Bispecific antibody for CD20 and CD3

A bispecific antibody and specific technology, applied in the field of biopharmaceuticals, can solve the problems of short half-life, poor stability, and low expression, and achieve the effect of long half-life, stable structure, and high expression

Active Publication Date: 2018-05-22
BEIJING DONGFANG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The anti-CD20 / CD3 bispecific antibody of the present invention overcomes the defects of poor stability, low affinity, short half-life, and low expression of bispecific antibodies in the prior art, and not only retains high affinity for the antigen and a longer half-life, At the same time, the Fc region has been specifically modified to make it more stable in vivo than bispecific antibodies using KiH technology. The antibody has a high expression level and can be used for industrial production

Method used

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  • Bispecific antibody for CD20 and CD3
  • Bispecific antibody for CD20 and CD3
  • Bispecific antibody for CD20 and CD3

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1. Construction of expression vector for bispecific antibody molecule

[0053] The constructed bispecific antibody molecule (antibody named JY052) contains the monovalent unit of anti-CD20 antibody and the single chain unit of anti-CD3 antibody, wherein the monovalent unit contains the light chain and heavy chain of anti-CD20 antibody, and the single chain unit contains anti-CD3 antibody The light chain and heavy chain are connected by linker, the configuration diagram is as follows figure 1 . PTSE was chosen as the expression vector to clone and express anti-CD20 light chain, heavy chain genes and anti-CD3 single chain genes. The light chain gene, heavy chain gene and single chain gene were added with Sall and BamHl restriction sites on both sides of the coding region sequence, and three genes were synthesized and cloned into the PUC19 vector. The three plasmids were transformed into TOP competence, and after small extraction kit for small extraction, the vector ...

Embodiment 2

[0054] Example 2. Expression and purification of bispecific antibody molecules

[0055] 1). Expression of bispecific antibodies

[0056] Use endotoxin-free large-scale extraction kit to carry out large-scale plasmid extraction, the specific operation steps are as follows:

[0057] (1) Take 200ul of activated bacteria solution and place it in a 500ml shake flask (containing 200ml amp+ LB medium), and cultivate overnight at 37°C, 220rpm shaker;

[0058] (2) Take 200ml of the bacterial solution cultured overnight, add it to the centrifuge tube, centrifuge at 7000rpm for 5 minutes to collect the bacteria, try to remove all the supernatant;

[0059] (3) Add 12.5ml Buffer P1 (RNase A has been added) to the centrifuge tube with bacterial pellet, mix well with a vortex shaker to suspend the bacterial pellet;

[0060] (4) Add 12.5ml Buffer P2 to the centrifuge tube, gently mix upside down and mix 8-10 times to fully lyse the bacteria, leave it at room temperature for 3-5 minutes, until the soluti...

Embodiment 3

[0074] Example 3. Binding of bispecific antibody molecules to cells overexpressing CD20 or CD3

[0075] The CD20-overexpressing lymphoma cell line (Raji) was used to detect the binding of the bispecific antibody to CD20 on the cell surface. The CD20 / CD3 bispecific antibody of patent CN 104640881A and patent CN1331888C was compared with JY052. The steps are as follows: The Raji cells were collected by centrifugation, and various antibodies were diluted at the same time, the highest concentration was 1uM, 3-fold gradient dilution; washed the collected cells with PBS+1% BSA three times, then added PBS+1% BSA to resuspend the cells, and then plate the cells on In 96-well plate, 1×10 per well 5 Add 100ul of the diluted bispecific antibody and incubate at room temperature for 1 hour; centrifuge to remove the supernatant, wash the cells three times with PBS, then resuspend the cells with the diluted Alexa488-labeled anti-human IgG-Fc antibody, avoid at room temperature Incubate for 1 ho...

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Abstract

The invention relates to the technical field of biological pharmacy and particularly discloses a bispecific antibody for CD20 and CD3. The bispecific antibody comprises univalent unit, a single-chainunit and a linker, wherein the univalent unit is a light chain-heavy chain pair with a specific binding capability for an antigen CD20 on the surface of a tumor cell; the single-chain unit can be usedfor specifically binding a CD3 antigen on the surface of an immune cell; the end N of a heavy chain and the end C of a light chain of the single-chain unit are connected by the linker to form a Fab-Fc form; the univalent unit is connected with the single-chain unit through a disulfide bond of a hinge region and a pestle mortar structure of a CH3 structural domain. According to the bispecific antibody, CD20 and CD3 can be specifically bound and the immune effector cell can be targeted to the tumor cell, so that the killing effect of the immune effector cell on the tumor cell is improved; the bispecific antibody can be used for treating CD20 antigen positive B-cell neoplasms, comprising non-Hodgkin lymphoma, chronic lymphocytic leukemia and the like, and has a broad application prospect inthe field of immunotherapy of tumors.

Description

Technical field [0001] The invention relates to the technical field of biopharmaceuticals, in particular to a bispecific antibody against CD20 and CD3, and its application in tumors and other immune diseases. Background technique [0002] Bispecific antibody (BsAb), also known as bifunctional antibody, can specifically bind to two different antigens or two different epitopes at the same time. Due to its specificity and bifunctionality, it has good application effects and prospects in tumor immunotherapy and autoimmune diseases. The present invention is a bispecific antibody against CD20 and CD3 antigens, which can simultaneously bind the CD20 antigen on malignant B cells and the CD3 antigen on immune cells, wherein the specific anti-tumor antigen part can bind to tumor cells and resist immunocompetent cells. The CD3 part has the effect of activating NK cells or T cells, targeting immune cells to tumor cells, increasing the local NK cell or T cell concentration, thereby enhancing...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46A61K39/395A61P35/00A61P35/02
CPCC07K16/2809C07K16/2887C07K2317/31C07K2317/52C07K2317/55C07K2317/73
Inventor 周海平张稳裴爽文圣梅白义
Owner BEIJING DONGFANG BIOTECH
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