Anti-cd56 antibody and dolastatin coupling complex and its preparation method and use

A dolastatin and compound technology, applied in chemical instruments and methods, anti-animal/human immunoglobulin, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can solve anti-tumor, etc. problem, to achieve the effect of improving utilization efficiency, improving therapeutic effect, and high proliferative activity

Active Publication Date: 2020-04-28
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 dolastatin coupling complex and its preparation method and use
  • Anti-cd56 antibody and dolastatin coupling complex and its preparation method and use
  • Anti-cd56 antibody and dolastatin coupling complex and its preparation method and use

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 variable regions of the light and heavy chains of the candidate clones are obtained, and the corresponding sequences of each clone are selected to construct a chimeric expression vector for human-mouse chimeric antibody transformation. The light chain variable region gene whose amino acid sequence is as shown in Seq ID NO: 7 was constructed on pTT5 expression vector, EcoRI-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, which is composed of EcoR I-signal peptide-V...

Embodiment 2

[0109] Example 2 Anti-CD56 antibody conjugated drug Promiximab-MMAE binding, targeting and affinity detection

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

[0111] TCEP reduction treatment of antibody promiximab and small-molecule chemotherapeutics according to the molar ratio of 1:6, room temperature coupling (40rpm) for 3h to obtain promiximab-MMAE ADCs complex. In order to prove that the CD56 antibody can still retain the original protein structure after this biological coupling process, we used SDS-PAGE to verify. Since the small molecule DUBA contributes little to the overall molecular weight of the entire antibody Promiximab-MMAE conjugate, if Promiximab After the bio-conjugation operation, the antibody still maintains the similar electrophoretic behavior with the unconjugated antibody, indicating that our bio-conjugation process does not have a huge impact on the protein structure of Promiximab antibody.

[0112] The experimental procedure is as f...

Embodiment 3

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

[0121] 1. Promiximab-MMAE in vitro cytotoxicity test

[0122] Culture human small cell lung cancer cells NCI-H446, NCI-H526, NCI-H524, NCI-H69 and NCI-H128 with RPMI 1640 medium containing 20% ​​fetal bovine serum, placed in a 5% CO2, 37℃ constant temperature cell incubator Culture; NK cells are freshly separated and extracted; small cell lung cancer cells in the logarithmic growth phase are collected, counted and cell density is adjusted; 96-well plates are laid, and the cell density of NCI-H128 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 is 10,000 cells / well and the volume is 100 μL / well; the cells are cultured in an incubator for 24 hours, and then treated with drugs.

[0123] The antibody and ADCs complexes were diluted with 20% fetal bovine serum RPMI 1640 medium to an initial concentration of 2.4μM, filtered with a 0.22μm...

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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 antibody-conjugated drug preparation, and specifically relates to an anti-CD56 antibody and docetoxin conjugated complex and a preparation method and application thereof. Background technique [0002] Antibody-drug conjugates (ADC) are a new type of conjugate that uses antibody targeting as a carrier and small molecule cytotoxic warheads as effector molecules. It retains the specific targeting of antibody drugs and the high-efficiency killing function of small-molecule chemotherapeutics. It has the advantages of high anti-tumor activity and low toxic and 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 biological coupling methods. CD56, also known as Neural Cell Adhesion Molecule 1 (NCAM1), is a type of neural cell adhesion molecule, a type I membrane glycoprotein, and a member of the immunoglobulin s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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