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Anti-CD19 humanized antibody and preparation method and application thereof

A humanized antibody and humanized technology, applied in the biological field, can solve the problems of long mouse transformation cycle and achieve the effect of improving the degree of humanization and retaining affinity

Active Publication Date: 2022-07-01
CUSABIO TECH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the early stage, it is necessary to transfer large fragments of the human antibody genome into mice, and the mouse transformation cycle is relatively long

Method used

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  • Anti-CD19 humanized antibody and preparation method and application thereof
  • Anti-CD19 humanized antibody and preparation method and application thereof
  • Anti-CD19 humanized antibody and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 Obtaining anti-CD19 humanized antibody

[0063] 1. Construction of phage display antibody library

[0064] 1. Lymphocyte isolation and antibody gene amplification

[0065] Take a 15ml centrifuge tube, first add the same amount of separation solution as the blood sample, carefully draw the blood sample and add it to the liquid surface of the separation solution, centrifuge at 450-650g for 20-30min; It is divided into four layers from the bottom, followed by plasma layer, annular milky white lymphocyte layer, transparent separation liquid layer and red blood cell layer; carefully suck the second annular milky white lymphocyte layer into another 15ml centrifuge tube with a pipette; Add 10ml of washing solution to the centrifuge tube, mix the cells, 250xg, and centrifuge for 10min; after centrifugation, check whether the supernatant is clear, if it is not clear enough, prolong the centrifugation time; wash the cells twice with 1xPBS, add trizol to lyse the cells ...

Embodiment 2

[0088] Example 2 Humanized Antibody Gene Antibody Expression

[0089] Place all reagents at room temperature for 10 minutes before transfection, use a 6-well culture dish for the following operations; dilute 3 μg of plasmid DNA into 250 μL of serum-free DMEM medium, pipette 3-4 times; dilute 5 μL of PEI reagent To 250μL of serum-free DMEM medium, pipette 3-4 times; Note: Serum-free DMEM medium is a diluent, and serum-containing medium cannot be used for DNA and diluted PEI transfection reagent Add it all at once to the diluted plasmid DNA, and immediately pipette 3-4 times; leave at room temperature for 10-15 minutes to form a PEI-DNA complex; 18-24 hours before transfection Count and plate so that at the time of transfection, adherent cells are approximately 80% confluent; discard medium in wells and add 1 ml of fresh DMEM complete medium; prepare PEI-DNA complexes; mix PEI-DNA The solution was evenly dropped into the cell culture medium, and the PEI-DNA complex was disperse...

experiment example 1

[0091] 1. Sequence Analysis of Humanized Antibody Gene

[0092] By software analysis, the Z-scores of the heavy and light chains were -1.5 and -1.0, respectively. After humanization, the Z-scores of the heavy and light chains of B5 were 1.6 and 0.4, respectively, and the Z-scores of the heavy and light chains of D12 were 1.2 and 0.7, respectively. The Z-score of the two antibodies obtained after humanization has been greatly improved. From the results in the figure, it can be seen that the two antibodies are basically fully human antibodies.

[0093] The results of FMC63 heavy chain analysis are as follows figure 1 As shown in (A), it shows that the unmodified antibody is a murine sequence, and the Z-score of the sequence analyzed by the software is distributed in the range of the murine antibody.

[0094] FMC63 light chain analysis results are as follows figure 1 (B) shows that the unmodified antibody is a murine sequence, and the Z-score of the sequence analyzed by the so...

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Abstract

The present invention provides an anti-CD19 humanized antibody, a preparation method and application thereof, the antibody contains a humanized heavy chain variable region VH and a humanized light chain variable region VL, and the VH and VL are determined by The cluster complementary region and the framework region are composed; the determinant complementary regions of the VH and the VL are all composed of CDR1, CDR2 and CDR3; the amino acid sequence of the CDR1 of the VH is selected from SEQ ID NO.1-4; the amino acid of the CDR2 of the VH The sequence is selected from SEQ ID NO.5-8; the amino acid sequence of CDR3 of VH is selected from SEQ ID NO.9-12; the amino acid sequence of CDR1 of VL is selected from SEQ ID NO.13-16; the amino acid sequence of CDR2 of VL is selected from SEQ ID NO.13-16; SEQ ID NO.17-20; the amino acid sequence of CDR3 of VL is selected from SEQ ID NO.21-24; the degree of humanization is high, and the affinity is high.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to an anti-CD19 humanized antibody and a preparation method and application thereof. Background technique [0002] CD19 is a glycoprotein on the surface of lymphocytes, which is expressed from myeloid progenitor B lymphocytes until B cell maturation and disappears in plasma cells. It is closely related to B cell activation, signal transduction and growth regulation. It is widely distributed on the surface of B lymphocytes and is a functional receptor molecule. In addition, CD19 is also expressed in malignant B-cell tumors, follicular dendritic cells, acute myeloid leukemia cells, and non-Hodgkin's lymphoma. In clinical practice, CD19 is commonly used as the immunophenotyping of B lymphocytic leukemia and lymphoma to provide a basis for clinical diagnosis. The high expression rate of CD19 in acute B lymphocytic leukemia reaches 100%. As a therapeutic target, CD19 protein has obtained id...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C12N15/13C12N5/10A61K39/395A61K47/68A61P35/00
Inventor 罗绍祥杨旭高霞
Owner CUSABIO TECH LLC
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