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HER2-resistant humanized antibody and relevant tumor-resistant composition thereof

A technology of humanized antibodies and compositions, which can be used in anti-tumor drugs, anti-animal/human immunoglobulins, antibodies, etc., and can solve the problem of low affinity

Active Publication Date: 2014-12-24
HEFEI HANKEMAB BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, whether it is chA21 or H1-2, there is a weakness of low affinity for antigen binding, and their affinity constants are at 1×10 -8 About M (Hu S, Zhu Z, Li L, Chang L, Li W, Cheng L, Liu J. (2008) Epitope mapping and structural analysis of an anti-ErbB2 antibody A21: Molecular basis for tumor inhibitory mechanism. Proteins 70:938 -949)

Method used

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  • HER2-resistant humanized antibody and relevant tumor-resistant composition thereof
  • HER2-resistant humanized antibody and relevant tumor-resistant composition thereof
  • HER2-resistant humanized antibody and relevant tumor-resistant composition thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0112] Example 1. Preparation of humanized fusion antibody HuA21-1 scFv-Fc

[0113] The amino acid sequence of the H1-2 humanized single-chain antibody disclosed in the Chinese invention patent application number 201010211554.8 was reconstructed to obtain the humanized single-chain antibody HuA21-1 scFv, and the humanized single-chain antibody HuA21-1 scFv was modified The humanized fusion antibody HuA21-1 scFv-Fc was constructed.

[0114] 1. Obtaining the amino acid sequence of the humanized single-chain antibody HuA21-1 scFv

[0115] This application restructures the amino acid sequence of the H1-2 humanized single-chain antibody disclosed in the Chinese Invention Patent Application No. 201010211554.8, and the restructured amino acid involves the heavy chain variable region V of the H1-2 humanized single-chain antibody H CDR1, CDR3 and light chain variable region V L CDR1, CDR2, and CDR3, and obtained a new humanized single-chain antibody HuA21-1 scFv, which was named huma...

Embodiment 2

[0142] Example 2. Binding properties of humanized fusion antibody HuA21-1 scFv-Fc to antigen

[0143] The binding properties of humanized fusion antibody HuA21-1 scFv-Fc to antigen were determined by ELISA method. The experiment was repeated three times, and the steps of each repeated experiment were as follows:

[0144] The HER2-ECD antigen (Beijing Yiqiao Shenzhou Company, product number 10004-H08H) was mixed with 50mM NaHCO at pH 9.6 3 The aqueous solution was diluted to 50ng / μL to obtain a 50ng / μL HER2-ECD antigen solution. Add 100 μL of 50 ng / μL HER2-ECD antigen solution to each well of the ELISA plate (Nunc Company) to coat the ELISA plate; incubate overnight at 4 ° C, add PBST (PBS+0.1% Tween 20) containing 3% skimmed milk powder Place at 37°C for blocking for 1 hour; add humanized fusion antibody HuA21-1 scFv-Fc, so that the final concentrations of humanized fusion antibody HuA21-1 scFv-Fc are 9000ng / mL, 3000ng / mL, 1000ng / mL, 333ng / mL, 111ng / mL, 37ng / mL, 12.33ng / mL...

Embodiment 3

[0148] Example 3. Affinity of Humanized Fusion Antibody HuA21-1 scFv-Fc to Antigen

[0149] Biacore 3000 Biomolecular Interaction Analyzer (GE Company) was used to measure the affinity of the humanized fusion antibody HuA21-1 scFv-Fc to the antigen. The experiment was repeated three times, and the steps of each repeated experiment were as follows:

[0150] The recombinant antigen HER2-ECD (Beijing Yiqiao Shenzhou Company, Cat. No. 10004-H08H) was used with EZ-Link TM-Sulfo-NHS-Biotin Labeling Kit (Pierce Company) was coupled to a biotin-labeled antigen. Dilute the biotin-labeled antigen with PBS to a concentration of 0.4 μg / mL to obtain a 0.4 μg / mL biotin-labeled HER2-ECD antigen solution. Flow 0.4 μg / mL biotin-labeled HER2-ECD antigen solution over the SA chip to bind the SA chip to the biotin-labeled HER2-ECD antigen. When the signal value is about 300 RU, the labeling of the antigen on the SA chip is completed, and the antigen is obtained. Labeled SA chips.

[0151] Hum...

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Abstract

The invention discloses an HER2-resistant humanized antibody and a relevant tumor-resistant composition thereof. The HER2-resistant humanized antibody in the tumor resistant composition provided by the invention is an HER2-resistant humanized antibody M1 containing a humanized heavy chain variable region M1-VH and a humanized light chain variable region M1-VL which both consist of a determinant complementary region and a framework region, the determinant complementary regions of the M1-VH and the M1-VL both consist of CDR1, CDR2 and CDR3; the amino acid sequences of the CDR1, the CDR2 and the CDR3 of the M1-VH are respectively shown as the 26th to 35th, the 50th to 66th, and the 99th to 109th of the SEQID No.1; the amino acid sequences of the CDR1, the CDR2 and the CDR3 of the M1-VL are respectively shown as the 24th to 40th, the 56th to 62th, and the 95th to 103th of the SEQID No.2.

Description

technical field [0001] The present invention relates to an anti-HER2 humanized antibody and its related anti-tumor composition in the field of biomedicine. Background technique [0002] HER2 (human epidermal growth factor receptor-2, human epidermal growth factor receptor-2, HER2), also known as ErbB2 / P185, is a tyrosine kinase receptor membrane glycoprotein encoded by the proto-oncogene erbB2 / Her2, which belongs to epidermal growth factor The second member of the EGFR family of factor receptors. HER2 forms a heterodimer with the other three members of the EGFR family, leading to the activation of signaling pathways such as MAPK and PI3K downstream of the receptor, thus playing a biological role. Although no ligand that can directly bind to HER2 has been found yet, since HER2 plays a central regulatory role in the signal transduction of the EGFR family, when HER2 is abnormally expressed, normal cells and tissues are prone to canceration, leading to tumor formation. It is k...

Claims

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Application Information

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IPC IPC(8): A61K39/395A61P35/00C07K16/30C07K16/46C12N15/13C12N15/62
Inventor 刘兢胡思怡黄辉胡冬梅
Owner HEFEI HANKEMAB BIOTECH CO LTD
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