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Screening method of CD22 nano antibody

A nanobody and screening method technology, which is applied in the field of CD22 nanobody screening, can solve problems such as application limitations, poor stability, and large volume of monoclonal antibodies, and achieve the effect of improving stability and safety

Pending Publication Date: 2021-04-30
河南创新生物科技研究院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] The advent of hybridoma technology in 1975 opened the prelude to the use of monoclonal antibodies in the diagnosis and treatment of human diseases. However, monoclonal antibodies are large in size, poor in stability, and immunogenic, which limits their clinical application.
Since the 1980s, new genetically engineered antibodies have emerged, including chimeric antibodies, humanized antibodies, fully humanized antibodies, and miniaturized genetically engineered antibodies. Single domain antibodies are miniaturized genetically engineered antibodies, but they are Stability, expression yield, protease resistance, and polymerization still need to be improved

Method used

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  • Screening method of CD22 nano antibody
  • Screening method of CD22 nano antibody
  • Screening method of CD22 nano antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 CD22 single domain antibody screening

[0037] 1.1 Preparation of human CD22 recombinant protein:

[0038] 1) CD22 recombinant protein sequence construction:

[0039] After obtaining the amino acid sequence of the extracellular segment of human CD22 from NCBI and Uniprot databases, artificial gene synthesis was performed to construct eukaryotic expression vectors;

[0040] 2) 22-sequence vector transfection and expression

[0041]Transfect the eukaryotic expression vector of CD22 sequence constructed above into CHO-S cells and express CD22 recombinant protein;

[0042] 3) Identification

[0043] The product was purified by an affinity column, and the protein activity was identified after purification.

[0044] 1.2 Single cell sorting

[0045] 1) Alpaca immunity

[0046] The human CD22 recombinant protein prepared above was used to immunize the alpaca that had been immunized four times in the previous stage;

[0047] 2) Blood collection and ELISA analysi...

Embodiment 2

[0059] Example 2 Preparation of humanized single domain antibody

[0060] 2.1 Design and gene preparation of humanized nanobody

[0061] Use the surface amino acid replacement design to humanize the candidate single-domain antibody, obtain and synthesize the humanized nanobody sequence, and construct the humanized single-domain antibody expression vector; perform a large amount of plasmid extraction on the expression vector prepared above, Use this to prepare transfection-grade plasmids.

[0062] 2.2 Expression and purification of humanized nanobody

[0063] The humanized Nanobody expression vector prepared above was transiently transfected into 293F cells, and the recombinant antibody was purified using Protein A, concentrated and then quantified using the BCA method.

[0064] 2.3 Binding specificity and affinity detection of humanized nanobodies:

[0065] flow experiment design

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Abstract

The invention relates to the technical field of biological medicine, in particular to a screening method of a CD22 nano antibody. In the screening process of the CD22 nano-antibody, the CD22 nano-antibody with high binding specificity is screened by adopting a mode of combining flow sorting with affinity detection, and the stability and safety of the CD22 nano-antibody are improved through humanized treatment modified by specific nucleotide mutation. The screened CD22 nano antibody can be used as an active pharmaceutical ingredient, or a combined structure of targeted drugs, or a targeted binding region sequence structure of immune effector cells, or a diagnostic reagent ingredient to prepare a pharmaceutical composition capable of preventing and treating tumors, or a diagnostic reagent capable of diagnosing tumors.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a screening method for CD22 nanobody. Background technique [0002] The advent of hybridoma technology in 1975 opened the prelude to the use of monoclonal antibodies in the diagnosis and treatment of human diseases. However, monoclonal antibodies are large in size, poor in stability, and immunogenic, which limits their clinical application. Since the 1980s, new genetically engineered antibodies have emerged, including chimeric antibodies, humanized antibodies, fully humanized antibodies, and miniaturized genetically engineered antibodies. Single domain antibodies are miniaturized genetically engineered antibodies, but they are Stability, expression yield, protease resistance, and polymerization still need to be improved. In 1993, Hamers-Casterman reported the discovery of an antibody that naturally lacks light chains in camelids, that is, heavy chain antibodies. It is the s...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13C12N15/85
CPCC07K16/2803C12N15/85C07K2317/22C07K2317/24C07K2317/569C07K2317/92Y02A50/30
Inventor 韦丹秦志华王贺李明志
Owner 河南创新生物科技研究院有限公司