Multi-specific single chain antibody and application thereof

A single-chain antibody and multi-specific technology, applied to specific peptides, peptides containing affinity tags, peptides containing His tags, etc., can solve the problems of affecting T cell activity, T cell damage, cumbersome process, etc., and achieve enhanced Effects of bonding force, damage reduction, and simplified separation steps

Pending Publication Date: 2021-12-10
广州长峰生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, the process of isolating mononuclear cells from whole blood requires repeated centrifugation, which is cumbersome and requires red blood cell lysate for cell lysis, which also has certain damage to T cells and affects the activity of T cells.

Method used

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  • Multi-specific single chain antibody and application thereof
  • Multi-specific single chain antibody and application thereof
  • Multi-specific single chain antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] 1. Expression and purification of anti-CD3CD28 bispecific single chain antibody

[0050] The sequence of the anti-CD3CD28 bispecific single-chain antibody molecule is shown in the table below. The relevant sequence was synthesized by nucleotides, and then cloned into the pcDNA3.1 vector, expressed by transfection into the 293T cell line, and the supernatant was collected and layered with nickel The expressed antibody was purified by column analysis, and the results after purification can be found in figure 1 .

[0051] according to figure 1 The results of expression and purification indicated that the anti-CD3CD28 bispecific single-chain antibody had been successfully obtained, and the molecular weight was also correct.

[0052]

[0053]

[0054]

[0055]

[0056] 2. Whole blood separation experiment

[0057] The expressed and purified anti-CD3CD28 bispecific single-chain antibody molecule was biotin-labeled with a biotin labeling kit, and labeled and pur...

Embodiment 2

[0064] The anti-CD3CD28 bispecific single chain antibody was expressed and purified by the method shown in Example 1.

[0065] The expressed and purified anti-CD3CD28 bispecific single-chain antibody molecules were biotin-labeled with a biotin labeling kit, and labeled and purified according to the manufacturer's instructions, so as to prepare for subsequent whole blood separation experiments to verify its feasibility. At the same time, a streptavidin protein-coated cellulose membrane is prepared, and the coated cellulose membrane is placed in a cell culture bag that has connected infusion tubes in the upper and lower directions.

[0066] Take 50mL of whole blood, add 100mL of normal saline to dilute, then add 50mg of biotin-labeled anti-CD3CD28 bispecific single-chain antibody molecules, mix well and incubate at room temperature for 5 minutes, then use gravity infusion to flow through the filled streptavidin-coated Cell culture bag with a cellulose membrane, unbound cells flo...

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PUM

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Abstract

The invention relates to a multispecific single-chain antibody and application thereof, the multispecific single-chain antibody sequentially comprises a signal peptide, a variable region sequence of a first antibody, a first connecting peptide and a variable region sequence of a second antibody from an N terminal to a C terminal, and does not comprise constant region sequences of the first antibody and the second antibody; the first antibody and the second antibody can be respectively and specifically combined with two surface marker antigens of T cells, the variable region sequence comprises a light chain variable region sequence and a heavy chain variable region sequence, and the light chain variable region sequence and the heavy chain variable region sequence are connected through a second connecting peptide. The multispecific single-chain antibody can effectively adsorb and separate T cells from whole blood, the separation step of the T cells is simplified, the damage to the T cells is reduced, the operation error risk and the equipment requirement caused by multi-step operation are reduced, and the probability of cross contamination and influence among different samples in T cell immunotherapy can be reduced.

Description

technical field [0001] The invention relates to the technical field of biochemical molecules, in particular to a multispecific single-chain antibody and its application. Background technique [0002] The separation of T cells is the first step in the preparation of CAR-T cells, and it is also an important technology in the preparation of T cell preparations. At present, when isolating T cells from whole blood, it is usually necessary to use lymphocyte separation medium or other density gradient centrifugation reagents to separate mononuclear cells from whole blood, and then use antibody separation technology to combine CD3, a characteristic molecule of T cells. , TCR molecules and other co-stimulatory molecules such as CD28 antibodies to sort the required T cells. Commonly used separation techniques include magnetic bead separation, flow sorting and other methods. [0003] However, the process of isolating mononuclear cells from whole blood requires repeated centrifugation...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N5/0783
CPCC07K16/2809C07K16/2818C12N5/0636C07K2317/31C07K2317/622C07K2317/56C07K2317/92C07K2319/02C07K2319/21C12N2509/00
Inventor 黄朝峰陈焕鹏韦凤娇
Owner 广州长峰生物技术有限公司
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