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Antibody-drug conjugates targeting TF and their preparation and use

A drug conjugate and conjugate technology, which is applied in the field of medicine, can solve the problems of cluttered electrophoresis of antibody conjugates, long synthesis routes of coupling reagents, poor chemical stability of coupling reagents, etc.

Inactive Publication Date: 2021-07-30
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, the above-mentioned technology has a long synthetic route of the coupling reagent, poor chemical stability of the coupling reagent, and messy electrophoresis of the antibody conjugate, suggesting that there may be side reactions in the coupling process, and the existing scheme does not solve the problem of in vivo circulation. Sulfhydryl exchange (reverse Michael addition reaction) and other issues

Method used

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  • Antibody-drug conjugates targeting TF and their preparation and use
  • Antibody-drug conjugates targeting TF and their preparation and use
  • Antibody-drug conjugates targeting TF and their preparation and use

Examples

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preparation example Construction

[0127] Antibody preparation

[0128] 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.

[0129] 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.

[0130] 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.

[0131] At present, the DNA sequence encoding the antibody of the ...

Embodiment 1

[0233] Synthesis and preparation of the compound shown in embodiment 1, formula Ic

[0234] 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

[0280] Embodiment 2, preparation of antibody conjugate

[0281] 2.1 Preparation of TF-mAb-H39-BL9-MMAE

[0282] The stock solution of the humanized antibody TF-mAb-H39 targeting TF was treated with 50 mM potassium dihydrogen phosphate-sodium hydroxide (KH 2 PO 4 -NaOH) / 150mM sodium chloride (NaCl) / 1mM diethyltriaminepentaacetic acid (DTPA), pH7.4 reaction buffer diluted to 5mg / mL, adding 10-fold excess molar ratio of tris(2-carboxyethyl ) Phosphine hydrochloride (TCEP), the reaction solution was stirred at 10°C for 4 hours.

[0283] The above reaction liquid was cooled to 5°C, an appropriate amount of diethylacetamide (DMA) was added without purification, and then compound Ic-1 prepared in Example 1 (10 mg / ml pre-dissolved in DMA Middle), ensure that the volume ratio of DMA in the reaction system does not exceed 10%, and agitate at 25°C for 2.5 hours for coupling.

[0284] The coupling reaction mixture was purified by histidine-acetic acid / sucrose gel filtration at pH 6.0 ...

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Abstract

The invention describes the anti-tissue factor (TF) antibody-drug conjugate, which uses a novel double-substituted maleimide linker to couple strong cytotoxic active substances and biomacromolecules. 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 TF 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 tissue factor (TF) and its preparation method and application. Background technique [0002] Tissue factor (TF) is a 47kDa transmembrane glycoprotein. Under normal physiological conditions, the expression of TF is mainly shielded in the sublayer of vascular endothelial cells. Once the blood vessels of the body are traumatized, TF is exposed to the blood flow, and activates the exogenous coagulation reaction by binding and activating factor VII. Studies have found that TF is abnormally activated and expressed in many tumor tissues, and plays an important role in the occurrence and development of tumors. Especially in the advanced stage of cancer, most patients are accompanied by spontaneous thrombosis, such as deep-vein thrombosis (Deep-vein thrombosis, DVT), disseminated intravascular coagulation (Disseminated intravascular coagulation, D...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K47/68A61P35/00A61P37/02A61P29/00C07K16/28
CPCA61K47/68A61P29/00A61P35/00A61P37/02C07K16/2896C07K2317/565C07K2317/92
Inventor 沈竞康余科孟韬马兰萍张永良王昕张学赛陈驎于霆
Owner SHANGHAI INST OF MATERIA MEDICA CHINESE ACAD OF SCI
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