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Anti-glypican 3 antibody having modified sugar chain

一种抗磷脂酰肌醇、聚糖的技术，应用在糖链改变的抗磷脂酰肌醇聚糖3抗体领域，能够解决没有公开抗磷脂酰肌醇聚糖3抗体等问题

Inactive Publication Date: 2007-11-07
CHUGAI PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, anti-glypican 3 antibodies that enhance ADCC activity due to changes in sugar chains have not been disclosed so far

Method used

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  • Anti-glypican 3 antibody having modified sugar chain
  • Anti-glypican 3 antibody having modified sugar chain
  • Anti-glypican 3 antibody having modified sugar chain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Preparation of mouse anti-GPC3 antibody

[0076]A soluble GPC3 protein lacking the C-terminal hydrophobic region (amino acids 564-580) was prepared as an immune protein for preparing anti-GPC3 antibodies, and immunized. Autoimmune disease mice MRL / MpJUmmCrj-lpr / lpr mice (hereinafter referred to as MRL / lpr mice, purchased from Charles River, Japan) were used as immunized animals. Immunization was started when the mice were 7 or 8 weeks old, and the preparation for the primary immunization was adjusted to a dose of 100 μg / mouse of soluble GPC3. Emulsions were prepared using Freund's complete adjuvant (FCA, Becton Dickinson) and injected subcutaneously. After 5 times of immunization, the last immunization dose was diluted with PBS to 50 μg per mouse, and administered intravenously through the tail vein. On the 4th day after the final immunization, spleen cells were taken out, mixed with mouse myeloma cells P3-X63Ag8U1 (hereinafter referred to as P3U1, purchased from ATCC...

Embodiment 2

[0080] Preparation of anti-GPC3 antibody mouse-human chimeric antibody

[0081] The H chain and L chain variable region sequences of anti-GPC3 antibody GC33 were linked to the human IgG1 and κ chain constant region sequences. PCR was performed using a synthetic oligonucleotide complementary to the 5' terminal nucleotide sequence of the antibody H chain variable region having a Kozak sequence and a synthetic oligonucleotide having an NheI site complementary to the 3' terminal nucleotide sequence . The obtained PCR product was cloned into a pB-CH vector in which the human IgG1 constant region was inserted into pBluescript KS+ vector (Toyobo Co., Ltd.). The variable region of the mouse H chain and the constant region of the human H chain (γ1 chain) are connected through an NheI site. The prepared H chain gene fragment was cloned into the expression vector pCXND3. In addition, a synthetic oligonucleotide complementary to the 5' terminal nucleotide sequence of the antibody L cha...

Embodiment 3

[0084] Preparation of Low Fucose Anti-GPC3 Chimeric Antibody

[0085] First, YB2 / 0 (ATCC, CRL-1662) cells were used as host cells and cultured in RPMI1640 medium containing 10% FBS. Then by electroporation, under the condition of 1.4kV, 25μF and 7.5×10 6 At a concentration of 1 cell / 0.75 mL PBS(-), 25 μg of the anti-GPC3 chimeric antibody expression vector prepared in Example 2 was introduced into YB2 / 0 cells (ATCCCRT-1662). After a recovery period of 10 minutes at room temperature, the electroporated cells were suspended in 40 mL of RPMI1640 medium containing 10% FBS. A 10-fold dilution was prepared with the same medium, and the cells were dispensed into 96-well culture plates at 100 μL / well. in CO 2 Incubator (5% CO 2 ) for 24 hours, Geneticin (Invitrogen) was added to a concentration of 0.5 mg / mL, and the cells were cultured for 2 weeks. A cell line expressing a high level of the chimeric antibody was screened by sandwich ELISA using an anti-human IgG antibody, and a c...

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PUM

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Abstract

The invention relates to an anti-glypican 3 antibody having a modified sugar chain, more specifically, an anti-glypican 3 antibody lacking fucose. This anti-glypican 3 antibody can be produced by a method for producing an antibody with the sugar chain modification as described above wherein a nucleic acid encoding an anti-glypican 3 antibody is transferred into host cells with lowered ability to add fucose (for example, YB2 / 0 cells) or fucose transporter-deficient cells and then the host cells are cultured. Because of having a high cytotoxic activity, this anti-glypican 3 antibody having a modified sugar chain is useful as a cell growth inhibitor such as an anticancer agent.

Description

technical field [0001] The present invention relates to antibodies corresponding to Glypican 3 antigens with enhanced cytotoxic activity, especially antibody-dependent cellular cytotoxicity (ADCC) (ie, anti-Glypican 3 antibodies) and methods for their preparation. Background technique [0002] Glypican 3 (GPC3) is a member of the heparan sulfate proteoglycan family present on the cell surface. It is suggested that GPC3 may be related to cell division in development and proliferation of cancer cells, but its function is still not very clear. [0003] Various antibodies that bind to GPC3 have been found to have the effect of inhibiting cell proliferation through ADCC (antibody-dependent cytotoxicity) activity and CDC (complement-dependent cytotoxicity) activity (WO2003 / 000883). GPC3 is cleaved in vivo and secreted into the blood in the form of soluble GPC3, suggesting that cancer diagnosis can be performed using antibodies that can detect the form of soluble GPC3. (WO2004 / 02...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18A61K39/395A61P35/00C12N15/09C12P21/08
CPCC07K2317/732C07K16/303C07K2317/72C07K2317/41C07K2317/56C07K2317/24A61P35/00A61P43/00A61K39/395C12N15/09
Inventor 中野清孝周乡泉杉本正道石黑敬弘田中美谷米饭岛成幸
Owner CHUGAI PHARMA CO LTD
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