence rate after radical hepatectomy is high, and the 5-year survival rate is only 10%. Furthermore, because most patients with HCC are diagnosed at a later stage of the disease, definitive treatments, including chemotherapy, chemoembolization, resection, and proton beam therapy, may often be ineffective. Sorafenib (Nexavar), the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/30C07K19/00C12N15/13C12N15/62C12N5/10A61K47/68A61K38/07A61P35/00
CPCA61K38/07A61K47/6851A61P35/00C07K16/303C07K2317/565C07K2319/00
Inventor 沈竞康孟韬康小强赖寿鹏马兰萍彭红丽王昕陈驎杜志彦王英于霆张永良
Owner SHANGHAI INST OF MATERIA MEDICA CHINESE ACAD OF SCI
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