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Anti-CD19 full humanized antibody or antibody segment as well as method and application thereof

A technology of fully human antibodies and antibody fragments, applied in the field of fully human antibodies or antibody fragments, can solve problems such as poor targeting of cellular immunotherapy

Active Publication Date: 2018-04-06
CHANGZHOU VELOX PHARMA SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The object of the present invention is to provide an anti-CD19 fully human antibody or antibody fragment and its method and application. Source CD19 protein binding, using chimeric antigen receptor T cell technology (CAR-T technology) to engineer the expression of human antibody fragments integrated in CAR and binding to CD19 in T cells, resulting in chimeric antigen receptor T cells Can be used to treat hematological cancers associated with CD19 expression

Method used

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  • Anti-CD19 full humanized antibody or antibody segment as well as method and application thereof
  • Anti-CD19 full humanized antibody or antibody segment as well as method and application thereof
  • Anti-CD19 full humanized antibody or antibody segment as well as method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0092] Experimental example 1 Screening of anti-CD19 single chain antibody

[0093] 1. Establish a phage antibody library of fully human single-chain antibody

[0094] Design primers to amplify the heavy chain variable region and light chain variable region of the antibody, connect the heavy chain variable region and light chain variable region with Linker by overlapping extension PCR, and obtain the full-length PCR product, use SfiI Digest the PCR product and the pCGMT phagemid vector, electrotransform the ligated transformation product into XL1-blue competent cells, then add 3 mL of SOC medium to the competent cells, culture at 30°C for 1 hour, and add a final concentration of 50 μg / mL of ampicillin, 20 mL of 10 μg / mL tetracycline solution, and shake at 37°C for 2 hours. Then add a concentration of 10 13 / mL VCSM13 helper phage 50 μL, incubate at room temperature for 1 h, and shake once every 10 min. Shake culture at 37°C for 2 hours, then add kanamycin at a final concen...

experiment example 2

[0108] Preparation of Experimental Example 2 Anti-CD19 Single Chain Antibody (Expression and Purification)

[0109] The monoclonal plasmid obtained by screening in Experimental Example 1 was digested by restriction endonuclease SfiI, ligated, and then the fragment was inserted into the pFUSE expression vector by the method of recombination (the pFUSE expression vector was digested by restriction endonuclease SfiI site), thereby obtaining the pFUSE expression vector of the antibody of the present invention.

[0110] Mix the 293Fectin transfection reagent with the eukaryotic antibody expression vector obtained above at a volume-to-mass ratio of 30 μL: 30 μg, add 30 μL of 293Freestyle suspension cells, culture on a shaker at 125 rpm at 37°C for 48-72 hours, and collect after centrifugation Supernatant, using HiTrap Protein A HP columns in The antibody protein was purified on a protein purification instrument to obtain an anti-CD19 single-chain antibody (anti-CD19 fully human sc...

experiment example 3

[0111] Experimental example 3 Detection of binding specificity and binding kinetics characteristics of anti-CD19 fully human scFv antibody

[0112] The affinity and kinetic characteristics of the antibody and antigen were detected by the method of multi-cycle kinetics, and the antibody was immobilized by the capture method. The human scFv sample is diluted and flows over the surface of the chip, diluted to a concentration of 7.5 μg / mL and 15 μg / mL, the anti-CD19 fully human scFv antibody will be captured by the CD19 antigen, and the signal will be detected and recorded after the antigen is combined with the antibody. Finally, the antibody and antigen samples on the surface of the sensor were all eluted with a regeneration reagent (glycine solution at pH 1.5), and a new round of detection was performed.

[0113] Experimental results:

[0114] Such as figure 2 Shown is the binding kinetics curve of anti-CD19 fully human scFv antibody to CD19, and each line represents differen...

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Abstract

The invention discloses an anti-CD19 full humanized antibody or antibody segment as well as a method and an application thereof. The antibody or the antibody segment contains a heavy chain and a lightchain, wherein the heavy chain and the light chain comprise variable regions; the variable regions comprise complementary determining regions; the complementary determining regions CDR1, CDR2 and CDR3 of the heavy chain are separately represented by HCDR1, HCDR2 and HCDR3; the complementary determining regions CDR1, CDR2 and CDR3 of the light chain are separately represented by LCDR1, LCDR2 and LCDR3; the amino acid sequence of the HCDR1 comprises SED ID NO: 3; the amino acid sequence of the HCDR2 comprises SEQ ID NO: 4; the amino acid sequence of the HCDR3 comprises SEQ ID NO: 5; the amino acid sequence of the LCDR1 comprises SEQ ID NO: 6; and the amino acid sequence of the LCDR2 comprises SEQ ID NO: 7. The antibody or the antibody segment can be combined with a humanized CD19 protein with high specificity, and by means of engineered expression integration by means of a chimeric antigen receptor cell technology in a cell T, the obtained chimeric antigen receptor cell T can be used for treating hematological cancers related to expression of CD19.

Description

technical field [0001] The invention belongs to the technical field of biological treatment of malignant tumors, and relates to an anti-CD19 antibody, in particular to an anti-CD19 fully human antibody or antibody fragment as well as a method and application thereof. Background technique [0002] CD19 is expressed on B lineage cells (excluding mature plasma cells) and follicular dendritic cells. It is an important signal transduction molecule that regulates the growth activation and activation of B lymphocytes, and regulates B lymphocyte antigen receptors or It plays an important role in the signal threshold of other surface receptors. It is an important membrane antigen related to the differentiation, activation, proliferation and antibody production of B lymphocytes. It is the best marker for diagnosing B lymphocyte tumors and identifying B lymphocytes. [0003] Many patients with B-cell malignancies cannot be cured using standard therapies. In addition, traditional treat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K19/00C12N15/13C12N5/10A61K35/17A61P35/02
CPCA61K35/17C07K14/7051C07K16/2803C07K2317/622C07K2317/92C07K2319/02C07K2319/33
Inventor 杨光闵晨雨
Owner CHANGZHOU VELOX PHARMA SCI & TECH CO LTD
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