Tissue factor-targeted antibody-drug conjugate

A technology of drug conjugates and antibody drugs, applied in the field of medicine, can solve the problems of low immunosensitivity and lack of high-specificity antibody-drug conjugates

Active Publication Date: 2017-07-11
FUDAN UNIV +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still many problems that must be considered and solved in the successful development of ADCs, such as the specific recognition of the lesion by the antibody, low immunosensitivity, and efficient and rapid endocytosis; the antibody-drug linker is stable in the blood It should be highly active and can be specifically activated in the target cells and release small molecule drugs efficiently; the coupled small molecule drugs should have strong cell killing ability, etc.
[0006] It can be seen that TF plays an important role in the development of tumors, and antibody-drug conjugates have unique characteristics and advantages. However, there is still a lack of highly specific antibody-drug conjugates against human TF.

Method used

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  • Tissue factor-targeted antibody-drug conjugate
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  • Tissue factor-targeted antibody-drug conjugate

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Experimental program
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Effect test

preparation example Construction

[0155] Antibody preparation

[0156] The sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by conventional techniques, such as PCR amplification or genomic library screening. In addition, the coding sequences for the light and heavy chains can be fused together to form single-chain antibodies.

[0157] Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

[0158] In addition, related sequences can also be synthesized by artificial synthesis, especially when the fragment length is relatively short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.

[0159] At present, the DNA sequence encoding the antibody of the ...

Embodiment 1

[0208] Example 1 Discovery and Preparation of Monoclonal Antibody Targeting Human TF

[0209] Step 1. Preparation of hybridoma cells:

[0210] First, the extracellular region of human TF protein (UniProtKB / Swiss-Prot: P13726.1, from the 34th to the 251st amino acid) was immunized to 8-week-old Balb / c female mice, and the amount of TF extracellular region protein was 100μg / monkey to prepare immune spleen cells; timely prepare mouse myeloma cells (SP2 / 0) and feeder cells for fusion.

[0211] After the preparation of the above three kinds of cells is completed, PEG-mediated fusion of immune spleen cells and SP2 / 0 cells, remove PEG, resuspend in HAT complete medium containing feeder cells, inoculate into 96-well plates for culture, and test positive by ELISA Well screening. Finally, the cells in the positive wells were cloned and cultured by the limiting dilution method, and the cells with high titer, good shape, and monoclonal growth were screened by ELSIA or immunofluorescence...

Embodiment 2

[0234] Embodiment 2 Preparation of human-mouse chimeric antibody

[0235] The human-mouse chimeric antibody was constructed on the basis of the highly active and specific murine TF-mAb-SC1.

[0236] Primers were designed to introduce EcoR I and Nhe I into the variable region of the heavy chain, and Age I and BsiW I restriction endonuclease sites were introduced into the variable region of the light chain, and then the above-mentioned obtained antibody heavy chain and light chain could be The variable region sequences were respectively cloned into vectors containing the human IgG1 heavy chain constant region and the Kappa chain constant region. After identification, the constructed chimeric The human-mouse chimeric antibody was expressed and purified, and the obtained human-mouse chimeric antibody was named TF-mAb-Ch.

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Abstract

The invention discloses a tissue factor-targeted antibody-drug conjugate. The invention also discloses a method for preparing the antibody-drug conjugate (ADC). The ADC can highly specifically bind TF antigen, and has high affinity, low immunogenicity, high cytotoxicity, and high antitumor activity.

Description

technical field [0001] The present invention relates to the field of medicine, in particular to an antibody targeting tissue factor and its antibody-drug conjugate, as well as its preparation method and application. Background technique [0002] Tissue factor (TF) is a 47kDa transmembrane glycoprotein. Under normal physiological conditions, the expression of TF is mainly shielded in the sublayer of vascular endothelial cells. Once the blood vessels of the body are traumatized, TF is exposed to the blood flow, and activates the exogenous coagulation reaction by binding and activating factor VII. [0003] Studies have found that TF is abnormally activated and expressed in many tumor tissues, and plays an important role in the occurrence and development of tumors. Especially in the advanced stage of cancer, most patients are accompanied by spontaneous thrombosis, such as deep venous thrombosis (Deep-veinthrombosis, DVT), disseminated intravascular coagulation (Disseminated int...

Claims

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Application Information

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Patent Type & AuthorityApplications(China)
IPC IPC(8): A61K47/68A61K31/537A61K31/40A61P35/00C07K16/36
CPCA61K31/40A61K31/537C07K16/36C07K2317/565A61K47/68033A61K47/68031A61P35/00A61K31/4025A61K31/5365A61K47/6849A61K47/68A61K47/6843A61K47/542A61K47/545C07K2317/24C07K2317/90
Inventor余科沈竞康孟韬马兰萍张学赛李晴柔
OwnerFUDAN UNIV