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Anti-CD56 antibody-duocarmycin conjugate complex, preparation method and uses thereof

A technology of duocamycin and complexes, which is applied in the field of antibody-conjugated drug preparation, can solve the problems of no duocamycin and anti-CD56 antibody coupling, anti-tumor, etc., achieve good application prospects, less toxic and side effects, and proliferative highly active effect

Active Publication Date: 2018-02-23
WEST VAC BIOPHARMA CO LTD
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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 duocarmycin and anti-CD56 antibody can be effectively anti-tumor

Method used

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  • Anti-CD56 antibody-duocarmycin conjugate complex, preparation method and uses thereof
  • Anti-CD56 antibody-duocarmycin conjugate complex, preparation method and uses thereof
  • Anti-CD56 antibody-duocarmycin conjugate complex, preparation method and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065]Example 1 Preparation of CD56 antibody-coupled drug Promiximab-DUBA

[0066] 1. Preparation of anti-CD56 antibody

[0067] 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:7 is constructed on the pTT5 expression vector, EcoR I-Kozak sequence-signal peptide-VH-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-CD56 antibody-VH, composed of EcoR I-signal peptide-VH-Iggamma-1chai...

Embodiment 2

[0109] Example 2 Anti-CD56 Antibody Conjugated Drug Promiximab-DUBA Binding Ability and Purity Detection

[0110] 1. SDS-PAGE verification of the structure and purity of promiximab-DUBA

[0111] The TCEP-reduced antibody promiximab was coupled with the small molecule chemotherapeutic drug at a molar ratio of 1:6, and the promiximab-DUBA ADCs complex was obtained after coupling at room temperature (40rpm) for 3 hours. In order to prove that the CD56 antibody can still retain the original protein structure after this bioconjugation process, we used SDS-PAGE for verification. Since the small molecule DUBA contributes little to the overall molecular mass of the entire antibody Promiximab-DUBA conjugate, if Promiximab After the antibody was bioconjugated, it still maintained similar electrophoretic behavior to the unconjugated antibody, indicating that our bioconjugation process did not have a great impact on the protein structure of the Promiximab antibody.

[0112] The experimenta...

Embodiment 3

[0120] Example 3 Anti-tumor activity of CD56 antibody conjugated drug

[0121] 1. In vitro cytotoxicity test of Promiximab-DUBA

[0122] Human small cell lung cancer cells NCI-H446, NCI-H526, NCI-H524, NCI-H69 and NCI-H128 were cultured in RPMI 1640 medium containing 20% ​​fetal bovine serum in a 5% CO2, 37°C constant temperature cell culture incubator Culture; NK cells are freshly isolated and extracted; small cell lung cancer cells in the logarithmic growth phase are collected, counted and adjusted for cell density; 96-well plates are spread, and the density of NCI-H128 cells is 1000-1500 cells / well according to the growth rate of different cells. The density of NCI-H526, NCI-H524, NCI-H69 and NK cells was 10,000 cells / well, and the volume was 100 μL / well; the cells were cultured in an incubator for 24 hours before drug treatment.

[0123] The antibody and ADCs complexes were diluted with RPMI 1640 medium containing 20% ​​fetal bovine serum to an initial concentration of 2....

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Abstract

The present invention belongs to the field of preparation of antibody-drug conjugates, particularly relates to an anti-CD56 antibody-duocarmycin conjugate complex, a preparation method and uses thereof, and provides an anti-CD56 antibody-duocarmycin conjugate complex, which comprises anti-CD56 antibody, duocarmycin and a linker for conjugating the antibody and the duocarmycin, wherein one end of the linker is conjugated to the free thiol of the anti-CD56 antibody through maleimide, and the other end of the linker is conjugated to the hydroxyl of the duocarmycin through an ester bond. Accordingto the present invention, the anti-CD56 antibody-duocarmycin conjugate complex retains the high-affinity targeted CD56 binding ability of the anti-CD56 antibody, further has the efficient cell killing ability of duocarmycin, can improve the treatment effect of the drug while reduce the side effect of the drug, can be used for preparing drugs for diagnosing and treating tumors and immune or nervous system diseases, and further has good application prospect.

Description

Technical field [0001] The invention belongs to the field of antibody conjugated drug preparation, and specifically relates to an anti-CD56 antibody and dokamycin conjugated complex and its preparation method and use. Background technique [0002] Antibody-chemotherapy drug conjugates, also called Antibody-drug conjugates (ADC), give full play to the targeted safety of antibodies and the efficient killing properties of chemical drugs, and have high anti-tumor activity and low toxic and side effects. The advantage is that it is the direction for the development of a new generation of antibody-targeted drugs for refractory malignant tumors. [0003] ADC drugs include three components: antibodies against tumor antigens, potent cytotoxic chemicals (warhead drugs), and linkers (linkers) that connect the antibodies and warhead drugs. The main function of monoclonal antibodies is to serve as drug carriers and also to confer targeting to ADCs. Their ideal tumor antigens are highly ...

Claims

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Application Information

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IPC IPC(8): A61K47/68A61K31/407A61K31/437C07K16/28G01N33/574G01N33/53A61P35/00A61P37/02A61P25/00
CPCA61K31/407A61K31/437C07K16/2803G01N33/53G01N33/574
Inventor 姚于勤杨金亮余琳
Owner WEST VAC BIOPHARMA CO LTD
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