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Fully human-anti-GPC3 all-molecule IgG antibody and applications thereof

A fully human and molecular technology, applied in the fields of application, antibodies, antibody medical components, etc., can solve the problems of unsatisfactory specificity and affinity, and achieve the effect of inhibiting proliferation, good affinity, and high specificity

Inactive Publication Date: 2016-11-09
SINOBIOWAY CELL THERAPY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most anti-GPC3 antibodies are prepared by hybridoma, such as the patent "GPC3 monoclonal antibody hybridoma cell line 7D11 and its preparation method and application" with application number 201210086009.X, but the specificity and affinity are often not ideal

Method used

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  • Fully human-anti-GPC3 all-molecule IgG antibody and applications thereof
  • Fully human-anti-GPC3 all-molecule IgG antibody and applications thereof
  • Fully human-anti-GPC3 all-molecule IgG antibody and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Preparation of fully human whole molecule IgG antibody against GPC3

[0034] 1) Using the GPC3 antigen in the human Fab phage library to undergo six rounds of "adsorption-elution-amplification" enrichment screening to obtain the anti-GPC3 Fab antibody, and then obtain the variable region sequence of the Fab antibody by PCR amplification and sequencing;

[0035] 2) Design primers according to the obtained antibody heavy and light chain variable region sequences;

[0036] 3) Amplify PA21 antibody heavy chain and light chain:

[0037] Using the human Fab template prepared above, use the above-mentioned upstream and downstream primers for the heavy chain and light chain to amplify the heavy chain and light chain genes of the whole molecule human antibody, respectively.

[0038] (1)PCR

[0039] The reaction system is as follows:

[0040]

[0041] The reaction conditions are as follows:

[0042]

[0043] (2) 2% agarose gel electrophoresis, observe the tar...

Embodiment 2

[0066] Example 2: Identification of functional activity of anti-GPC3 antibodies

[0067] 1) ELISA

[0068] Coat the ELISA 96-well plate with coating solution (0.1M carbonate buffer, pH9.6) GPC3 protein to 2 μg / mL, add 100 μL to each well, overnight at 4°C; PBST (PBS containing 0.5% Tween20) 5% degreased Block with milk-washing buffer, incubate at 37°C for 2 hours; after washing with PBST for 5 times, add 100 μL of PA21 antibody (2 μg / mL initial concentration, 14 concentration gradient dilutions) to each well for 2 hours at 37°C; dilute at 1:4000 Add 100 μL / well of goat anti-human secondary antibody to the well, and incubate at 37°C for 1 hour; 100 μL / well of peroxidase substrate chromogenic solution, stop the reaction with 2M sulfuric acid after 10 minutes at room temperature, and use dual wavelengths for colorimetry on the machine 450nm / 690nm.

[0069] The result is as figure 2 shown by figure 2 It can be seen that the anti-GPC3 antibody can react with the GPC3 protein,...

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Abstract

The invention discloses a fully human-anti-GPC3 all-molecule IgG antibody. The amino acid sequence of a light chain variable region is shown by SEQ ID NO.3, or is a conservative variant obtained by conservative mutation of the sequence through adding, deleting, replacing and modifying one or more amino acids; and the amino acid sequence of heavy chain variable region is shown by SEQ ID NO.4, or is a conservative variant obtained by conservative mutation of the sequence through adding, deleting, replacing and modifying one or more amino acids. The invention further discloses applications of the fully human-anti-GPC3 all-molecule IgG antibody. The invention also discloses a gene coded with the fully human-anti-GPC3 all-molecule IgG antibody. The invention further discloses an expression vector comprising the gene and an expression regulation sequence operatively connected with the gene. The invention also discloses a host cell which is transformed by the expression carrier.

Description

technical field [0001] The present invention relates to the technical field of biological immunity, in particular to a fully human anti-GPC3 full-molecular IgG antibody and its application, and also relates to its coding gene, its coding gene expression vector, and host cells. Background technique [0002] The incidence of primary hepatocellular carcinoma (HCC) ranks fifth in the world among malignant tumors, while the death rate ranks third. my country is one of the high-incidence areas of liver cancer. More than 100,000 people die of primary liver cancer every year. Only 10-20% of HCC patients can be diagnosed at an early stage and receive surgical treatment. Most patients are diagnosed at an advanced stage. Poor, and treatment options are few. In addition, primary liver cancer is prone to metastasis. Even if the small liver cancer is radically resected, the recurrence rate of metastasis is still as high as 50% within 5 years after operation. HCC mostly passes through blo...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N15/13A61K39/395A61P35/00G01N33/574
CPCA61K39/00C07K16/18C07K2317/52C07K2317/56C07K2317/565C07K2317/73
Inventor 冯振卿唐奇许国贞刘振云朱进熊四平唐小军
Owner SINOBIOWAY CELL THERAPY CO LTD
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