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Coupled compound of anti-5T4 antibody and maytansine derivative DM4, preparation method and application

A technology of maytansine derivatives and complexes, which can be used in drug combinations, chemical instruments and methods, and anti-animal/human immunoglobulins, etc., can solve the problem of no anti-tumor, and achieve less toxic side effects and high proliferation activity. , the effect of good application prospects

Active Publication Date: 2018-06-22
GUANGDONG ZHONGSHENG PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no relevant report on whether the coupling of maytansine derivative DM4 to anti-5T4 antibody can effectively fight tumors

Method used

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  • Coupled compound of anti-5T4 antibody and maytansine derivative DM4, preparation method and application
  • Coupled compound of anti-5T4 antibody and maytansine derivative DM4, preparation method and application
  • Coupled compound of anti-5T4 antibody and maytansine derivative DM4, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 Preparation of 5T4 antibody conjugated drug H6-DM4

[0063] 1. Preparation of anti-5T4 antibody

[0064] Through sequencing and sequence analysis, the amino acid sequences of the light and heavy chain variable regions of the candidate clones were obtained, and the corresponding sequences of each clone were selected to construct chimeric expression vectors for human-mouse chimeric antibody transformation. The light chain variable region gene whose amino acid sequence is shown in Seq ID NO:3 is constructed on the pTT5 expression vector, EcoR I-Kozak sequence-signal peptide-VL-constant region-stop codon-Hind III, light chain constant region The gene sequence is: NCBI Reference Sequence: NG_000834.1, EcoRI-signal peptide-VL-Ig kappa chain C region-Hind III. The heavy chain variable region gene whose amino acid sequence is shown in Seq ID NO:1 is cloned into the expression vector pTT5 / anti-5T4 antibody-VH, composed of EcoR I-signal peptide-VH-Iggamma-1chain C regi...

Embodiment 2

[0085] Example 2 Preparation of Anti-5T4 Antibody and Maytansine Derivative DM4 Coupling Complex H6-DM4

[0086] 1. Explore the reaction conditions

[0087]The antibody and the maytansine derivative DM4 are in a certain molar ratio, and the SPDB-DM4 dissolved in dimethylformamide (DMF) is directly added to the antibody, and the linker molecule that couples the antibody to the maytansine derivative DM4 is added to fully After mixing, react at room temperature and 80 rpm for a certain period of time. The specific reaction molar ratio and reaction time are explored in Table 2.

[0088] Table 2 explores the reaction condition setting table

[0089]

[0090]

[0091] Take out the reaction solution, and use a desalting column HiTrap TM Desalting 5mL to remove the remaining unreacted small molecules in the reaction system; add the collected eluate to the rinsed ultrafiltration tube, centrifuge at 4000rpm / min for 15-20min and pour Remove the liquid in the lower layer of the ul...

Embodiment 3

[0095] Example 3 Detection of Binding Ability and Purity of Anti-5T4 Antibody Conjugated Drug H6-DM4

[0096] 1. Detection of 5T4 antibody conjugated drug H6-DM4 targeting by flow cytometry

[0097] Culture 5T4-positive small cell lung cancer cell lines, collect gastrointestinal tumor cell lines in the logarithmic growth phase, and then divide the cells into several groups on average, and the number of cells in each group is 1×10 6 ; Cell grouping: negative control group (PBS group), experimental group (H6-DM4); resuspend the cells in each group with PBS buffer salt, centrifuge at 3500rmp at 4°C for 3min, absorb the supernatant with the tip of a gun; Add 100 μL PBS to the negative control group, add 100 μL antibody H6-DM4 (concentration: 10 μg / mL) to the experimental group, resuspend, and incubate at 4°C for 30 min; centrifuge the cells in each group at 3500 rpm for 3 min, and absorb the supernatant. Add 300 μL PBS buffer salt to each group, resuspend the cells twice, then ce...

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Abstract

The invention belongs to the field of preparation of antibody coupled medicines, and particularly relates to a coupled compound of an anti-5T4 antibody and a maytansine derivative DM4, a preparation method and an application. The coupled compound of the anti-5T4 antibody and the maytansine derivative DM4 is characterized by comprising the anti-5T4 antibody, the maytansine derivative DM4, and a joint molecule coupling the antibody and the maytansine derivative DM4, wherein one terminal of the joint molecule coupling the antibody and the maytansine derivative DM4 is coupled with amino of the anti-5T4 antibody via succinimide activated carboxylic ester; and the other terminal of the joint molecule forms a disulfide bond to be coupled with sulfydryl of the DM4. The antibody coupled compound reserves high affinity of the anti-5T4 antibody in target binding with 5T4, and high-efficiency cell killing ability of the maytansine derivative DM4; a medication effect is improved; toxic and side effects of a medicine on an organism are reduced at the same time; and the coupled compound can be used for preparing a medicine for tumor diagnosis and treatment and has good application prospects.

Description

technical field [0001] The invention belongs to the field of preparation of antibody-coupled drugs, and in particular relates to a conjugated complex of an anti-5T4 antibody and a maytansine derivative DM4, a preparation method and an application. Background technique [0002] Antibody-chemotherapy drug conjugates, also known as antibody-drug conjugates (ADC), give full play to the targeting safety of antibodies and the high-efficiency lethality of chemical drugs, and have high anti-tumor activity and low toxic and side effects. The advantages are the direction of the development of a new generation of antibody-targeted drugs for refractory malignant tumors. [0003] ADC drugs consist of three components: antibodies against tumor antigens, potent cytotoxic chemicals (warhead drugs), and linker molecules (linkers) that link antibodies and warhead drugs. The main function of the monoclonal antibody is that the drug carrier also confers targeting to the ADC. The ideal tumor an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/68A61K31/5365C07K16/30A61P35/00
CPCA61K31/5365C07K16/30C07K2317/92C07K2317/73Y02P20/55
Inventor 杨金亮汪瑞雪姚于勤陈小新龙超峰陈俐娟杨莉
Owner GUANGDONG ZHONGSHENG PHARMA
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