Unlock instant, AI-driven research and patent intelligence for your innovation.

Construction and application of a bispecific antibody cd19×cd3

A bispecific antibody and specific technology, which is applied in the field of construction and preparation of bispecific antibodies, can solve the problems of limited curative effect of solid tumors, and achieve the effect of improving efficacy

Active Publication Date: 2019-03-12
WUHAN YZY BIOPHARMA CO LTD
View PDF3 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, immunotherapy can only remove a small amount of scattered tumor cells, and has limited efficacy for advanced solid tumors

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Construction and application of a bispecific antibody cd19×cd3
  • Construction and application of a bispecific antibody cd19×cd3
  • Construction and application of a bispecific antibody cd19×cd3

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1: Construction of expression vectors for bispecific antibodies (CD19×CD3, M902)

[0063] 1. Bispecific antibody sequence design

[0064] The bispecific antibody targeting CD19 and CD3 is named CD19×CD3, such as figure 2 The anti-CD19 side is in ScFv-Fc form, including anti-CD19 VH, VL, and Fc domains; the anti-CD3 side is in IgG form, including anti-CD3 heavy chain and light chain, including Fab and Fc domains. Among them, ScFv-Fc undergoes KKW transformation on one side of Fc, and one side of IgG form undergoes LDY transformation on Fc. For the specific Fc transformation process, refer to PCT / CN2012 / 084982, so that they are not easy to form homodimers, but easy to form hybrid dimers, namely CD19×CD3 bispecific antibody. At the same time, in order to express the diabody in CHO cells and secrete it into the culture medium, the leader peptide sequence of the mouse kappa chain was selected as the secretion signal peptide. The amino acid sequence and nucleic ac...

Embodiment 2

[0089] Example 2: Expression and purification of bispecific antibodies

[0090] 1. Expression of bispecific antibodies

[0091] The endotoxin-free large-scale extraction kit (Qiagen, 12391) was used for large-scale extraction of plasmids, and the specific operation was carried out according to the instructions provided by the manufacturer. CHO-S cells were cultured in CD CHO medium (Gibco, 10743-029) according to the instructions provided by the manufacturer, placed at 37°C, 5% CO 2 Cultured in a cell culture incubator, after the cells were prepared, the plasmid pCHO1.0-anti-CD3-HL-LDY and pCHO1.0-hygromycin-anti-CD19 -ScFv-Fc-KKW were co-transfected together into CHO-S cells, and the co-transfection of these two plasmids was designed to express bispecific antibodies against CD19×CD3.

[0092] On the second day after transfection, the culture temperature was lowered to 32° C., and 3.5% FeedA was added every day. After 14 days of culture, the expression supernatant was harves...

Embodiment 3

[0095] Example 3: Determination of the binding activity of bispecific antibodies to cells (FACS)

[0096] The bispecific antibody of the invention binds to the target antigen on the corresponding cell. In the present invention, Raji (purchased from ATCC, CCL-86) is used as CD19-positive cells, Jurkat (Jurkat, TIB-152) is used as CD3-positive cells, and the cell-binding activity is determined by the diabody prepared in the present invention.

[0097] 1. Detection of binding activity of bispecific antibody to Raji cells by flow cytometry

[0098] Sufficient Raji cells were cultured, and the cells were collected by centrifugation. Dilute the bispecific antibody at the same time, the concentration starts from 1000nmol, and 3-fold gradient dilution is obtained to obtain 12 concentration gradients for later use. Wash the collected cells twice with PBS+1%FBS, then resuspend the cells in PBS+1%FBS to 4×10 6 cells / ml, the cells were plated in a 96-well plate, 50ul per well (2×10 5 ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides a bispecific antibody which is composed of a single-chain unit and a mono-valent unit, wherein the mono-valent unit, aiming to surface antigen CD3 of immune cells, has a specifically combining capability while the single-chain unit, aiming to surface antigen CD19 of tumor cells, has a specifically combining capability. The single-chain unit includes a single-chain variable fragment (ScFv) fused with an Fc fragment. The mono-valent unit includes a light chain-heavy chain pair. The application also provides a preparation of the bispecific antibody and medicinal applications of these antibodies.

Description

technical field [0001] The present invention relates to the technical field of immunology. Specifically, it relates to the construction and preparation methods of bispecific antibodies. Background technique [0002] Bispecific antibody (bispecific antibody, BiAb) is an artificial antibody containing two specific antigen-binding sites, which can build a bridge between target cells and functional molecules (cells) to produce oriented effector functions. BiAb has broad application prospects in biomedicine, especially in tumor immunotherapy. Killing tumor cells through BiAb-mediated cytotoxicity is a hot topic in current immunotherapy application research. Sexual killing. The following is an introduction to the background technology of the studied immune cell antigens and tumor cell antigens, as well as the development of related technologies. [0003] 1. CD3 [0004] The CD3 molecule is composed of 4 subunits: δ, ε, γ, ζ, the molecular masses are 18.9k Da, 23.1k Da, 20.5kD...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/46C07K16/30C12N15/85A61K39/395A61P35/00
Inventor 张敬刘晓燕付思思刘金刚范克索周鹏飞
Owner WUHAN YZY BIOPHARMA CO LTD
Features
  • R&D
  • Intellectual Property
  • Life Sciences
  • Materials
  • Tech Scout
Why Patsnap Eureka
  • Unparalleled Data Quality
  • Higher Quality Content
  • 60% Fewer Hallucinations
Social media
Patsnap Eureka Blog
Learn More

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2025 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com