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Antibody drug conjugate, an intermediate, a preparation method and application of antibody drug conjugate

An antibody drug and conjugate technology, applied in the field of biomedicine, can solve the problem of a single type of antibody drug conjugate, and achieve the effects of good inhibitory effect, good druggability, and good targeting.

Pending Publication Date: 2021-01-19
上海复旦张江生物医药股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] The technical problem to be solved by the present invention is to overcome the single defect of existing antibody-drug conjugates, and provide an antibody-drug conjugate, its intermediate, preparation method and application

Method used

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  • Antibody drug conjugate, an intermediate, a preparation method and application of antibody drug conjugate
  • Antibody drug conjugate, an intermediate, a preparation method and application of antibody drug conjugate
  • Antibody drug conjugate, an intermediate, a preparation method and application of antibody drug conjugate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0108] Example 1: Preparation of anti-Claudin18.2 antibody IMAB362, anti-Trop2 antibody RS7, anti-CD30 antibody CAC10 and anti-Her2 antibody Trastuzumab

[0109] In the present invention, the monoclonal antibody IMAB362 with high affinity and specificity targeting Claudin18.2 is selected, the amino acid sequence of its light chain is shown in SEQ ID NO:1, and the amino acid sequence of its heavy chain is shown in SEQ ID NO:2. IMAB362 light and heavy chain nucleotide sequences were obtained by total gene synthesis (Suzhou Jinweizhi), and were separately constructed into pV81 vectors (such as figure 1 ), transformed into Trans 1-T1 competent cells (purchased from Beijing Quanshijin Biology, CD501-03) by ligation, from which clones were selected for PCR identification and submitted for inspection, sequencing confirmation, cultured and amplified positive clones for plasmid pumping, so as to obtain the antibody light chain eukaryotic expression plasmid IMAB362-L / pV81 and the antibo...

Embodiment 2

[0113] Embodiment 2: the synthesis of compound CLB-SN38

[0114] Step A:

[0115]

[0116] 10 g (25.5 mmol) of SN38 (purchased from Bailingwei) was suspended in 200 mL of anhydrous dichloromethane, 5 mL of pyridine and 11.3 g (51 mmol) of Boc-anhydride were added, and the reaction was carried out overnight at room temperature. After the reaction was completed, the solvent was distilled off under reduced pressure to obtain a light yellow solid, which was washed with petroleum ether to obtain 9.8 g of the final product, with a yield of 78%. MS[M+H] + : 493.2.

[0117] Step B:

[0118]

[0119] Dissolve 5g (10.1mmol) of compound Boc-SN38 in 100mL of anhydrous dichloromethane, add 2mL of pyridine, 2.47g (20.2mmol) of DMAP, cool down to 0°C, and then dissolve 1.5g (5.0mmol) of triphosgene in dichloromethane Slowly add methane, react at 0°C for 1 h, add 11.3 g (20.0 mmol) of raw material VII (purchased from Suzhou Lianning Biology), and raise to room temperature for 5 h. ...

Embodiment 3

[0137] Embodiment 3: Preparation of antibody drug conjugate

[0138] A: IMAB362-CLB-SN38-1 conjugate

[0139] IMAB362 antibody solution, concentration 15mg / ml, 12 equivalents of TECP (purchased from Sigma-Aldrich) was added, stirred at 37°C for 1 hour to completely open the disulfide bonds between the antibody chains, and then the excess TCEP was removed through a G25 desalting column. Dissolve 12 equivalents of compound CLB-SN38-1 in DMSO, add dropwise to the reduced antibody solution, and add DMSO to a final concentration of 10% (v / v), and stir the reaction at 25°C under sealed conditions for 1 Hours, after the reaction was complete, the sample was filtered using a 0.22 μm membrane. Use TFF (tangential flow ultrafiltration system) to purify and remove uncoupled small molecules. The buffer is 50mM PB, 1.0mM EDTA solution (pH6.0), and the replacement volume is 15 times the volume. After purification, add a final concentration of 6% sucrose and place it in Store in -20°C refr...

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Abstract

The invention relates to an antibody drug conjugate, and an intermediate, a preparation method and application of the antibody drug conjugate. Specifically, the invention discloses the antibody drug conjugate, and the structure of the antibody drug conjugate is shown as Formula I. The antibody drug conjugate has a very strong targeting property, achieves a very good inhibition effect on various tumor cells expressing Claudin 18.2, CD30, Trop2, Her2 and the like, and also has very good druggability and high safety. The invention also discloses an intermediate of the antibody drug conjugate. Theintermediate can be coupled with various antibodies.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to an antibody-drug conjugate, an intermediate, a preparation method and an application thereof. Background technique [0002] Antibody-drug conjugates (ADCs) are a new generation of antibody-targeted therapeutic drugs, which are mainly used in the treatment of cancer tumors. ADC drug consists of three parts: small molecule cytotoxic drug (Drug), antibody (Antibody) and linker (Linker) connecting antibody and cytotoxic drug. Small molecule cytotoxic drug is bound to antibody protein by chemical coupling method. ADC drugs use antibodies to specifically recognize and guide small molecule drugs to cancer cell targets expressing cancer-specific antigens, and enter cancer cells through endocytosis. The linker part is broken under the action of intracellular low pH value environment or lysosomal protease, releasing small molecular cytotoxic drugs, so as to achieve the effect of specifically ki...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/68A61K31/4745A61P35/00C08G65/333C08G65/334
CPCA61K47/6851A61K31/4745A61P35/00C08G65/33396C08G65/3348
Inventor 吴劲松郭青松鲍彬高贝蔡俊立杨彤沈毅珺
Owner 上海复旦张江生物医药股份有限公司
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