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Anti-CD19 humanized antibody as well as preparation method and application thereof

A humanized antibody and humanized technology, applied in the biological field, can solve the problems of long transformation cycle of mice

Active Publication Date: 2021-04-20
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 as well as preparation method and application thereof
  • Anti-CD19 humanized antibody as well as preparation method and application thereof
  • Anti-CD19 humanized antibody as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 Obtaining anti-CD19 humanized antibody

[0063] 1. Phage display antibody library construction

[0064] 1. Lymphocyte isolation and antibody gene amplification

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

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 and pipette 3-4 times; dilute 5 μL of PEI reagent Into 250 μL of serum-free DMEM medium, pipette 3-4 times; Note: Serum-free DMEM medium is a diluent, do not use serum-containing medium for DNA and the diluted PEI transfection reagent Add all of it to the diluted plasmid DNA at one time, and immediately pipette 3-4 times; place at room temperature for 10-15 minutes to form PEI-DNA complexes; carry out cell incubation 18-24 hours before transfection Count and plate so that the confluence of adherent cells is about 80% at the time of transfection; discard the original medium in the well and add 1ml of fresh DMEM complete medium; prepare PEI-DNA complexes; mix PEI-DNA Evenly drop the solution into the cell culture med...

experiment example 1

[0091] 1. Humanized antibody gene sequence analysis

[0092] By software analysis, the Z-scores of the heavy chain and light chain were -1.5 and -1.0, respectively. After humanization, the heavy chain and light chain Z-scores of B5 were 1.6 and 0.4, respectively, and the heavy chain and light chain Z-scores 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] FMC63 heavy chain analysis results are as follows figure 1 As shown in (A), it shows that the unmodified antibody is a mouse sequence, and the Z-score of the sequence analyzed by the software is distributed in the range of the mouse antibody.

[0094] The results of FMC63 light chain analysis are as follows: figure 1 As shown in (B), it indicates that the unmodified antibody is a mouse-derived sequence, and the Z-score of the seq...

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Abstract

The invention provides an anti-CD19 humanized antibody as well as 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 the VL respectively consist of a determinant complementary region and a framework region; the determinant complementary region of the VH and the determinant complementary region of the VL both consist 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 sequence of the CDR2 of the VH is selected from SEQ ID NO.5-8; the amino acid sequence of the CDR3 of the VH is selected from SEQ ID NO. 9-12; the amino acid sequence of the CDR1 of the VL is selected from SEQ ID NO. 13-16; the amino acid sequence of the CDR2 of the VL is selected from SEQ ID NO.17-20; and the amino acid sequence of the CDR3 of the VL is selected from SEQ ID NO. 21-24. The humanization degree 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 its preparation method and application. Background technique [0002] CD19 is a glycoprotein on the surface of lymphocytes. It is expressed from progenitor B lymphocytes in the bone marrow until B cells mature and disappears in plasma cells. It is closely related to B cell activation, signal transduction and growth regulation. Widely distributed on the surface of B lymphocytes, it 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. Clinically, CD19 is commonly used as the immunotype of B lymphocyte leukemia and lymphoma, and provides a basis for clinical diagnosis. The high expression rate of CD19 in acute B lymphocytic leukemia reaches 100%. As the target of treatment, CD19 protein has achieved satisfactory results ...

Claims

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Application Information

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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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