Construction and application of bispecific antibody CD19*CD3

A bispecific antibody and antibody technology, applied in the direction of antibody, anti-animal/human immunoglobulin, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can solve the problem of limited curative effect of solid tumors , to achieve the effect of improving the efficacy

Active Publication Date: 2015-08-12
WUHAN YZY BIOPHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, immunotherapy can only remove a small amount of scatt...

Method used

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  • Construction and application of bispecific antibody CD19*CD3
  • Construction and application of bispecific antibody CD19*CD3
  • Construction and application of bispecific antibody CD19*CD3

Examples

Experimental program
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Embodiment 1

[0067] Example 1: Construction of expression vectors for bispecific antibodies (CD19×CD3, M904)

[0068] 1. Bispecific antibody sequence design

[0069] The bispecific antibody targeting CD19 and CD3 is named CD19×CD3, M904, as shown in the figure ( figure 2 ), both anti-CD19 and anti-CD3 are in the form of ScFv-Fc, including fusion peptides of anti-CD3, containing VH, VL, and Fc domains. Among them, anti-CD19 undergoes KKW transformation on one side of Fc, and one side of antibody CD3 undergoes LDY transformation on Fc. For the specific Fc transformation process, see PCT / CN2012 / 084982, so that they are not easy to form homodimers, but easy to form hybrid dimers, that is, CD19 ×CD3 bispecific antibody. At the same time, in order to express the diabody in Chinese hamster ovary cells (CHO) 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 acid sequence...

Embodiment 2

[0090] Example 2: Expression and purification of bispecific antibodies

[0091] 1. Expression of bispecific antibodies

[0092] The endotoxin-free large-scale extraction kit (Qiagen, 12391) was used for large-scale extraction of plasmids, and the specific operation was performed 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, and placed in a 5% CO2 cell incubator at 37 ° C. After the cells were prepared, they were cultured according to the manufacturer’s instructions ( Maxcyte), the plasmid pCHO1.0-anti-CD19-SCFV-FC-KKW was co-transfected with pCHO1.0-hygromycin-L2K-ScFv-Fc-LDY into CHO-S cells using a Maxcyte STX electroporator, Co-transfection of these two plasmids was designed to express the bispecific antibody M904 to CD19×CD3.

[0093] On the second day after transfection, the culture temperature was lowered to 32° C., and 3.5% FeedA was ...

Embodiment 3

[0096] Example 3: Determination of the binding activity of bispecific antibodies to cells (FACS)

[0097] 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.

[0098] 1. Detection of binding activity of bispecific antibody to Daji cells by flow cytometry

[0099] 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 5c...

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Abstract

The invention provides a bispecific antibody which is composed of a single-chain unit A and a single-chain unit B, wherein the single-chain unit A, aiming to surface antigen CD19 of immune cells, has a specifically combining capability while the single-chain unit B, aiming to surface antigen CD3 of tumor cells, has a specifically combining capability. The single-chain unit A and the single-chain unit B both include a single-chain variable fragment (ScFv) fused with an Fc fragment. 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, this application relates to the construction and application 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 consists of four subunits: δ, ε, γ, and ζ, with molecular masses of 18.9 kDa, 23.1 k...

Claims

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Application Information

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IPC IPC(8): C07K16/46C07K16/30C12N15/85A61K39/395A61P35/00
Inventor 方丽娟杨锦霞戴晴陈婷王涛范克索周鹏飞
Owner WUHAN YZY BIOPHARMA CO LTD
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