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Antibody L3H3 of CD20-resistant antigen and application of antibody L3H3

A DNA molecule, sequence listing technology, applied in applications, antibodies, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can solve problems such as affecting function

Active Publication Date: 2016-02-24
安徽中生安兰健康产业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although RTX can enhance the biological effects of antibodies, because it is a human-mouse chimeric antibody, it

Method used

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  • Antibody L3H3 of CD20-resistant antigen and application of antibody L3H3
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  • Antibody L3H3 of CD20-resistant antigen and application of antibody L3H3

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1, construction recombinant plasmid

[0032] 1. Insert the DNA molecule shown in sequence 2 of the sequence listing into the pcDNA-3.3 vector to obtain the recombinant plasmid pcDNA-3.3-L3.

[0033] The DNA molecule shown in Sequence 2 of the sequence listing encodes a leader peptide from the 20-79th nucleotide at the 5' end, the 80-412th nucleotide codes VL, the 413-718th nucleotide codes CL, and the 719th nucleotide Nucleotide -721 is a stop codon.

[0034] 2. Insert the DNA molecule shown in sequence 4 in the sequence listing into the pOptiVEC vector to obtain the recombinant plasmid pOptiVEC-H3.

[0035] The DNA molecule shown in sequence 4 of the sequence listing encodes a leader peptide from the 14th to 70th nucleotides at the 5' end, the 71st to 436th nucleotides encode VH, the 437th to 730th nucleotides encode CH1, and the 731st -1118 nucleotides are introns, 1119-1163 nucleotides code Hinge, 1164-1281 nucleotides are introns, 1282-1614 nucleotides ...

Embodiment 2

[0036] Example 2, preparation of L3H3 antibody

[0037] 1. Take 10 μg of recombinant plasmid pcDNA-3.3-L3 and 10 μg of recombinant plasmid pOptiVEC-H3, and use OptiPRO TM The volume of SFM medium was adjusted to 500 μl, and left at room temperature for 5 minutes.

[0038] 2. Take 20μl of liposomes and use OptiPRO TM The volume of SFM medium was adjusted to 500 μl, and left at room temperature for 5 minutes.

[0039] 3. Mix the solution of step 1 and step 2, and let it stand at room temperature for 20 minutes.

[0040] 4. Inoculate CHO-DG44 cells (about 3 × 10 6 cells / flask), centrifuge at 800rpm for 4min before transfection, discard the culture supernatant, and add 4ml fresh CDDG44Medium to each bottle to resuspend.

[0041] 5. Add the solution obtained in step 3 dropwise into the cell culture flask that completed step 4, mix well, and 2 Shake culture at 37°C and 150rpm for 5 days in a shaker (first cultivate for 6-8 hours, then centrifuge at 800rpm for 4min, discard the su...

Embodiment 3

[0050] Embodiment 3, affinity detection

[0051] Entrust Beijing Konuo Xincheng Technology Co., Ltd. to complete, the brief process is as follows: Using Fortebio instrument, biotinylated CD20 epitope (the amino acid sequence of CD20 epitope is: CEP ANPs EKNSPSTQ YCYSI Q) Immobilize on the Steptavidin chip, and flow through 5 different concentrations of antibody solutions to be tested (dilute the L3H3 solution or rituximab prepared in Example 1 with pH 7.2, 0.01M PBS buffer solution to obtain Different concentrations of antibody solutions to be tested, the protein concentrations in each antibody solution to be tested are 0, 25, 50, 75 or 100nmol / L), and the binding (Kon) and dissociation (Koff) values ​​are detected to calculate the affinity (Kd) .

[0052] The affinity (Kd) of rituximab is 6.48×10 -9 M, the affinity (Kd) of L3H3 antibody is 3.28×10 -9 M.

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Abstract

The invention discloses an antibody L3H3 of a CD20-resistant antigen and application of the antibody L3H3. The CD20-resistant antigen provided by the invention is named the antibody L3H3 and is IgG (Immunoglobulin G) which is formed by a light chain and a heavy chain; CDR1, CDR2 and CDR3 in a light chain variable region in the light chain are sequentially an amino acid residue ranging from 61st to 79th, an amino acid residue ranging from 89th to 102nd and an amino acid residue ranging from 112th to 123rd from an N tail end in a sequence I of a sequence list; CDR1, CDR2 and CDR3 in a heavy chain variable region in the heavy chain are sequentially an amino acid residue ranging from 50th to 60th, an amino acid residue ranging from 82nd to 97th and an amino acid residue ranging from 107th to 120th from an N tail end in a sequence III of the sequence list. The application of the antibody L3H3 which is used for preparing medicines for treating B cell lymphoma is protected. According to the antibody L3H3 and the application, disclosed by the invention, great application value for treating B cell lymphoma is obtained.

Description

technical field [0001] The invention relates to an anti-CD20 antigen antibody L3H3 and its application. Background technique [0002] Non-Hodgkin's lymphoma (NHL) is a malignant tumor of the lymphatic system with different biological characteristics and histological forms, and it is one of the ten malignant tumors that threaten human life. % are derived from B cells. [0003] CD20 molecule is a B cell differentiation antigen, which is expressed in more than 95% of B cell lymphomas, but not expressed in hematopoietic stem cells, plasma cells and other normal tissues. The molecular weight of CD20 is 33KD. It is a non-glycosylated protein composed of 279 amino acid residues. It is initially expressed in the pre-B cell stage and ends when the B cell terminal differentiates into plasma cells. It has always been considered as the surface of B lymphocytes. Unique logo. The CD20 molecule has four transmembrane regions, and there is a loop region composed of 43 amino acid residues...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13A61K39/395A61P35/00
Inventor 张部昌朱林戴维·威孚曹诚靳彦文于庆卓汪苏曼
Owner 安徽中生安兰健康产业有限公司