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Anti-CD56-antibody and aplysiatoxin coupled composite, and preparation method and application thereof

A technology of dolastatin and complex, applied in chemical instruments and methods, anti-animal/human immunoglobulin, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc. problem, to achieve the effect of improving utilization efficiency, improving treatment effect, and good affinity

Active Publication Date: 2018-03-02
WEST VAC BIOPHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, there is no relevant report on whether the coupling of alastatin and anti-CD56 antibody can effectively anti-tumor

Method used

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  • Anti-CD56-antibody and aplysiatoxin coupled composite, and preparation method and application thereof
  • Anti-CD56-antibody and aplysiatoxin coupled composite, and preparation method and application thereof
  • Anti-CD56-antibody and aplysiatoxin coupled composite, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1 Preparation of CD56 Antibody Conjugated Drug Promiximab-MMAE

[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-1c...

Embodiment 2

[0109] Example 2 Anti-CD56 Antibody Conjugated Drug Promiximab-MMAE Binding Ability, Targeting and Affinity Detection

[0110] 1. SDS-PAGE verification of the structure and purity of Promiximab-MMAE

[0111] The TCEP-reduced antibody promiximab was coupled with the small molecule chemotherapeutic drug at a molar ratio of 1:6, and was coupled at room temperature (40rpm) for 3 hours to obtain the promiximab-MMAE ADCs complex. 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-MMAE 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 expe...

Embodiment 3

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

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

[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 invention belongs to the field of preparation of antibody-coupled-medicines, and particularly relates to an anti-CD56-antibody and aplysiatoxin coupled composite, and a preparation method and an application thereof. The anti-CD56-antibody and aplysiatoxin coupled composite includes the anti-CD56-antibody and aplysiatoxin, and a connection molecule which couples the anti-CD56-antibody with theaplysiatoxin, wherein one end of the connection molecule is coupled with a free sulfydryl group on the anti-CD56-antibody via maleimide, while the other end is coupled with a hydroxyl group on the aplysiatoxin via an ester bond. The anti-CD56-antibody coupled composite not only maintains the high-affinity capability of targeted combination between the anti-CD56-antibody and CD56 but also has high-effective cell killing capability of the aplysiatoxin, so that the composite can improve therapy effect of a medicine and also reduces toxic and side effects on human body due to the medicine. The composite can be used for preparing medicines for diagnosing and treating tumors, immunosystem diseases or nervous system diseases, and has great application prospect.

Description

technical field [0001] The invention belongs to the field of preparation of antibody-coupled drugs, and in particular relates to an anti-CD56 antibody-dolastatin-coupled complex and a preparation method and application thereof. Background technique [0002] Antibody-drug conjugates (ADC) are a new type of conjugates with antibody-targeted as carrier and small molecule cytotoxic warhead as effector molecule. It retains the specific targeting of antibody drugs and the efficient killing function of small molecule chemotherapy drugs, and has the advantages of high anti-tumor activity and less toxic side effects in vivo, and is a research hotspot of anti-tumor targeted drugs. [0003] The design of ADC drugs needs to consider the target, cytotoxic small molecules and bioconjugation methods. CD56, also known as Neural Cell Adhesion Molecule 1 (NCAM1), is one of the nerve cell adhesion molecules, a type I membrane glycoprotein, and a member of the immunoglobulin superfamily. Its ...

Claims

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Application Information

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IPC IPC(8): A61K47/68A61K38/07A61K38/08C07K16/28A61P35/00A61P37/02A61P25/00
CPCA61K38/07A61K38/08C07K16/2803C07K2317/33C07K2317/56C07K2317/73C07K2317/77C07K2317/92
Inventor 余琳姚于勤杨金亮
Owner WEST VAC BIOPHARMA CO LTD
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