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Antibody capable of binding to transforming growth factor alpha and having antiproliferative activity on cancer having Ras gene mutation

A ras gene and antibody technology, applied in the direction of transforming growth factor, growth factor/inducer, anti-growth factor immunoglobulin, etc., can solve the problem that there is no effective means for Ras mutant cancer, and achieve the effect of excellent proliferation inhibitory activity

Active Publication Date: 2013-03-27
MEDICAL & BIOLOGICAL LAB CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, until now, no effective means exist for the treatment of Ras-mutated cancers

Method used

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  • Antibody capable of binding to transforming growth factor alpha and having antiproliferative activity on cancer having Ras gene mutation
  • Antibody capable of binding to transforming growth factor alpha and having antiproliferative activity on cancer having Ras gene mutation
  • Antibody capable of binding to transforming growth factor alpha and having antiproliferative activity on cancer having Ras gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0364] [Example 1] Acquisition of anti-TGFα monoclonal antibody

[0365] As the immunogen, proTGFα-mFc alone or proTGFα-mFc and proTGFα-mH were used. A total of 60 mice ( figure 1 a and b). The reactivity with the immunogen or the antigen for screening was evaluated by antigen solid-phase ELISA using hybridoma supernatants fused with lymphocytes and myeloma from immunized mice. Next, TGFα / 293T cells that transiently express the full length of TGFα in 293T cells were used for flow cytometry analysis, and hybridoma supernatants that showed reactivity with membrane-type TGFα were selected, and those capable of immunoprecipitating the secreted antigen proTGFα-mH were selected. Hybridoma supernatant ( Figure 4 ). After the hybridomas were monoclonalized by the limiting dilution method, immunogen solid-phase ELISA and immunoprecipitation were performed again, and a total of 39 anti-TGFα monoclonal antibodies were selected (Table 1). In order to confirm the reactivity of ...

Embodiment 2

[0366] [Example 2] Selection of TGFα neutralizing antibodies that inhibit the binding of TGFα to EGFR

[0367] In order to select an antibody (neutralizing antibody) having the activity of inhibiting the binding of TGFα to EGFR from the obtained anti-TGFα antibody, the following experiment was performed using EGFR highly expressing cancer cell A431. For A431 cultured in serum-free medium, the tyrosine phosphorylation level of EGFR transiently increased when TGFα was added. When A431 was treated after reacting the TGFα solution with each anti-TGFα antibody in advance, an antibody that did not increase the phosphorylation level of EGFR compared with the PBS control was searched. As a result, it was found that MBL352-34, MBL292-1, MBL184-6, MBL016-8, MBL144-1, MBL023-1, and MBL018-1 phosphorylated EGFR tyrosine (P- Tyr1173) inhibits more than 70%-100% ( Figure 5a and b). On the other hand, TGFα neutralizing antibody 189-2130 (Non-Patent Document 50) and EGFR-blocking ant...

Embodiment 3

[0368] [Example 3] Study on membrane-type TGFα or membrane-bound TGFα of cancer cells

[0369] Next, in order to verify the presence of TGFα on the cell surface of cancer cells having a K-Ras gene mutation, flow cytometry analysis was performed using MBL259-3, which has relatively good reactivity to TGFα / 293T cells. As a result, SW620 derived from colorectal cancer, which is a K-Ras gene G12V homomutated cancer cell line, and A427, which is a K-Ras gene G12D heteromutated cell line derived from lung cancer, reacted with MBL259-3 ( Image 6 ).

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Abstract

Among antibodies exhibiting reactivity to natural TGF[alpha], antibodies having low reactivity to G79A-substituted TGF[alpha] are identified as exhibiting superior antiproliferative activity against cancer cells having a mutation in the Ras gene. Further identified is that many of the antibodies also inhibit the tyrosine phosphorylation of EGFR and / or inhibit the induction of vascular endothelial cells.

Description

technical field [0001] The present invention relates to a monoclonal antibody that binds to transforming growth factor alpha (hereinafter referred to as "TGFα") and has an effect of inhibiting the proliferation of cancer cells having a Ras protein mutation in vivo, and an anticancer agent containing the same . Background technique [0002] In early 1980, the group of Spawn (スポーン) and Roberts (Roberts) etc., from the culture supernatant of mouse 3T3 cells transformed with mouse sarcoma virus, was used as a method for inducing normal rat kidney fibroblasts in soft agar. One of the two factors necessary for colony formation of cells (NRK cells) was isolated and purified (Non-Patent Documents 1-6). [0003] TGFα is biosynthesized as a precursor polypeptide consisting of 160 amino acids, and after intracellular sugar chain modification and palmitoylation, it is expressed on the cell membrane surface as a type I membrane-penetrating protein (membrane-type TGFα) (Non-Patent Docume...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09A61K39/395A61P35/00C07K14/495C07K16/24C07K16/42C12N5/10C12N15/02C12P21/08G01N33/574
CPCC07K14/495C07K16/22A61K2039/505C07K2317/76C07K2317/24A61P35/00
Inventor 金田诚藤井庆宏早田芳弘岸义朗矢原一郎
Owner MEDICAL & BIOLOGICAL LAB CO LTD
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