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Anti-GPC3 antibody and pharmaceutical composition containing anti-GPC3 antibody

A composition and antibody technology, applied in the directions of drug combinations, antibodies, anti-tumor drugs, etc., can solve the problems of insufficient ADCC effect, and achieve the effect of improving the killing effect, improving the anti-tumor activity, and enhancing the ADCC effect.

Active Publication Date: 2021-09-28
SUZHOU PRO HEAL PHARM TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Insufficient ADCC effect of the antibody is an important reason for the clinical failure of GC33

Method used

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  • Anti-GPC3 antibody and pharmaceutical composition containing anti-GPC3 antibody
  • Anti-GPC3 antibody and pharmaceutical composition containing anti-GPC3 antibody
  • Anti-GPC3 antibody and pharmaceutical composition containing anti-GPC3 antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Molecular construction of anti-GPC3 antibody and anti-VEGF antibody

[0069] The light and heavy chain amino acid sequences of the antibodies in Table 1 were codon-optimized according to the human host cells and the genes were conventionally synthesized, adding 5'(EcoRI) and 5'UTR([SEQ ID NO 11]) and 3'UTR([TGATGA]) and 3'(HindIII), clone the gene into the vector pTT5 through 5'EcoRI and 3'HindIII (the vector map is as follows Figure 4 shown), the pTT5 vector sequence is as SEQ ID NO 12. Select the clones for sequencing, select the correctly sequenced bacteria for preservation and expand the culture of the bacteria, and the expanded bacteria are used for plasmid extraction.

[0070] Table 1 The sequence combination of anti-GPC3 antibody and anti-VEGF antibody

[0071] Antibody light chain sequence heavy chain sequence Anti-GPC3 SEQ ID NO 6 SEQ ID NO 7 Bevacizumab SEQ ID NO 8 SEQ ID NO 9 Anti-GPC3 (WT) SEQ ID NO 6 SEQ...

Embodiment 2

[0072] Example 2 Expression and purification of anti-GPC3 antibody and anti-VEGF antibody

[0073] Transfect the extracted plasmid into HEK293 cells and separate and purify the protein as follows

[0074] 1. Measure the cell density, the viability should be greater than 95%, adjust the cell density to 3×106 cells / mL with (preheated) HEK293 cell culture medium, shake gently and distribute the cells (transfection system 90%) , the volume of cells in the shake flask should not exceed 1 / 3 of the size of the shake flask, and put it into a shaker for later use.

[0075]2. Calculate the volume of transfection buffer opti-MEM according to the volume of transfected cells, which is 1 / 10 of the transfection system; calculate the amount of transfection reagent Polyethyleneimine, whose ratio is 3 μL / mL transfected cells; calculate the volume of transfected DNA The total amount, the ratio is 1 μg / mL transfected cells.

[0076] The specific transfection procedure is as follows:

[0077] T...

Embodiment 3

[0088] Example 3 Affinity Detection of Anti-GPC3 and Anti-GPC3 (WT) and Human CD16A

[0089] Using Gator TM Anti-GPC3 and Anti-GPC3 (WT) expressed above were tested for affinity with human CD16A protein (purchased from Novoprotein, Cat. No. CS11) by a non-labeled bioanalyzer, and Protein A biosensor was selected to capture the antibody sample, and then the The kinetic detection of binding and dissociation between the captured antibody sample and human CD16A protein. Kinetics were analyzed by fit using a 1:1 binding model. The brief steps are shown in Table 4 below:

[0090] Table 4

[0091] step time protein loading 200s combine 180s dissociate 300s regeneration 30s

[0092] Use Gator TM The affinity data determined by the instrument are shown in Table 5 below:

[0093] table 5

[0094]

[0095]

[0096] The affinity of the engineered antibody Anti-GPC3 to human CD16A protein is about 10.8 times higher than that of the unmod...

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Abstract

The invention belongs to the technical field of anti-cancer drugs, and particularly relates to an anti-GPC3 antibody and a pharmaceutical composition comprising the anti-GPC3 antibody. A light chain variable region and a heavy chain variable region of the anti-GPC3 antibody respectively include amino acid sequences as shown in SEQ ID NO 3 and SEQ ID NO 4, or respectively include sequences which have at least 90% consistency with the amino acid sequences shown in SEQ ID NO 3 and SEQ ID NO 4. According to the novel anti-human GPC3 antibody, the ADCC effect of the anti-GPC3 antibody is enhanced by enhancing the affinity of the antibody and Fc [gamma] RIIIa (CD16A), so that the killing effect of the antibody on tumors, especially hepatocellular tumors, is greatly improved.

Description

technical field [0001] The invention belongs to the technical field of anticancer drugs, and in particular relates to an anti-GPC3 antibody and a pharmaceutical composition comprising the antibody. Background technique [0002] Glypican-3 (GPC3) (SEQ ID NO 1) core protein consists of an N-terminal protein with a relative molecular mass of 40KD and a membrane-bound C-terminal protein of 30KD. The C-terminus is anchored on the cell surface by glycosyl-phosphatidylinositol anchor (GPI), which can bind heparin-binding proteins, such as growth factors, and is an extracellular matrix component that regulates cell growth, proliferation, Behaviors such as differentiation, adhesion, and migration may also be involved in inhibiting or regulating the growth of most mesoderm tissues and organs. GPC3 gene expression varies greatly in different tissues, its high expression in hepatocellular carcinoma tissue, but in non-neoplastic liver tissue, hepatocellular adenoma, cholangiocarcinoma, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/30A61K39/395A61P35/00
CPCC07K16/303A61P35/00C07K2317/56A61K2039/505
Inventor 张鹏马琳郭树华
Owner SUZHOU PRO HEAL PHARM TECH CO LTD
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