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Therapeutic agent for tumors identified by phosphorylation of proto-oncogene protein belonging to vav family

A proto-oncogene and therapeutic agent technology applied to the field of therapeutic agents for tumors identified through the phosphorylation of proto-oncogene proteins belonging to the VAV family can solve problems such as clinical development and achieve high effectiveness

Inactive Publication Date: 2019-07-05
UNIV OF TSUKUBA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Dasatinib also has an inhibitory effect on tyrosine kinases other than ABL, but clinical development using this effect has not yet been carried out

Method used

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  • Therapeutic agent for tumors identified by phosphorylation of proto-oncogene protein belonging to vav family
  • Therapeutic agent for tumors identified by phosphorylation of proto-oncogene protein belonging to vav family
  • Therapeutic agent for tumors identified by phosphorylation of proto-oncogene protein belonging to vav family

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0131] (1) Jurkat cells (T cells) expressing wild-type RHOA cDNA by TET-ON or the coding region of RHOA (hereinafter referred to as G17V RHOA cDNA. Leukemia cell line) each 2×10 5 / mL was inoculated into 3 15 cm petri dishes in RPMI (10% FCS, 1% PS). Add doxycycline in such a way that the final concentration is 2 μg / mL. The next day, the cells were recovered temporarily, and centrifuged at 6×10 5 / mL each was inoculated into 3 15 cm Petri dishes in RPMI (serum-free). After 4 hours, the cells were recovered, sterilized, washed once with phosphate-buffered saline (PBS), and adjusted to 2×10 7 / ml, transferred to a 15ml tube, and incubated at 37°C for 5 minutes. Join LEAF TM Purify anti-human CD3 Ab (BioLegend), anti-mouse IgG antibody Ab (each added in an amount of 2 μg / ml). Incubate at 37 °C for 5 min or 30 min. Add 10ml of cooled PBS, centrifuge, and remove the supernatant. Lysis buffer (with complete protease inhibitors and PhosSTOP added beforehand) was added at 1000...

Embodiment 2

[0137] Using lentivirus, the SU9T01 cells (adult T-cell leukemia / lymphoma cell lines) expressing wild-type RHOA or G17V RHOA cDNA through TET-ON were divided into 2×10 5 / mL each was inoculated into 3 15 cm Petri dishes in RPMI (10% FCS, 1% PS). Add doxycycline in such a way that the final concentration is 2 μg / mL. After the cells were recovered the next day, they were washed once with PBS. Add 1000 μl / tube of lysis buffer to the pellet. Thereafter, experiments were performed in the same manner as the Jurkat cells of Example 1. show the result in image 3 , 4 .

Embodiment 3

[0139] LEAF was diluted with sterile phosphate-buffered saline (PBS) TM Purified anti-human CD3 Ab (BioLegend) was adjusted to 10 μg / ml, and 50 μL / well was dispensed into each 96-well flat-bottom plate, and incubated overnight at 4°C. After removing the antibody solution, wash 3 times with sterilized PBS.

[0140] Take 5×10 4 Seed Jukat cells per well into 24-well plates. The next day, X-tremeGENE HP DNA transfection reagent was used to transiently transfect the pGL4.30 vector (Promega) inserted with the NFAT response sequence and the cDNA encoding firefly luciferase, and the pGL4.30 vector (Promega) inserted with the cDNA encoding Renilla luciferase. phRL vector (Promega), pEF vector inserted with wild-type or G17VRHOA cDNA. 24 hours after transfection, the cultured cell suspension was dispensed at 150 μl / well.

[0141] After 7 hours, the cultured cell solution was recovered into the tube, and 200 μl / well of PBS was further dispensed into the culture plate, transferred to...

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PUM

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Abstract

The present invention provides a therapeutic agent, etc., for various tumors, including angioimmunoblastic T-cell lymphoma (AITL), which is a rare disease. The present invention pertains to a therapeutic agent, etc., for tumors identified by phosphorylation of a proto-oncogene protein belonging to the VAV family, the therapeutic agent including dasatinib, a prodrug thereof, a pharmacologically acceptable salt of these, or a hydrate or solvate of these as an active ingredient.

Description

technical field [0001] The present invention relates to a therapeutic agent for a tumor identified by phosphorylation of a proto-oncogene protein belonging to the VAV family, a method for examining the effectiveness of dasatinib and the like in a patient to be administered, and the like. Background technique [0002] Dasatinib (please refer to Non-patent Document 1 for compounds, and refer to Non-Patent Document 2 for clinical trials) exerts high effectiveness in chronic myelogenous leukemia by inhibiting the ATP binding site of tyrosine kinase BCR-ABL. Dasatinib also has an inhibitory effect on tyrosine kinases other than ABL, but clinical development utilizing this effect has not been carried out so far. [0003] Angioimmunoblastic T-cell lymphoma (Angioimmunoblastic T-cell lymphoma: AITL) is a blood tumor that is extremely difficult to treat, and the 5-year survival rate is about 20%. In addition to lymphadenopathy, there are often characteristic clinical manifestations ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/506A61P35/00A61P35/02A61P43/00
CPCA61K31/506A61P35/00A61P35/02C12Q1/485G01N33/573G01N33/57426G01N2440/14G01N2800/52G01N33/5748
Inventor 千叶滋柳元麻实子
Owner UNIV OF TSUKUBA
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