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An anti-glypican-3 antibody and an application thereof

A technology of phosphatidylinositol and proteoglycan, applied in the field of tumor immunotherapy or diagnosis, can solve the problems of curative effect, side effect, long-term survival, allergic reaction, etc.

Active Publication Date: 2017-02-15
CARSGEN THERAPEUTICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing studies have shown that different antibodies may have different immunogenicity, which will lead to the efficacy and side effects of the antibody or its derived therapeutic preparations
For example, CAR T cells based on mouse anti-mesothelin antibodies can cause allergic reactions and affect their long-term survival in humans

Method used

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  • An anti-glypican-3 antibody and an application thereof
  • An anti-glypican-3 antibody and an application thereof
  • An anti-glypican-3 antibody and an application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0141] Example 1. Preparation of a specific single-chain antibody (scFv) that binds to human GPC3

[0142] 1.1 Screening of GPC3-specific binding antibodies based on phage display

[0143] Using phage display technology, human GPC3 (hereinafter referred to as huGPC3)-specific antibodies were screened from a fully human natural antibody library. For this purpose, 400ml of 2×YT / ampicillin medium was inoculated with glycerol bacteria (purchased from Shanghai Ruijin Biotechnology Co., Ltd.) displaying a natural library of fully human single-chain antibody by phage, so that the cell density reached OD 600 =0.1, shake culture at 37°C and 200rpm until the cell density reaches OD 600 = 0.5. use 10 12 pfu of M13KO7 helper phage (purchased from Invitrogen) was infected and incubated at 30° C. and 50 rpm for 30 minutes. After adding 50mg / L kanamycin, shake and culture at 37°C and 200rpm for 30 minutes, separate the precipitate by centrifugation (15 minutes, 1600×g, 4°C), resuspend in...

Embodiment 2

[0149] Example 2, Expression and purification of single-chain antibody against GPC3

[0150] The scFv was amplified from the plasmid (pCantab 5E-P1B12E) of the screened clone P1B12E using the primer pair V5-P1B12E-F (SEQ ID NO:5) and V5-P1B12E-R (SEQ ID NO:6) according to standard protocols - P1B12E fragment, scFv was amplified from the plasmid (pCantab 5E-P7D4) of clone P7D4 obtained by screening using the primer pair V5-P7D4-F (SEQ ID NO: 7) and V5-P7D4-R (SEQ ID NO: 8) -P7D4 fragment, through NheI / BamHI (purchased from NEB) double digestion, with T4DNA ligase (purchased from NEB) in the same with NheI / BamHI double digestion vector plasmid pCMV-V5-Fc (the vector is in the multiple cloning site Downstream fusion expresses the Fc fragment of human antibody IgG1, hereinafter referred to as V5-Fc, which was purchased from Shanghai Ruijin Biotechnology Co., Ltd.) and transformed into the host strain TOP10. The positive clones were picked and identified by PCR and confirmed by seq...

Embodiment 3

[0152] Example 3, flow cytometry analysis of the binding of each cell line to the anti-GPC3 single-chain antibody

[0153] The binding ability of the antibodies scFv-P1B12E-Fc and scFv-P7D4-Fc to the GPC3-positive liver cancer HepG2 cell line (ATCC) was analyzed by fluorescence activated cell sorting (FACS) (BD Company, FACSCalibur).

[0154] The specific method is as follows:

[0155] 1). Inoculate the HepG2 cell line of liver cancer in the logarithmic growth phase into a 6cm plate, the inoculated cell density is about 90%, and cultivate overnight in a 37°C incubator.

[0156] 2). Use 10mM EDTA to digest the cells, and collect the cells by centrifugation at 200g×5min. Take 1×10 6 ~1×10 7 The concentration per mL was resuspended in 1% phosphate buffered saline (NBS PBS) containing calf serum, and added to a flow-type tube in an amount of 100 ul / tube.

[0157] 3). Centrifuge at 200g×5min, discard the supernatant.

[0158] 4). The antibodies to be tested, scFv-P1B12E-Fc and...

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PUM

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Abstract

The invention relates to an anti-glypican-3 (GPC3) antibody and an application thereof. The novel antibody capable of specifically recognizing the GPC3 is disclosed, includes a single chain antibody and a humanized antibody, and can be applied for preparing targeting antitumor medicines and tumor diagnosing medicines.

Description

technical field [0001] The present invention relates to the field of tumor immunotherapy or diagnosis, and more specifically relates to an antibody specifically recognizing phosphatidylinositol proteoglycan-3 (GPC3) and its application. Background technique [0002] At present, adoptive immunotherapy based on immune effector cells has achieved certain effects in some tumors, and this immunotherapy method can overcome the above-mentioned defects of antibody therapy, but the efficacy in most tumors is still unsatisfactory[Grupp SA , et al.Adoptive cellular therapy.Curr Top Microbiol Immunol., 2011; 344:149-72.]. In recent years, based on the discovery that cytotoxic T lymphocytes (cytotoxic lymphocytes, CTLs) recognize target cells specifically depends on T cell receptors (T Cell Receptors, TCRs), the scFv of antibodies against tumor cell-associated antigens was combined with Intracellular signal activation motifs such as CD3ζ or FcεRIγ of T lymphocyte receptors are fused int...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K16/30C12N15/13C12N15/63C12N5/10C12N7/01C07K19/00G01N33/68G01N33/574A61K39/395A61K39/44A61K47/68A61K38/19A61K38/20A61K38/21A61P35/00
CPCA61K38/191A61K38/208A61K38/2086A61K38/215A61K39/44C07K14/7051C07K14/70517C07K14/70521C07K14/70578C07K14/70596C07K16/28G01N33/68A61K2039/505C07K2319/03C07K2317/56C07K2317/565C07K2317/622C12N2740/15043C07K16/303C12N15/86C12N5/0638A61K47/6813A61K47/6817A61K47/6849A61K39/001102A61K39/001138A61K39/00114A61K39/001141A61P35/00G01N33/57484C07K2317/24C07K2319/33C07K2319/74C12N2800/107C12N2510/00A61K2039/5156A61K2300/00A61K39/395A61K48/00C12N5/10C12N15/63A61K39/0011C07K14/7151C07K2317/21C07K2317/31C07K2317/73C07K2317/92A61P1/16A61P15/08A61P25/00A61K2039/5158Y02P20/55C07K16/30C07K19/00C07K16/46C12N15/62A61K47/6803A61K35/17C07K16/2809C12N5/0636G01N33/574A61K47/6879G01N33/57438
Inventor 王华茂宋波
Owner CARSGEN THERAPEUTICS
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