Anti-phosphatidylinositol proteoglycan 3 complete humanized antibody

A technology of phosphatidylinositol and proteoglycan, which is applied in the direction of antibodies, anti-animal/human immunoglobulins, anti-tumor drugs, etc., and can solve problems such as the patent application of Glypican 3 antibody that has not yet been seen

Inactive Publication Date: 2015-11-11
BEIJING BIYANG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] A total of 12 domestic patent databases and glypican-3 therapeutic antibody-related patents were searched, which were issued by Chugai Pharmaceutical Co., Ltd. CN102850455B, authorization announcement number: CN101287492B, authorization announcement number: CN101809162B, authorization announcement number: CN101186650B, authorization announcement number: CN101014367B, authorization announcement number: CN1842540B, authorization announcement number: CN1314803C), of which authorization announcement number: CN102850455B and Zanke Co., Ltd. Co-owned by the company; owned by 3 foreign companies including Midles (authorized announcement number: CN101815726B)

Method used

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  • Anti-phosphatidylinositol proteoglycan 3 complete humanized antibody
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  • Anti-phosphatidylinositol proteoglycan 3 complete humanized antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1, Synthesis of Glypican 3 (glypican-3) C-terminal polypeptide

[0035] Based on the analysis of the C-terminal structural characteristics of glypican-3, Shanghai Taopu Biotechnology Co., Ltd. was entrusted to artificially synthesize 40 amino acid peptides "AELAYDLDVDDAPGNSQQATPKDNEISTFHNLGNVHSPLK" (524A-563K) 5mg with a purity greater than 95% , for the screening of fully humanized antibodies.

Embodiment 2

[0036] Example 2. Screening of anti-glypican-3 antibody from a fully human large-capacity phage antibody library

[0037] The large-capacity phage antibody library used for antibody screening has a capacity of 4×10 8 Fab antibody library. First, the phage antibody library was amplified, and the peptides were enriched by coating at 10ug / well. After 6 rounds of elutriation, the enriched phage infected bacteria, and about 1,000 single clones were picked. After a small amount of induction, the supernatant was detected by ELISA, and positive clones were picked, and about 100 positive clones were obtained. The heavy and light chains of these positive clones were re-amplified to establish a peptide-positive phage antibody library, and the peptides were re-enriched for 2 rounds. The enriched phages were used to infect bacteria, plated, and about 200 single clones were picked, and a small amount of induced supernatant was tested by ELISA. The positive clones were picked for sequencin...

Embodiment 3

[0052] Example 3, Biacore3000 detects the affinity of Fab antibody and synthetic peptide

[0053] 1. Fixation: Dilute the synthetic peptide to a concentration of 70 μg / ml. Amino coupling method was used to covalently immobilize on carboxymethyl dextran-coated CM5 chip (General Electric product) via primary amine, immobilization buffer 10mM sodium acetate (ph5.0), immobilization amount: 180RU.

[0054] 2. Kinetic analysis:

[0055] PBST (PBS, 0.005% Tween 20) is Runningbuffer; use KineticAnalysisWizard mode; Fab dilution is 37.5, 75, 150, 300, 600, 1200nM concentration gradient; injection time: 1min, dissociation time: 2min, flow rate: 40ul / min

[0056] 3. Regeneration conditions:

[0057] Regeneration solution: 8mMNaOH, injection time: 30s, flow rate: 40ul / min, buffer: 40siniection

[0058] 4. Result analysis:

[0059] Fitting software: BIAevaluation4.1software, fitting model: 1:1bindingmodel. ka(1 / Ms)=1.03×10 5 , kd(1 / s)=6.73×10 -3 , KD(M)=6.54×10 -8 , anti-glypican...

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Abstract

The invention discloses a complete humanized antibody which is selected from humanized high-capacity phage antibody library and is high-affinity-combined with phosphatidylinositol proteoglycan 3. The invention includes a selection method of the antibody, an antibody coding sequence and corresponding amino acid residue sequence, and especially includes three CDR-zone sequences respectively at a heavy chain and a light chain, combination characters of an antigen and the construction method of the complete antibody. The antibody is a complete humanized antibody, is used for treatment in human body, is low in immunogenicity, is less in toxic and side effects, and has a potential value of treating liver cancer and melanin tumor.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a screening and preparation method of a fully human antibody that binds to glypican-3 (GPC3) with high affinity and its potential use in liver cancer and Applications in the treatment of melanoma. Background technique [0002] Liver cancer is a malignant tumor with a wide range of hazards. In the world, liver cancer ranks the fifth in males and the seventh in females among all malignant tumors, and it is the second in males and sixth in females in the death of malignant tumors. In developing countries, it ranks the third in the incidence of malignant tumors in men, the sixth in women, the second in the death of malignant tumors in men, and the fifth in women. The number of hepatitis B patients in our country ranks first in the world, which directly leads to the highest number of liver cancer patients in our country in the world. According to the survey, in 1973-1975, 1990-1992 and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18A61K39/395A61P35/00
Inventor 胡品良何芸司少艳白洁洪伟东邹敬宋凌云杨泽荣
Owner BEIJING BIYANG BIOTECH
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