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Anti-cd19 antibody and its application

An antibody and chimeric antigen receptor technology, applied in the field of medicine and biology, can solve the problems of limited survival time of CAR-T cells and high recurrence rate of patients

Active Publication Date: 2020-09-15
GUANGDONG PANGUARD CELL BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] However, some of the patients receiving CD19-CAR chimeric antigen receptor immunotherapy have limited survival time of CAR-T cells, and these patients have a higher recurrence rate after treatment

Method used

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  • Anti-cd19 antibody and its application
  • Anti-cd19 antibody and its application
  • Anti-cd19 antibody and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Antibody humanization

[0059] According to the amino acid sequence of the mouse monoclonal antibody targeting human CD19 of our company, the 3D structure of the antibody was simulated by Accelrys Discoverystudio software, and the similarity of the mouse monoclonal antibody in the human antibody library was compared using the DomainGapAlign tool of the IMGT database Yes, select the human antibody heavy chain and light chain framework regions with the highest similarity as the framework, replace the selected human antibody complementarity determining region with the mouse antibody complementarity determining region, the sequence of the mouse antibody heavy chain and light chain is SEQ1, SEQ2, respectively, The obtained amino acid sequences of different humanized antibody heavy chains and light chains were named as SEQ3, SEQ4, SEQ5, SEQ6, SEQ7, and SEQ8. The above sequence was synthesized by artificial gene synthesis, and combined by overlapping PCR through (G4S...

Embodiment 2

[0062] Example 2 Expression and purification of single-chain antibody

[0063] The CHO-K1 cell line was recovered from liquid nitrogen, and cultured in F12K, 10% FBS medium continuously for about 2 weeks, so that the cells were in the logarithmic growth phase. Prepare 10 cells in 15 cm culture dishes, respectively, and transfect the above obtained single-chain antibody expression vectors into CHO-K1 cell lines to secrete and express the single-chain antibody. The transfection process was as follows: take out 100 μM PEI from the refrigerator, and heat it in a 60° C. water bath for 15 minutes until the PEI is completely dissolved. Take out the scFv expression plasmid from the refrigerator, thaw at room temperature, and mix completely by blowing up and down with a pipette gun. Remove the PBS or HBSS buffer and warm to room temperature. Prepare PEI / DNA complexes (the following volume or plasmid mass is based on the amount of a 15cm dish): take 2mL PBS to one well of a 6-well pla...

Embodiment 3

[0065] Example 3 Analysis of Binding Ability of Single Chain Antibody to Target Protein CD19

[0066]The CHO-K1-CD19 recombinant cell line was recovered from liquid nitrogen, and cultured continuously for about 2 weeks using F12K and 10% FBS medium, so that the cells were in the logarithmic growth phase. Take 1*10 6 For each cell, resuspend in 100uL PBS, then add 1ug of the above-mentioned purified single-chain antibody, incubate at room temperature for 30 minutes, add 1mL PBS to wash the cells 3 times, then resuspend the cells in 100uL PBS, add 5uL PE-labeled Anti-human IgG Antibody, after incubating at room temperature in the dark for 30 minutes, add 1mL PBS to wash three times, and finally resuspend the cells in 500uL PBS, and use flow cytometry to analyze the binding ability of the humanized single-chain antibody to the target protein CD19. A mouse monoclonal antibody that was not humanized was used as a control. The result is as figure 1 As shown, the single-chain anti...

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Abstract

The present invention relates to an anti-CD19 antibody and its application. The heavy chain sequences of the anti-CD19 antibody or its antigen-binding part of the present invention are respectively shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, and the sequences of the light chains are shown in SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or a sequence whose heavy chain and light chain satisfy at least one of the following two compared with the aforementioned sequence: a) Bind to the same antigenic epitope; b) the identity is greater than 70%, 80%, 85%, 90% or 97%; at the same time, based on the anti-CD19 antibody or its antigen-binding part, the present invention provides it as a nucleic acid molecule, vector, host cell , conjugates, compositions and uses in the preparation of drugs for preventing or treating tumors.

Description

technical field [0001] The present invention relates to the field of medicine and biology, in particular to an anti-CD19 antibody and its application. Background technique [0002] CD19, also known as B4 or Leu-12, is specifically expressed on the membrane surface of normal and malignant B lymphocytes, as well as on the membrane surface of follicular dendritic cells. It is a member of the immunoglobulin (Ig) superfamily. On the short arm of the chromosome, it contains 15 exons, encoding a type I transmembrane glycoprotein of 556 amino acids. CD19 is one of the most reliable surface markers of B cells, and it is first expressed in late pro-B cells and early pre-B cells when immunoglobulin gene recombination occurs. CD19 is highly expressed throughout the development and maturation of B cells, until the B cells differentiate into plasma cells, the expression level is down-regulated, and its expression in mature B cells is 3 times that of immature cells. [0003] CD19 establi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C12N15/13C12N15/867A61K39/395A61P35/00
CPCA61K2039/505C07K16/2803C07K2317/24C07K2317/622C12N15/86C12N2740/15043A61K39/0011A61K2039/5156A61K2039/5158C07K14/7051C07K2319/03
Inventor 李相鲁谢海涛张严冬李莉李俊马利雅
Owner GUANGDONG PANGUARD CELL BIOLOGICAL TECH CO LTD