(19)
(11)EP 3 536 321 A1

(12)EUROPEAN PATENT APPLICATION
published in accordance with Art. 153(4) EPC

(43)Date of publication:
11.09.2019 Bulletin 2019/37

(21)Application number: 17866638.4

(22)Date of filing:  19.10.2017
(51)Int. Cl.: 
A61K 31/506  (2006.01)
A61P 35/02  (2006.01)
A61P 35/00  (2006.01)
A61P 43/00  (2006.01)
(86)International application number:
PCT/JP2017/038595
(87)International publication number:
WO 2018/084053 (11.05.2018 Gazette  2018/19)
(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
Designated Extension States:
BA ME
Designated Validation States:
MA MD

(30)Priority: 02.11.2016 JP 2016215521

(71)Applicant: University of Tsukuba
Tsukuba-shi, Ibaraki 305-8577 (JP)

(72)Inventors:
  • CHIBA, Shigeru
    Tsukuba-shi Ibaraki 305-8577 (JP)
  • YANAGIMOTO, Mamiko
    Tsukuba-shi Ibaraki 305-8577 (JP)

(74)Representative: J A Kemp 
14 South Square Gray's Inn
London WC1R 5JJ
London WC1R 5JJ (GB)

  


(54)THERAPEUTIC AGENT FOR TUMORS IDENTIFIED BY PHOSPHORYLATION OF PROTO-ONCOGENE PROTEIN BELONGING TO VAV FAMILY


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


Description

TECHNICAL FIELD



[0001] The present invention relates to a therapeutic agent for tumors identified by phosphorylation of a proto-oncogene protein belonging to the VAV family, a method for testing the efficacy of dasatinib or other form thereof in a patient who is a subject to be administered therewith, etc.

BACKGROUND ART



[0002] Dasatinib (see Non-patent Document 1 for details of the compound and Non-patent Document 2 for its clinical trial) exerts high efficacy in chronic myelogenous leukemia through inhibition of the ATP-binding site in tyrosine kinase BCR-ABL. Dasatinib also has an inhibitory effect against tyrosine kinases other than ABL, but no effort has been made to pursue clinical development based on this effect.

[0003] Angioimmunoblastic T-cell lymphoma (AITL) is an extremely intractable blood cancer and its five-year survival rate is about 20%. AITL often presents characteristic clinical features including lymph node swelling, as well as fervescence, exanthema, autoimmune disease-like alterations, hypergammaglobulinemia and so on. The inventors of the present invention have reported the genomic analysis of AITL indicating that a RHOA gene mutation (c.50G>T) causing a glycine to valine substitution at amino acid position 17 of the RHOA protein (p.G17V, hereinafter referred to as a "G17V RHOA mutation") is observed in 70% of AITL cases (Non-patent Document 3: Sakata-Yanagimoto M, et al. Nat Genet. 2014 Feb; 46(2):171-5). Thus, there have been demands not only for elucidating the mechanism of oncogenesis due to these genomic alterations, but also for developing targeted therapies.

Prior Art Documents


Non-patent Documents



[0004] 

Non-patent Document 1: Shah NP., et al., Overriding imatinib resistance with a novel ABL kinase inhibitor., Science, vol. 305, p. 399-401, 2004

Non-patent Document 2: Talpaz M, et al., N. Engl. J. Med., 2006 Jun 15; 354(24):2531-41

Non-patent Document 3: Sakata-Yanagimoto M, et al., Nat. Genet., 2014 Feb; 46(2):171-5


SUMMARY OF THE INVENTION



[0005] Under these circumstances, there has been a demand for the development of pharmaceutical formulations and therapeutic methods useful in the treatment of various tumors including angioimmunoblastic T-cell lymphoma (AITL), which is an orphan disease. In particular, for the treatment of AITL, combination chemotherapy has been used conventionally, but AITL is often refractory to conventional therapy; and hence the development of novel therapeutic agents and therapeutic methods has been awaited.

[0006] The present invention has been made in consideration of the above situation and aims to provide a therapeutic agent for tumors identified by phosphorylation of a proto-oncogene protein belonging to the VAV family, a pharmaceutical composition for the treatment of these tumors, the use of dasatinib or other form thereof for the manufacture of a pharmaceutical agent for the treatment of these tumors, a therapeutic method for these tumors, and a therapeutic kit for these tumors, as well as a method for testing the efficacy of dasatinib or other form thereof in a patient who is a subject to be administered therewith, etc., as shown below.
  1. (1) A therapeutic agent for a tumor identified by phosphorylation of a proto-oncogene protein belonging to the VAV family,
    wherein the therapeutic agent comprises dasatinib or a prodrug thereof or a pharmacologically acceptable salt thereof or a hydrate or solvate thereof as an active ingredient.
  2. (2) The therapeutic agent according to (1) above, wherein the tumor is a tumor identified to show enhanced phosphorylation of a proto-oncogene protein belonging to the VAV family.
  3. (3) The therapeutic agent according to (1) or (2) above, wherein the protein is the VAV1 protein.
  4. (4) A therapeutic agent for a tumor carrying a gene mutation or gene fusion in the RHOA gene and/or in a proto-oncogene belonging to the VAV family,
    wherein the therapeutic agent comprises dasatinib or a prodrug thereof or a pharmacologically acceptable salt thereof or a hydrate or solvate thereof as an active ingredient.
  5. (5) The therapeutic agent according to any one of (1) to (4) above, wherein the tumor is at least one selected from the group consisting of T-cell lymphoma, gastric cancer, pancreatic cancer, skin tumor, colorectal cancer, uterine cancer and nervous system tumor.
  6. (6) The therapeutic agent according to any one of (1) to (5) above, wherein the tumor is T-cell lymphoma.
  7. (7) The therapeutic agent according to any one of (1) to (6) above, wherein the T-cell lymphoma is angioimmunoblastic T-cell lymphoma or peripheral T-cell lymphoma, not otherwise specified.
  8. (8) A pharmaceutical composition for the treatment of a tumor identified by phosphorylation of a proto-oncogene protein belonging to the VAV family, which comprises dasatinib or a prodrug thereof or a pharmacologically acceptable salt thereof or a hydrate or solvate thereof.
    The present invention also encompasses a pharmaceutical composition for the treatment of a tumor carrying a gene mutation or gene fusion in the RHOA gene and/or in a proto-oncogene belonging to the VAV family, which comprises dasatinib or a prodrug thereof or a pharmacologically acceptable salt thereof or a hydrate or solvate thereof.
  9. (9) The use of dasatinib or a prodrug thereof or a pharmacologically acceptable salt thereof or a hydrate or solvate thereof for the manufacture of a pharmaceutical agent for the treatment of a tumor identified by phosphorylation of a proto-oncogene protein belonging to the VAV family.
    The present invention also encompasses the use of dasatinib or a prodrug thereof or a pharmacologically acceptable salt thereof or a hydrate or solvate thereof for the manufacture of a pharmaceutical agent for the treatment of a tumor carrying a gene mutation or gene fusion in the RHOA gene and/or in a proto-oncogene belonging to the VAV family.
  10. (10) A therapeutic method for a tumor identified by phosphorylation of a proto-oncogene protein belonging to the VAV family, which comprises administering a subject with dasatinib or a prodrug thereof or a pharmacologically acceptable salt thereof or a hydrate or solvate thereof.
    The present invention also encompasses a therapeutic method for a tumor carrying a gene mutation or gene fusion in the RHOA gene and/or in a proto-oncogene belonging to the VAV family, which comprises administering a subject with dasatinib or a prodrug thereof or a pharmacologically acceptable salt thereof or a hydrate or solvate thereof.
  11. (11) A therapeutic kit for a tumor identified by phosphorylation of a proto-oncogene protein belonging to the VAV family, which comprises dasatinib or a prodrug thereof or a pharmacologically acceptable salt thereof or a hydrate or solvate thereof.
    The present invention also encompasses a therapeutic kit for a tumor carrying a gene mutation or gene fusion in the RHOA gene and/or in a proto-oncogene belonging to the VAV family, which comprises dasatinib or a prodrug thereof or a pharmacologically acceptable salt thereof or a hydrate or solvate thereof.
  12. (12) A method for testing the efficacy of dasatinib or a prodrug thereof or a pharmacologically acceptable salt thereof or a hydrate or solvate thereof in a patient who is a subject to be administered therewith, wherein the efficacy is indicated by the level of phosphorylation of a proto-oncogene protein belonging to the VAV family in an analyte taken from the patient.
  13. (13) A method for testing the efficacy of dasatinib or a prodrug thereof or a pharmacologically acceptable salt thereof or a hydrate or solvate thereof in a patient who is a subject to be administered therewith, wherein the efficacy is indicated by the presence or absence of a gene mutation or gene fusion in the RHOA gene in an analyte taken from the patient.
  14. (14) A method for testing the efficacy of dasatinib or a prodrug thereof or a pharmacologically acceptable salt thereof or a hydrate or solvate thereof in a patient who is a subject to be administered therewith, wherein the efficacy is indicated by the presence or absence of a gene mutation or gene fusion in the VAV1 gene in an analyte taken from the patient.
  15. (15) A method for testing the efficacy of dasatinib or a prodrug thereof or a pharmacologically acceptable salt thereof or a hydrate or solvate thereof in a patient who is a subject to be administered therewith, wherein the efficacy is indicated by the presence or absence of a gene mutation or gene fusion in the VAV2 gene in an analyte taken from the patient.
  16. (16) A method for testing the efficacy of dasatinib or a prodrug thereof or a pharmacologically acceptable salt thereof or a hydrate or solvate thereof in a patient who is a subject to be administered therewith, wherein the efficacy is indicated by the presence or absence of a gene mutation or gene fusion in the VAV3 gene in an analyte taken from the patient.

EFFECTS OF THE INVENTION



[0007] The present invention enables the provision of a pharmaceutical formulation and a pharmaceutical composition, each being useful in the treatment of tumors identified by phosphorylation of a proto-oncogene protein belonging to the VAV family, a method useful in the treatment of these tumors, and a therapeutic kit for these tumors, etc. Particularly in the treatment of intractable diseases such as angioimmunoblastic T-cell lymphoma (AITL), dasatinib can be expected to produce a more specific and efficacious therapeutic effect because dasatinib inhibits the activation of VAV protein molecules, per se, which are activated by a mutation or the like in the RHOA gene and/or in a proto-oncogene belonging to the VAV family.

[0008] Moreover, the present invention also enables the provision of a method for testing the efficacy of dasatinib or other form thereof in a patient who is a subject to be administered therewith.

BRIEF DESCRIPTION OF THE DRAWINGS



[0009] 

Figure 1 indicates that in acute T-cell leukemia cell line Jurkat cells, a G17V RHOA mutant binds to VAV1, which is a molecule important for T cell receptor (TCR) signaling.

Figure 2(A) indicates that in Jurkat cells, G17V RHOA mutant expression enhances Y174 phosphorylation which is indicative of VAV1 activation, and that this effect is enhanced upon anti-CD3 antibody stimulation.

Figure 2(B) indicates that in Jurkat cells, G17V RHOA mutant expression enhances phospholipase C (PLC)-gamma 1 phosphorylation under anti-CD3 antibody stimulation.

Figure 3 indicates that in adult T-cell leukemia/lymphoma cell line SU9T01 cells, a G17V RHOA mutant binds to VAV1, which is a molecule important for T cell receptor (TCR) signaling.

Figure 4(A) indicates that in SU9T01 cells, G17V RHOA mutant expression enhances Y174 phosphorylation which is indicative of VAV1 activation.

Figure 4(B) indicates that in SU9T01 cells, G17V RHOA mutant expression enhances phospholipase C (PLC)-gamma 1 phosphorylation.

Figure 5 indicates that in Jurkat cells, G17V RHOA mutant expression enhances NFAT (nuclear factor of activated T-cells) activity.

Figure 6 indicates that in Jurkat cells, G17V RHOA mutant expression enhances interleukin-2 mRNA expression.

Figure 7(A) indicates that in Jurkat cells, dasatinib cancels the enhanced Y174 phosphorylation induced by G17V RHOA mutant expression.

Figure 7(B) indicates that in Jurkat cells, dasatinib cancels the enhanced PLC-gamma 1 phosphorylation induced by G17V RHOA mutant expression.

Figure 8 indicates that in Jurkat cells, dasatinib cancels the enhanced interleukin-2 mRNA expression induced by G17V RHOA mutant expression.

Figure 9 indicates that there are VAV1 gene mutations in AITL and peripheral T-cell lymphoma, not otherwise specified.

Figure 10 indicates that in Jurkat cells, VAV1 mutant expression enhances Y174 phosphorylation.

Figure 11 indicates that in Jurkat cells, VAV1 mutant expression enhances PLC-gamma 1 phosphorylation.

Figure 12 indicates that in Jurkat cells, VAV1 mutant expression enhances NFAT activity.

Figure 13 indicates that in Jurkat cells, VAV1 mutant expression enhances interleukin-2 mRNA expression.

Figure 14 indicates that in Jurkat cells, VAV1 mutant expression enhances interleukin-2 secretion into the supernatant.

Figure 15 indicates that in Jurkat cells, dasatinib cancels the enhanced Y174 phosphorylation induced by VAV1 mutant expression.

Figure 16 indicates that in Jurkat cells, dasatinib cancels the enhanced NFAT activity induced by VAV1 mutant expression.

Figure 17 is a graph comparing the survival rate ("Probability" in the figure) between groups where nude mice (AITL model mice) were intraperitoneally injected with donor mouse swollen lymph node-derived cells and then administered with dasatinib (the dasatinib group: "Dasatinib" in the figure) and not administered with dasatinib (the control group: "Vehicle" in the figure).


DESCRIPTION OF EMBODIMENTS



[0010] The present invention will be further described in more detail below. The scope of the present invention is not limited by the following description, and any embodiments other than those illustrated below may also be carried out with appropriate modifications without departing from the spirit of the present invention. It should be noted that this specification incorporates the specification of Japanese Patent Application No. 2016-215521 (filed on November 2, 2016) in its entirety, based on which the present application claims priority. Moreover, all publications cited herein, including prior art documents, patent gazettes and other patent documents, are incorporated herein by reference.

1. Overview of the present invention



[0011] Proto-oncogene proteins belonging to the VAV family, such as VAV1, are molecules critically important for T cell receptor signaling and are known to be activated upon phosphorylation by Src family kinases. The inventors of the present invention have found that a G17V RHOA mutant binds to VAV1 and enhances the phosphorylation (tyrosine phosphorylation) thereof to thereby activate the downstream signaling of VAV1, and that in some cases with no G17V RHOA mutation, a gene mutation is found in VAV1 per se and this mutation causes VAV1 activation. The inventors of the present invention have further found that VAV1 activation (abnormal activation) caused by such a G17V RHOA mutant or VAV1 mutant is inhibited by dasatinib. Moreover, the inventors of the present invention have found that the NFAT activity-enhancing effect of the VAV1 mutant under T cell receptor signaling is inhibited by dasatinib, and further that interleukin-2 (IL-2) production by the G17V RHOA mutant under T cell receptor signaling is inhibited by dasatinib.

[0012] Based on these findings, the inventors of the present invention have found out the present invention directed to a therapeutic agent for AITL comprising dasatinib, more particularly a therapeutic agent for tumors identified by phosphorylation (tyrosine phosphorylation) of a proto-oncogene protein belonging to the VAV family, etc.

2. Therapeutic agent, pharmaceutical composition and others for tumor treatment



[0013] The therapeutic agent of the present invention for a tumor identified by phosphorylation of a proto-oncogene protein belonging to the VAV family (hereinafter also simply referred to as "the therapeutic agent of the present invention") and the pharmaceutical composition of the present invention for the treatment of a tumor identified by phosphorylation of a proto-oncogene protein belonging to the VAV family (hereinafter also simply referred to as "the pharmaceutical composition of the present invention") are each characterized by comprising dasatinib or a prodrug thereof or a pharmacologically acceptable salt thereof or a hydrate or solvate thereof (hereinafter also referred to as "dasatinib or other form thereof') as an active ingredient, as described above.

[0014] It should be noted that the present invention also encompasses: (i) a therapeutic method for a tumor identified by phosphorylation of a proto-oncogene protein belonging to the VAV family, which comprises using dasatinib or other form thereof, e.g., more specifically administering an effective amount of dasatinib or other form thereof to a subject (i.e., a patient with a tumor identified by phosphorylation of a proto-oncogene protein belonging to the VAV family or a patient at the risk thereof, or alternatively, such a non-human mammal); (ii) the use of dasatinib or other form thereof for the manufacture of a pharmaceutical agent for the treatment of such a tumor; (iii) the use of dasatinib or other form thereof for the treatment of such a tumor; and (iv) dasatinib or other form thereof for use in the treatment of such a tumor.

[0015] In the present invention, the treatment of such a tumor more specifically also includes, for example, suppression of tumor progression, improvement of prognosis, and/or prevention of recurrence, etc.

[0016] In the present invention, the tumor to be treated is a tumor identified by phosphorylation (tyrosine phosphorylation) of a proto-oncogene protein belonging to the VAV family. As intended herein, the tumor identified by phosphorylation of such a protein is not limited in any way and may be, for example, a tumor in a state identified to show enhanced (or improved) phosphorylation of the protein when compared to that in the tissue of normal subjects. Moreover, in the present invention, other tumors to be treated are preferably exemplified by tumors carrying a gene mutation or gene fusion in the RHOA gene and/or in a proto-oncogene belonging to the VAV family. More specifically, these tumors to be treated are preferably exemplified by T-cell lymphoma (preferably angioimmunoblastic T-cell lymphoma (AITL), peripheral T-cell lymphoma, not otherwise specified, etc.), gastric cancer, pancreatic cancer, skin tumor, colorectal cancer, uterine cancer, and nervous system tumor, etc.

[0017] Examples of a proto-oncogene protein belonging to the VAV family include VAV1, VAV2 and VAV3. In particular, in the case of VAV1, its activation induced by phosphorylation (abnormal activation induced by enhanced tyrosine phosphorylation) is a factor responsible for causing T-cell lymphoma, particularly angioimmunoblastic T-cell lymphoma (AITL).

[0018] In the present invention, examples of a gene mutation in the RHOA gene (SEQ ID NO: 1 (NCBI GenBank Accession No. NM_001664.3): cDNA covers nucleotides at positions 281 to 862 among 1943 nucleotides) include, but are not limited to, those shown in Table 1 below, including a gene (DNA) mutation (c.50G>T) which results in an amino acid mutation p.G17V (G17V RHOA mutation) in the RHOA protein (SEQ ID NO: 2). Further examples include gene mutations resulting in amino acid mutations p.A161E, p.A161P, p.A161V and p.A118E in this protein.

[0019] As to the notation of gene mutations used herein, for example, "c.50G>T" refers to a G to T substitution at nucleotide position 50 of cDNA, and "c.C518_529del" refers to C deletions at positions 518 and 529 of cDNA.

[0020] Likewise, as to the notation of amino acid mutations in the protein, for example, "p.G17V" refers to a G (glycine) to V (valine) substitution at position 17 of the amino acid sequence encoded by cDNA, "p.E175V/L" refers to a E (glutamic acid) to V (valine) or L (leucine) substitution at position 175 of the amino acid sequence, "p.173_177del" refers to deletion of residues at positions 173 and 177 of the amino acid sequence, and "p.R24*" refers to a mutation where the codon encoding R (arginine) at position 24 of the amino acid sequence is converted into a stop codon.
[Table 1]
RHOA gene mutationAmino acid mutation in RHOA protein
c.50G>T p.G17V
c.125A>G p.Y42C
c.46T>C p.C16R
c.14G>A p.R5Q
c.14G>A p.G17E


[0021] Moreover, in the present invention, examples of a gene mutation in a proto-oncogene belonging to the VAV family include, but are not limited to, gene mutations in the VAV1 gene (SEQ ID NO: 3 (NCBI GenBank Accession No. NM_005428.3): cDNA covers nucleotides at positions 141 to 2678 among 2944 nucleotides), which result in amino acid mutations in the VAV1 protein (SEQ ID NO: 4) as shown in Table 2 below. Further examples include gene mutations resulting in amino acid mutations p.E157K, p.Y174C, p.E175V/L, p.L177R, p.K494R, p.Q487K/R, p.M501R/L/V, p.E556K, p.P615L, p.R790C, p.D797N/H, p.R798P, p.J815E and p.R822L in this protein.
[Table 2]
VAV 1 gene mutationAmino acid mutation in VAV1 protein
c.C518_529del p.173_177del
c.C494_520del p.165_174del
c.1668G>C p.E556D
c.1844C>T p.P615L
c.1211A>G p.K404R
c.2465G>A p.R822Q
c.388C>T p.P130S
c.2393G>A p.R798Q
c.1696C>T p.R566*


[0022] Likewise, in the case of the VAV2 gene (SEQ ID NO: 5 (NCBI GenBank Accession No. NM_001134398.1): cDNA covers nucleotides at positions 47 to 2683 among 4865 nucleotide), examples include gene mutations resulting in amino acid mutations in the VAV2 protein (SEQ ID NO: 6) as shown in Table 3 below. Further examples include gene mutations resulting in amino acid mutations p.R103Q, p.R76H, p.D760N, p.G854D, p.P657S, p.L88F, p.D170E/G, p.R700Q, p.A362T and p.P130L in this protein.
[Table 3]
VAV2 gene mutationAmino acid mutation in VAV2 protein
c.763C>T p.L255L
c.748C>T p.L250L
c.686C>T p.P229L
c.701C>T p.P234L
c.308G>A p.R103Q


[0023] Likewise, in the case of the VAV3 gene (SEQ ID NO: 7 (NCBI GenBank Accession No. NM_006113.4): cDNA covers nucleotides at positions 55 to 2598 among 4776 nucleotides), examples include gene mutations resulting in amino acid mutations in the VAV3 protein (SEQ ID NO: 8) as shown in Table 4 below. Further examples include gene mutations resulting in amino acid mutations p.R24*, p.V275M, p.A213T, p.E53D, p.V65I, p.W277*, C98*, p.L198*, p.K187*, p.W112* and p.G79fs in this protein. As used here, "p.G79fs" refers to a frameshift mutation where the codon encoding G (glycine) at position 79 of the amino acid sequence is translated as another codon.
[Table 4]
VAV3 gene mutationAmino acid mutation in VAV3 protein
c.1309G>A p.D437N
c.1037C>T p.T346I
c.578G>A p.R193Q
c.2382C>T p.I794I


[0024] Moreover, in the present invention, examples of a gene fusion in a proto-oncogene belonging to the VAV family include, but are not limited to, VAV1-STAP2, VAV1-GSS, VAV1-MYO1F and so on in the case of the VAV1 gene.

[0025] Dasatinib for use as an active ingredient in the therapeutic agent or pharmaceutical composition of the present invention may be a known commercially available product, but is not limited thereto, and may also be synthesized, extracted and purified independently for this purpose.

[0026] It should be noted that dasatinib is officially known as N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1-piperazinyl]-2-methyl-4-pyrimidinyl]amino]-5-thiazolecarboxamide and is represented by the following structural formula.



[0027] As an active ingredient in the therapeutic agent or pharmaceutical composition of the present invention, a dasatinib derivative may also be used in combination with dasatinib or in place of dasatinib. Such a derivative is not limited in any way as long as it is considered to be a derivative of dasatinib on the basis of common knowledge shared among those skilled in the art, e.g., in terms of having a chemical structure derived from dasatinib, and preferred is a derivative having the ability to suppress the phosphorylation-induced activation of a proto-oncogene protein belonging to the VAV family (e.g., VAV1) at the same level as dasatinib.

[0028] Examples of dasatinib or a derivative thereof for use in the present invention include not only those which undergo in vivo metabolism such as oxidation, reduction, hydrolysis or conjugation, but also compounds which produce dasatinib or derivatives thereof upon in vivo metabolism such as oxidation, reduction or hydrolysis (i.e., so-called prodrugs). In the present invention, such a prodrug refers to a compound prepared by modifying its parent compound with a pharmacologically acceptable group commonly used in prodrugs, and is exemplified by a compound which is provided with properties such as improved stability and sustainability and can be expected to exert the intended effect when converted into the parent compound in the intestinal tract or elsewhere. For example, a prodrug of a compound such as dasatinib can be prepared in a standard manner by using a prodrug-forming reagent such as a corresponding halide to introduce a prodrug-constituting group(s) as appropriate in a standard manner into any one or more groups selected from among the groups in this compound, which can be used for prodrug formation (e.g., a hydroxyl group, an amino group, other groups), optionally followed by isolation and purification. As intended here, the above prodrug-constituting groups preferably include, but are not limited to, lower alkyl-CO-, lower alkyl-O-lower alkylene-CO-, lower alkyl-OCO-lower alkylene-CO-, lower alkyl-OCO-, and lower alkyl-O-lower alkylene-OCO-, etc.

[0029] As an active ingredient in the therapeutic agent or pharmaceutical composition of the present invention, a pharmacologically acceptable salt of dasatinib or a dasatinib derivative or a prodrug thereof may also be used in combination with dasatinib or a dasatinib derivative or a prodrug thereof or in place of dasatinib or a dasatinib derivative or a prodrug thereof.

[0030] Examples of a pharmacologically acceptable salt of dasatinib or a derivative thereof preferably include, but are not limited to, halogenated hydroacid salts (e.g., hydrochloride salt, hydrobromide salt, and hydroiodide salt), inorganic acid salts (e.g., sulfate salt, nitrate salt, perchlorate salt, phosphate salt, carbonate salt, and bicarbonate salt), organic carboxylic acid salts (e.g., acetate salt, trifluoroacetate salt, maleate salt, tartrate salt, fumarate salt, and citrate salt), organic sulfonic acid salts (e.g., methanesulfonate salt, trifluoromethanesulfonate salt, ethanesulfonate salt, benzenesulfonate salt, toluenesulfonate salt, and camphorsulfonate salt), amino acid salts (e.g., aspartate salt, and glutamate salt), quaternary amine salts, alkali metal salts (e.g., sodium salt, and potassium salt), alkaline earth metal salts (e.g., magnesium salt, and calcium salt), etc.

[0031] Dasatinib or other form thereof for use in the present invention includes all isomers possible in terms of the compound's structure (e.g., geometrical isomers, optical isomers based on asymmetric carbons, rotational isomers, stereoisomers, and tautomers) and mixtures of two or more of these isomers, and is not limited to the descriptions about the structural formula shown for convenience' sake. Moreover, dasatinib or other form thereof may be in S-configuration, R-configuration or RS-configuration, and is not limited in any way. Further, dasatinib or other form thereof may be present in the form of a hydrate or solvate, depending on its type. In the present invention, such a hydrate or solvate also falls within dasatinib or other form thereof, and may be used as an active ingredient in the therapeutic agent or pharmaceutical composition of the present invention. Examples of such a solvate include, but are not limited to, a solvate with ethanol, etc.

[0032] In the therapeutic agent or pharmaceutical composition of the present invention, the content of dasatinib or other form thereof as an active ingredient is not limited in any way and may be set as appropriate, for example, to be within the range of 0.01% to 99% by weight relative to the total amount of the therapeutic agent or pharmaceutical composition, and preferably to be within the range of 0.01% to 30% by weight, more preferably 0.05% to 20% by weight, and even more preferably 0.1% to 10% by weight. When the active ingredient content is within the above range, the therapeutic agent or pharmaceutical composition of the present invention will be able to suppress the phosphorylation-induced activation of a proto-oncogene protein belonging to the VAV family (e.g., VAV1) and thus exert a sufficient therapeutic effect on tumors identified by this phosphorylation.

[0033] The therapeutic agent or pharmaceutical composition of the present invention may further comprise other ingredients in addition to dasatinib or other form thereof, as long as the effect of the present invention is not significantly impaired. For example, in the present invention, dasatinib or other form thereof may be used in combination with one or more pharmaceutical agents which are known as therapeutic agents for T-cell lymphoma or under development for this purpose, as exemplified by prednisolone, dexamethasone, cyclophosphamide, doxorubicin, vincristine, azacytidine, poteligeo, romidepsin, adcetris, pralatrexate and so on, without being limited thereto. Further, the therapeutic agent or pharmaceutical composition of the present invention may comprise, for example, ingredients commonly used in pharmaceutical production as described later, etc.

[0034] The therapeutic agent or pharmaceutical composition of the present invention may be administered to a human or non-human mammalian subject (e.g., rat, rabbit, sheep, pig, cattle, cat, dog, monkey) by various routes of administration, as specifically exemplified by oral administration or parenteral administration (e.g., intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, intrarectal administration, percutaneous administration). Thus, dasatinib or other form thereof for use in the present invention may be not only used alone, but also formulated with a pharmaceutically acceptable carrier into an appropriate dosage form in a manner commonly used, depending on the intended route of administration.

[0035] Dosage forms for oral formulations may be exemplified by tablets, powders, fine granules, granules, coated tablets, capsules, solutions for internal use, suspensions, emulsions, syrups and troches, etc., while dosage forms for parenteral formulations may be exemplified by injections (including drops), inhalants, ointments, nose drops, and liposomes, etc. It should be noted that when formulated into various oral formulations mentioned above, the therapeutic agent or pharmaceutical composition of the present invention may also be used as supplements (e.g., corresponding to functional food products) in some cases.

[0036] Examples of carriers which may be used to formulate these formulations include commonly used excipients, binders, disintegrants, lubricants, colorants, and correctives, as well as optionally stabilizers, emulsifiers, absorbefacients, surfactants, pH adjusters, antiseptics, antioxidants, extenders, humectants, surface active agents, dispersants, buffering agents, preservatives, solvent aids, and soothing agents, etc., which may be blended with known ingredients available for use as source materials for pharmaceutical formulations and then formulated in a standard manner.

[0037] Non-toxic ingredients available for this purpose may be exemplified by animal and vegetable oils including soybean oil, beef tallow, and synthetic glycerides; hydrocarbons including liquid paraffin, squalane, and hard paraffin; ester oils including octyldodecyl myristate, and isopropyl myristate; higher alcohols including cetostearyl alcohol, and behenyl alcohol; silicone resin; silicone oil; surfactants including polyoxyethylene fatty acid esters, sorbitan fatty acid esters, glycerin fatty acid esters, polyoxyethylenesorbitan fatty acid esters, polyoxyethylene hydrogenated castor oil, and polyoxyethylene-polyoxypropylene block copolymers; water-soluble polymers including hydroxyethylcellulose, polyacrylic acid, carboxyvinyl polymers, polyethylene glycol, polyvinylpyrrolidone, and methylcellulose; lower alcohols including ethanol, and isopropanol; polyhydric alcohols (polyols) including glycerin, propylene glycol, dipropylene glycol, sorbitol, and polyethylene glycol; sugars including glucose, and sucrose; inorganic powders including silicic anhydride, magnesium aluminum silicate, and aluminum silicate; inorganic salts including sodium chloride, and sodium phosphate; purified water, etc., and these ingredients may be in salt or hydrate form.

[0038] Preferred examples of excipients include lactose, fructose, corn starch, sucrose, glucose, mannitol, sorbit, crystalline cellulose, and silicon dioxide. Preferred examples of binders include polyvinyl alcohol, polyvinyl ether, methylcellulose, ethylcellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropylmethylcellulose, hydroxypropylcellulose, polyvinylpyrrolidone, polypropylene glycol-polyoxyethylene block polymers, and meglumine. Preferred examples of disintegrants include starch, agar, gelatin powder, crystalline cellulose, calcium carbonate, sodium bicarbonate, calcium citrate, dextrin, pectin, and carboxymethylcellulose calcium. Preferred examples of lubricants include magnesium stearate, talc, polyethylene glycol, silica, and hydrogenated vegetable oils. Preferred examples of colorants include those approved for addition to pharmaceutical products. Preferred examples of correctives include cocoa powder, menthol, aromatic powder, peppermint oil, borneol, and cinnamon powder. These ingredients may be in salt or hydrate form.

[0039] The dose of the therapeutic agent or pharmaceutical composition of the present invention may generally be determined extensively as appropriate for the age and body weight of a subject (patient) to be administered, the type and progression of disease, the route of administration, the frequency of administration (per day), the period of administration, etc., in consideration of the ratio of the active ingredient (dasatinib or other form thereof) incorporated into the formulation.

[0040] A detailed explanation will be given below for the case where the therapeutic agent or pharmaceutical composition of the present invention is used as a parenteral formulation or as an oral formulation.

[0041] For use as a parenteral formulation, the therapeutic agent or pharmaceutical composition of the present invention may usually be formulated into any dosage form. In the case of various types of injections, for example, they may be provided in the form of unit dose ampules or multi-dose containers or as freeze-dried powders which are dissolved again in a diluent before use. Such a parenteral formulation may comprise not only dasatinib or other form thereof serving as an active ingredient, but also various known excipients and/or additives as appropriate for each dosage form as long as the effect of the above active ingredient is not impaired. In the case of various types of injections, examples of excipients and/or additives include water, glycerol, propylene glycol, and aliphatic polyalcohols such as polyethylene glycol, etc.

[0042] The dose (daily dose) of such a parenteral formulation is not limited in any way. For example in the case of various types of injections, the dose may generally be set such that dasatinib or other form thereof serving as an active ingredient can be taken in an amount of 0.01 to 1000 mg, 0.05 to 500 mg or 0.1 to 50 mg, per kg body weight of a subject to be applied (e.g., a subject, a patient), or alternatively, can be taken in an amount of 0.5 to 20 mg or can be taken in an amount of 1 to 10 mg.

[0043] For use as an oral formulation, the therapeutic agent or pharmaceutical composition of the present invention may usually be formulated into any dosage form among those mentioned above, or alternatively, may be formulated into a freeze-dried product which is dissolved again before use. Such an oral formulation may comprise not only dasatinib or other form thereof serving as an active ingredient, but also various known excipients and/or additives as appropriate for each dosage form as long as the effect of the above active ingredient is not impaired. Examples of excipients and/or additives include binders (e.g., syrup, gum arabic, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone), fillers (e.g., lactose, sugar, corn starch, potato starch, calcium phosphate, sorbitol, glycine), lubricants (e.g., magnesium stearate, talc, polyethylene glycol, silica), disintegrants (e.g., various types of starches), and wetting agents (e.g., sodium lauryl sulfate), etc.

[0044] The dose (daily dose) of such an oral formulation may generally be set such that dasatinib or other form thereof serving as an active ingredient can be taken in an amount of 0.05 to 5000 mg, 0.1 to 1000 mg or 0.1 to 100 mg, per kg body weight of a subject to be applied (e.g., a subject, a patient), or alternatively, can be taken in an amount of 0.5 to 50 mg or can be taken in an amount of 1 to 10 mg. Moreover, the ratio of the active ingredient incorporated into the oral formulation is not limited in any way and may be set as appropriate in consideration of the frequency of administration per day, etc.

3. Kit



[0045] In the treatment of tumors identified by phosphorylation of a proto-oncogene protein belonging to the VAV family, a kit comprising dasatinib or other form thereof can be used for this purpose (as specifically exemplified by a kit comprising the therapeutic agent or pharmaceutical composition of the present invention mentioned above).

[0046] In such a kit, dasatinib or other form thereof may be contained in any state, but may be provided, for example, in a dissolved state in consideration of its stability (storage quality) and easiness of use, etc.

[0047] Such a kit may comprise not only dasatinib or other form thereof, but also other constituent elements, as appropriate.

[0048] Such a kit is required to comprise at least the above dasatinib or other form thereof as a constituent element. Thus, the kit may be configured to comprise all constituent elements essential for the treatment of the above tumors, either together with or separately from dasatinib or other form thereof, without being limited thereto.

4. Method for testing the efficacy of pharmaceutical agent in patient



[0049] In the present invention, there is provided a method for testing the efficacy of dasatinib or a prodrug thereof or a pharmacologically acceptable salt thereof or a hydrate or solvate thereof (dasatinib or other form thereof) in a patient who is a subject to be administered therewith (hereinafter also referred to as "the test method of the present invention"). In more detail, this method is a test method for determining (or deciding) a patient for whom the administration of dasatinib or other form thereof is effective.

[0050] It should be noted that the present invention also provides a method for determining or deciding whether the administration of dasatinib or other form thereof is effective (i.e., the efficacy of this administration) in a patient who is a subject to be administered.

[0051] Each method mentioned above does not require any judgment or diagnosis made by a physician and is intended to test or determine (or decide), in advance of administration, whether dasatinib or other form thereof has an effect (i.e., a therapeutic effect on tumors) when administered to each patient, in accordance with the procedures (i) to (iii) described later.

[0052] In each method mentioned above, the efficacy is not limited in any way, but is exemplified by the efficacy in tumor treatment.

[0053] Preferred examples of the test method of the present invention include: (i) a method characterized in that the efficacy is indicated by the level of phosphorylation of a proto-oncogene protein belonging to the VAV family (e.g., VAV1, VAV2, VAV3) in an analyte taken from the above patient; (ii) a method characterized in that the efficacy is indicated by the presence or absence of a gene mutation or gene fusion in the RHOA gene in the analyte; and (iii) a method characterized in that the efficacy is indicated by the presence or absence of a gene mutation or gene fusion in a proto-oncogene belonging to the VAV family (e.g., VAV1 gene, VAV2 gene, VAV3 gene) in the analyte.

[0054] In the above methods (i) to (iii), any analyte may be taken from a patient, but preferred examples include tumor tissues (e.g., lymph node lesions and extralymphatic lesions), serum, plasma, blood cells, pleural effusion, ascites, spinal fluid and so on.

[0055] In the case of the above method (i), the indicator of the test, i.e., the level of phosphorylation (tyrosine phosphorylation) of a proto-oncogene protein belonging to the VAV family may be measured or determined, for example, by immunohistological staining, Western blotting and other techniques. If the level of phosphorylation is positive in a certain percentage or higher of cells (e.g., 10% or higher, preferably 20% or higher, more preferably 40% or higher, based on cell counts), the patient to be administered from which the analyte was taken can be determined to be a patient for whom the administration of dasatinib or other form thereof is effective. In more detail, this determination can also be made if phosphorylated VAV1/VAV2/VAV3 can be detected by Western blotting, if cells where VAV1/VAV2/VAV3 is more strongly stained than in normal lymph nodes can be found by immunostaining, or if positively stained cells can be found at a higher percentage than in normal lymph nodes.

[0056] In the case of the above method (ii), the indicator of the test, i.e., the presence or absence of a gene mutation or gene fusion in the RHOA gene may be detected, for example, by direct sequencing, allele-specific PCR, sequencing with a next-generation sequencer, PNA-LNA PCR clamp, digital PCR, WAVE and other techniques for gene mutation detection, or by reverse transcription-polymerase chain reaction (RT-PCR), genomic PCR, chromosomal analysis, fluorescence in situ hybridization (FISH), total RNA sequencing and other techniques for gene fusion detection.

[0057] Examples of a gene mutation in the RHOA gene include, but are not limited to, those shown in Table 5 below, etc.
[Table 5]
RHOA gene mutationAmino acid mutation in RHOA protein
c.50G>T p.G17V
c.125A>G p.Y42C
c.46T>C p.C16R
c.14G>A p.R5Q
c.14G>A p.G17E


[0058] In the case of the above method (iii), the presence or absence of a gene mutation or gene fusion in a proto-oncogene belonging to the VAV family (e.g., VAV1 gene, VAV2 gene, VAV3 gene) may be detected, for example, by direct sequencing, sequencing with a next-generation sequencer, digital PCR, WAVE and other techniques for gene mutation detection, or by reverse transcription-polymerase chain reaction (RT-PCR), genomic PCR, chromosomal analysis, fluorescence in situ hybridization (FISH), total RNA sequencing and other techniques for gene fusion detection.

[0059] Examples of a gene mutation in a proto-oncogene belonging to the VAV family include, but are not limited to, those in the VAV1 gene, as exemplified by VAV1 gene mutations resulting in amino acid mutations in the VAV1 protein as shown in Table 6 below.
[Table 6]
VAV1 gene mutationAmino acid mutation in VAV1 protein
c.C518_529del p.173_177del
c.C494_520del p.165_174del
c.1668G>C p.E556D
c.1844C>T p.P615L
c.1211A>G p.K404R
c.2465G>A p.R822Q
c.388C>T p.P130S
c.2393G>A p.R798Q
c.1696C>T p.R566*


[0060] Likewise, in the case of the VAV2 gene, examples include VAV2 gene mutations resulting in amino acid mutations in the VAV2 protein as shown in Table 7 below.
[Table 7]
VAV2 gene mutationAmino acid mutation in VAV2 protein
c.763C>T p.L255L
c.748C>T p.L250L
c.686C>T p.P229L
c.701C>T p.P234L
c.308G>A p.R103Q


[0061] Likewise, in the case of the VAV3 gene, examples include VAV3 gene mutations resulting in amino acid mutations in the VAV3 protein as shown in Table 8 below.
[Table 8]
VAV3 gene mutationAmino acid mutation in VAV3 protein
c.1309G>A p.D437N
c.1037C>T p.T346I
c.578G>A p.R193Q
c.2382C>T p.I794I


[0062] Moreover, examples of a gene fusion in a proto-oncogene belonging to the VAV family include, but are not limited to, VAV1-STAP2, VAV1-GSS and so on in the case of the VAV1 gene.

[0063] The test method of the present invention enables, in advance of treatment, the selection and determination of a patient for whom the use of dasatinib or other form thereof is highly likely to provide an effective outcome in tumor treatment. This in turn enables further reduction of therapeutic burdens (i.e., economic, time and physical burdens) in patients.

EXAMPLES



[0064] The present invention will be further described in more detail by way of the following examples, although the present invention is not limited only to these examples.

1. Data indicating VAV 1 activation induced by G17V RHOA mutant (Examples 1 to 4) [Example 1]



[0065] 
  1. (1) Jurkat cells (T-cell leukemia cell line) engineered with a lentivirus-mediated Tet-On system to express wild-type RHOA cDNA or a RHOA coding region modified to comprise a G to T substitution at position 50 of the coding region (hereinafter referred to as G17V RHOA cDNA) were seeded in RPMI (10% FCS, 1% PS) at 2 × 105 cells/mL in 15 cm petri dishes in triplicate. Doxycycline was added to give a final concentration of 2 µg/mL. On the following day, the cells were collected and centrifuged, and then seeded in RPMI (serum-free) at 6 × 105 cells/mL in 15 cm petri dishes in triplicate. After 4 hours, the cells were collected and washed once with sterile phosphate buffered saline (PBS), and then adjusted to 2 × 107 cells/ml and transferred to 15 ml tubes, followed by incubation at 37°C for 5 minutes. LEAF™ purified anti-human CD3 Ab (BioLegend) and anti-mouse IgG antibody Ab (at 2 µg/ml each) were added. The cells were incubated at 37°C for 5 minutes or 30 minutes. Cold PBS (10 ml) was added to each tube, followed by centrifugation to remove the supernatants. Lysis buffer (supplemented in advance with cOmplete protease inhibitor and PhosSTOP) was added in a volume of 1000 µl/tube. The tubes were incubated on ice for 20 minutes and centrifuged to collect the supernatants. Laemuli's buffer and DTT were added, followed by incubation at 95°C for 5 minutes.
  2. (2) Immunoprecipitation
    The remaining supernatants were each taken in a volume of 500 µL × two tubes, to which anti-flagM2 antibody and anti-HA antibody as a control (5 µg each) were added respectively, followed by rotation at 4°C for 2 hours. Protein G was tapped and 50 µL of which was then transferred to another tube. Protein G was washed twice with 1% triton TBS.
    The tubes were centrifuged at 10000 rpm at 4°C for 10 seconds to remove the supernatants (repeated twice). The washed 50% protein G was added in 50 µL volumes to the supernatants, followed by rotation at 4°C for 1 hour. The tubes were centrifuged at 1700 rpm at 4°C for 2 minutes to remove the supernatants. To the pellets, 1% Triton TBS (500 µL) was added, followed by rotation at 4°C for 5 minutes and centrifugation at 1700 rpm at 4°C for 2 minutes to remove the supernatants. This operation was repeated three times in total. 1 × Flag peptide (diluted to 500 µg/ml with TBS) was added in 50 µL volumes, followed by rotation at room temperature for 10 minutes and centrifugation at 1700 rpm at 4°C for 2 minutes. The supernatants were collected, and Laemulis buffer and DTT were added thereto, followed by incubation at 95°C for 5 minutes.
  3. (3) After each of the procedures shown in (1) and (2) above, the samples were electrophoresed on an acrylamide gel and then blotted on Immobilon P (Promega), and then stained with anti-VAVl antibody (Abcam or Cell signaling), anti-VAV1 Y174 antibody, anti-PLC-gamma 1 antibody (Cell signaling) or anti-phospho PLC-gamma 1 antibody (Cell signaling) as a primary antibody and HRP-labeled anti-rabbit IgG antibody (Dako) as a secondary antibody, or with anti-flag M2 antibody (Sigma) as a primary antibody and HRP-labeled anti-mouse IgG antibody (Dako) as a secondary antibody, followed by color development using Immobilon Western chemiluminescence HRP substrate (Millipore) and photography. The results obtained are shown in Figures 1 and 2.

[Example 2]



[0066] SU9T01 cells (adult T-cell leukemia/lymphoma cell line) engineered with a lentivirus-mediated Tet-On system to express wild-type RHOA or G17V RHOA cDNA were seeded in RPMI (10% FCS, 1% PS) at 2 × 105 cells/mL in 15 cm petri dishes in triplicate. Doxycycline was added to give a final concentration of 2 µg/mL. On the following day, the cells were collected and then washed once with PBS. To the pellets, Lysis buffer was added in a volume of 1000 µl/tube. The same experiments as conducted on the Jurkat cells in Example 1 were then repeated. The results obtained are shown in Figures 3 and 4.

[Example 3]



[0067] In a 96-well flat-bottomed plate, LEAF™ purified anti-human CD3 Ab (BioLegend) adjusted to 10 µg/ml with sterile phosphate buffered saline (PBS) was dispensed in a volume of 50 µL/well and incubated overnight at 4°C. After removal of the antibody solution, each well was washed three times with sterile PBS.

[0068] Jurkat cells were seeded at 5 × 104 cells/well in a 24-well plate. On the following day, using X-tremeGENE HP DNA transfection reagent, the cells were transiently transfected with pGL4.30 vector designed to carry an NFAT response element and cDNA encoding firefly luciferase (Promega), phRL vector designed to carry cDNA encoding renilla luciferase (Promega) and pEF vector designed to carry wild-type or G17V RHOA cDNA. At 24 hours after transfection, the cultured cell suspensions were each dispensed in a volume of 150 µl/well.

[0069] After 7 hours, the cultured cell suspensions were collected into tubes, and PBS was further dispensed into the culture plate in a volume of 200 µl/well, followed by pipetting and collection into the tubes. This operation was repeated to completely collect the cells. The cells were washed once with PBS.

[0070] In accordance with the protocol of Dual Luciferase Reporter Assay (Promega), firefly luciferase activity and renilla luciferase activity were each measured, and the value of firefly luciferase was divided by the value of renilla luciferase to correct the transfection efficiency. In more detail, the cells were mixed with Passive Lysis Buffer (PLB) in a volume of 100 µl/tube, followed by freezing at -80°C to lyse the cells. After centrifugation, the supernatants were collected. In a 96-well plate, LARII was dispensed in a volume of 100 µl/well. 20 µl of each cell lysate was added. The plate was mounted on a luminometer and Stop & Glo® Reagent was added in 100 µL volumes, followed by measurement again with the luminometer. The results obtained are shown in Figure 5.

[Example 4]



[0071] Dynabeads T-activator CD3/CD28 (Veritas) were mixed with buffer (PBS supplemented with 0.1% BSA + 2 mM EDTA, pH 7.4) and allowed to stand on a magnetic rack to remove the supernatant.

[0072] Jurkat cells engineered with a lentivirus-mediated Tet-On system to express wild-type or G17V RHOA cDNA were seeded in RPMI (10% FCS, 1% PS) at 2 × 105 cells/mL in 15 cm petri dishes in triplicate. Doxycycline was added to give a final concentration of 2 µg/mL. On the following day, the cells were collected and seeded in a 24-well plate. The Dynabeads T-activator CD3/CD28 (Veritas) washed above were added. After 3 and 6 hours, the cells were collected. The cells were washed once with PBS and their RNA was extracted with an RNeasy Mini Kit (Qiagen). SuperscriptIII (Thermo Fisher Scientific) was used to synthesize cDNA. IL-2 mRNA was measured with the primer/probe sets of Taqman Gene Expression Assays (Thermo Fisher Scientific). rRNA was measured with TaqMan® Ribosomal RNA Control Reagents (Thermo Fisher Scientific) and used for correction. The results obtained are shown in Figure 6.

2. Data indicating that dasatinib cancels VAV1 activation induced by G17V RHOA mutant and downstream signaling (Examples 5 and 6)


[Example 5]



[0073] Jurkat cells engineered with a lentivirus-mediated Tet-On system to express wild-type RHOA or G17V RHOA cDNA were seeded in RPMI (10% FCS, 1% PS) at 2 × 105 cells/mL in 15 cm petri dishes in triplicate. Doxycycline was added to give a final concentration of 2 µg/mL. On the following day, the cells were collected and centrifuged, and then seeded in RPMI (serum-free) at 6 × 105 cells/mL in 15 cm petri dishes in triplicate. After 3.5 hours, dasatinib was added at a final concentration of 0 to 10 nM. After 30 minutes, the cells were collected. The same experiments as conducted in Example 1 were then repeated. The results obtained are shown in Figure 7.

[Example 6]



[0074] Jurkat cells engineered with a lentivirus-mediated Tet-On system to express wild-type RHOA or G17V RHOA cDNA were seeded in RPMI (10% FCS, 1% PS) at 2 × 105 cells/mL in 15 cm petri dishes in triplicate. Doxycycline was added to give a final concentration of 2 µg/mL. On the following day, the cells were collected and seeded in a 24-well plate. Dasatinib was added at a final concentration of 0 to 10 nM. Dynabeads T-activator CD3/CD28 (Veritas) washed in the same manner as shown in Example 4 were added. After 3 hours, the cells were collected. The cells were washed once with PBS and their RNA was extracted with an RNeasy Mini Kit (Qiagen). SuperscriptIII (Thermo Fisher Scientific) was used to synthesize cDNA. IL-2 mRNA was measured with the primer/probe sets of Taqman Gene Expression Assays (Thermo Fisher Scientific). rRNA was measured with TaqMan® Ribosomal RNA Control Reagents (Thermo Fisher Scientific) and used for correction. The results obtained are shown in Figure 8.

3. Discovery of VAV1 gene mutations by genomic analysis (Examples 7 to 11) [Example 7]



[0075] A library was prepared using an AmliSeq system (Thermo Fisher Scientific) for regions comprising exons of the VAV1 gene or prepared using an Ion Plus Fragment Library kit (Thermo Fisher Scientific) for PCR amplicons amplified by genomic PCR using KOD Plus neo (TOYOBO). After sequencing was conducted with an Ion Torrent PGM sequencer (Thermo Fisher Scientific) in accordance with a standard protocol for 300 base pairs, mutation candidates were analyzed by Variant caller software and their results were confirmed by direct sequencing. The results obtained are shown in Figure 9.

[Example 8]



[0076] Jurkat cells engineered with a lentivirus-mediated Tet-On system to express wild-type VAV1 or VAV-STAP2 cDNA were seeded in RPMI (10% FCS, 1% PS) at 2 × 105 cells/mL in 15 cm petri dishes in triplicate. Doxycycline was added to give a final concentration of 2 µg/mL. The same experiments as conducted on the Jurkat cells engineered to express wild-type or G17V RHOA cDNA were then repeated by reference to Example 1, etc. The results obtained are shown in Figures 10 and 11.

[Example 9]



[0077] Using X-tremeGENE HP DNA transfection reagent, Jurkat cells were transfected with pGL4.30 vector designed to carry an NFAT response element and cDNA encoding firefly luciferase (Promega), phRL vector designed to carry cDNA encoding renilla luciferase (Promega) and pEF vector designed to carry wild-type or mutant VAV cDNA. The cells were then stimulated with LEAF™ purified anti-human CD3 Ab (BioLegend) and measured for firefly luciferase activity and renilla luciferase activity, as in the case of wild-type or G17V RHOA cDNA, by reference to Example 3, etc. The results obtained are shown in Figure 12.

[Example 10]



[0078] In a 24-well flat-bottomed plate, LEAF™ purified anti-human CD3 Ab (BioLegend) adjusted to 10 µg/ml with PBS was dispensed in a volume of 200 µL/well and incubated overnight at 4°C. After removal of the antibody solution, each well was washed three times with sterile PBS.

[0079] Jurkat cells engineered with a lentivirus-mediated Tet-On system to express wild-type VAV1 or various mutant VAV cDNAs were seeded. After 3 and 6 hours, the cells were collected, and the same experiments as in the case of wild-type or G17V RHOA were repeated by reference to Example 4, etc. The results obtained are shown in Figure 13.

[Example 11]



[0080] Jurkat cells engineered with a lentivirus-mediated Tet-On system to express wild-type VAV1 or VAV-STAP2 cDNA were seeded in a 24-well plate at 5 × 104 cells/well in a volume of 500 µl/well. Doxycycline (2 µg/ml) was added and the cells were collected after 24 hours, and the cell suspensions were each dispensed at 5 × 104 cells/50 µl/well in a 96-well plate which had been coated with LEAF™ purified anti-human CD3 Ab (BioLegend) in the same manner as shown in Example 3 above. At 0, 8 and 24 hours after stimulation, the culture supernatants were collected. IL-2 was measured using a BD cytometric beads array. The results obtained are shown in Figure 14.

4. Dasatinib-induced inhibition of VAV1 mutation activation (Examples 12 and 13) [Example 12]



[0081] Jurkat cells engineered with a lentivirus-mediated Tet-On system to express wild-type VAV1 or VAV1-STPA2G cDNA were seeded in RPMI (10% FCS, 1% PS) at 2 × 105 cells/mL in 15 cm petri dishes in triplicate. Doxycycline was added to give a final concentration of 2 µg/mL. On the following day, the cells were collected and centrifuged, and then seeded in RPMI (serum-free) at 6 × 105 cells/mL in 15 cm petri dishes in triplicate. After 3.5 hours, dasatinib was added at a final concentration of 0 to 10 nM. After 30 minutes, the cells were collected. The same procedures as shown above were then repeated to conduct a stimulation experiment with anti-CD3 antibody. The results obtained are shown in Figure 15.

[Example 13]



[0082] In a 96-well flat-bottomed plate, LEAF™ purified anti-human CD3 Ab (BioLegend) adjusted to 10 µg/ml with PBS was dispensed in a volume of 50 µL/well and incubated overnight at 4°C. After removal of the antibody solution, each well was washed three times with sterile PBS.

[0083] Jurkat cells were seeded at 5 × 104 cells/well in a 24-well plate. On the following day, using X-tremeGENE HP DNA transfection reagent, the cells were transiently transfected with pGL4.30 vector designed to carry an NFAT response element and cDNA encoding firefly luciferase (Promega), phRL vector designed to carry cDNA encoding renilla luciferase (Promega) and pEF vector designed to carry wild-type or G17V RHOA cDNA. At 24 hours after transfection, dasatinib was added at a final concentration of 0 to 10 nM. After 30 minutes, the cells were collected and then stimulated with LEAF™ purified anti-human CD3 Ab (BioLegend) and measured for firefly luciferase activity and renilla luciferase activity by reference to Example 3 described above, etc. The results obtained are shown in Figure 16.

5. Tumor suppressive effect upon administration of dasatinib to therapeutic model mice (Example 14)


[Example 14]


(1) Preparation of model mice



[0084] 80% or more of angioimmunoblastic T-cell lymphoma (AITL) cases show loss-of-function mutations in the TET2 gene, and a G17V RHOA gene mutation is also found in about 70% of all cases. To prepare an animal model of this disease, the following genetically modified mice were prepared.

[0085] Mice were genetically modified as follows at the fertilized egg stage.
  1. i) Fertilized eggs are engineered to have a lox sequence inserted into the Tet2 gene, whereby mice are designed to disrupt the Tet2 gene by the action of Cre recombinase.
  2. ii) Fertilized eggs are engineered to have a DNA sequence (Cre expression cassette) composed of Cre recombinase ligated downstream of the Mx promoter. In cells responsive to interferon, the Mx promoter is activated upon interferon stimulation to thereby cause the expression of Cre recombinase. For this reason, upon administration of poly-inosilic:poly-cytidilic acid (pIpC) capable of inducing interferon production, Cre recombinase is expressed in these cells responsive to interferon (which also include hematopoietic stem cells). Namely, i) and ii) mean that mice are designed such that the Tet2 gene is disrupted and inactivated in hematopoietic stem cells upon pIpC administration.
  3. iii) Independently of i) and ii) above, fertilized eggs are engineered to have a DNA sequence (expression cassette) composed of G17V mutant human RHOA cDNA ligated downstream of the CD2 promoter. Since the CD2 promoter is specifically activated in T cells, a G17V mutant RHOA protein is expressed only in T cells.


[0086] When the thus prepared mice were intraperitoneally administered four times with 20 mg/kg pIpC every two days starting from 4 weeks of postnatal age, multiple lymph node swelling and splenomegaly were observed in many mice at about 40 weeks of age to 50 weeks of age, and these mice died at 45 weeks of age or later. For detailed analysis, mice showing splenomegaly or hyposthenia at 35 weeks of age or later were sacrificed for further analysis. Histological observation of swollen lymph nodes and spleens showed not only 10% to 30% infiltration of T cells regarded as tumor cells, but also infiltration of lymphocytes and other various inflammatory cells, thus resulting in a pathological image where tumor and inflammation were difficult to distinguish from each other. T cells growing in a tumor-like fashion were found to have characteristics of follicular helper T cells, as seen from their expression pattern of cell surface antigens. When DNA was prepared from swollen lymph nodes to analyze T cell receptor reconstitution, it was indicated that T cell receptor reconstitution was mono- or oligo-clonal, thus yielding the results that were not inconsistent with the tumor-like growth of T cells. Moreover, some of the mice also showed tumor formation in their liver, lungs and other organs, so that tumor-like infiltration of lymphocyte-like cells was observed histologically.

[0087] In AITL patients, tumor cells are known to have characteristics of follicular helper T cells. Moreover, the ratio of tumor cells in their tumor tissue is low, and the great majority of cells are various kinds of inflammatory cells in most cases. AITL patients cannot be pathologically diagnosed as having tumor and are therefore often diagnosed as having inflammation. Thus, the disease occurring in the mice in this example has the same characteristics as seen in AITL patients. (This mouse model is not regarded as a perfect model of AITL, but there has been no report showing that tumor having such characteristics was modeled in mice; and hence there is no doubt that this model is most similar to AITL patients.)

(2) Transplant model



[0088] An AITL model is regarded as having been established, but a long incubation period is required to observe oncogenesis after pIpC administration. For this reason, this genetically modified mouse model is not suitable for use in therapeutic experiments. Thus, an attempt was made to establish a transplant model. When all cells prepared from swollen lymph nodes were allowed to float and then intraperitoneally transplanted into many nude mice, lymph node swelling was observed in almost all of the host mice within about one month. Their histological study showed that their swollen lymph nodes were substantially the same as those in the mouse used as a donor, while their cytological study also showed an increase in T cells having characteristics of follicular helper T cells. It should be noted that inflammatory cells migrating to the tumor tissue were composed of donor mouse-derived cells and host mouse-derived cells in admixture.

[0089] On the other hand, even when only cells having characteristics of follicular helper T cells (i.e., CD4+ ICOS+ cells) were isolated from swollen lymph node cells of the donor mouse and provided for transplantation into nude mice, lymphoma did not occur (*).

(3) Therapeutic model



[0090] Donor mouse swollen lymph node-derived cells (2 × 107 cells) were all intraperitoneally injected into nude mice and, after 2 weeks (after lymph node swelling was observed by echography in some mice), 5 mg/kg of dasatinib dissolved in a solution of propylene glycol: distill water adjusted to 1:1 was orally administered to the mice for successive 14 days (the dasatinib group: "Dasatinib" in Figure 17). As a control, nude mice under completely the same conditions were administered with the same volume of the solution alone (propylene glycol : distill water = 1:1) according to the same schedule (the control group: "Vehicle" in Figure 17). The results of these administration experiments are show in Figure 17. In the control group, 77.8% (14/18) of the mice showed progression of lymphoma (body weight loss and hyposthenia) at 20 days, whereas in the dasatinib group, only 33.3% (6/18) of the mice showed progression of lymphoma during the observation period of 150 days, so that progression was significantly suppressed and survival was prolonged in the dasatinib group.

[0091] In addition, donor mouse swollen lymph node-derived cells (2 × 107 cells) were all intraperitoneal injected into nude mice and, after 2 weeks, dasatinib or a control solution was administered three times daily for 2 days in accordance with the above procedure. On the following day, the nude mice were sacrificed, and thin section specimens were prepared from their swollen lymph nodes and then stained with anti-phosphorylated VAV1 antibody. As a result, phosphorylated VAV1 was stained in swollen lymph nodes from the mice of the group receiving the control solution, whereas phosphorylated VAV1 was not stained in swollen lymph nodes from the mice of the group receiving dasatinib (this result supports the hypothesis that the tumor suppressive effect of dasatinib is mediated by VAV1 phosphorylation suppression).

(4) Contribution of microenvironmental cells to AITL development



[0092] As seen from the description indicated with * in (2) above, even when only cells having characteristics of follicular helper T cells (i.e., CD4+ ICOS+ cells) were isolated from swollen lymph node cells of the donor mouse and provided for transplantation into nude mice, lymphoma did not occur. However, this suggests that donor-derived microenvironmental cells contribute to AITL development.

[0093] Simultaneously with the preparation of model mice in (1) above, mice differing from these model mice only in ii) below were also prepared.
  1. i) Fertilized eggs are engineered to have a lox sequence inserted into the Tet2 gene, whereby mice are designed to disrupt the Tet2 gene by the action of Cre recombinase (completely the same as in the case of the model mice in (1) above).
  2. ii) Fertilized eggs are engineered to have a DNA sequence (Cre expression cassette) composed of Cre recombinase ligated downstream of the CD4 promoter. The CD4 promoter used here is known to be activated primarily in T cells and to induce the downstream gene expression. Namely, i) and ii) mean that mice are designed such that the Tet2 gene is disrupted and inactivated in T cells.
  3. iii) Independently of i) and ii) above, fertilized eggs are engineered to have a DNA sequence (expression cassette) composed of G17V mutant human RHOA cDNA ligated downstream of the CD2 promoter. Since the CD2 promoter is specifically activated in T cells, a G17V mutant RHOA protein is expressed only in T cells (completely the same as in the case of the model mice in (1) above).


[0094] In this mouse model, lymph node swelling was not observed at all until 80 weeks of age. Taken together with the description indicated with * in (2) above, this result was deemed to strongly suggest that Tet2 inactivation was required to occur in inflammatory cells other than T cells, and these Tet2-inactivated inflammatory cells would contribute to oncogenesis in tissue.
























































Claims

1. A therapeutic agent for a tumor identified by phosphorylation of a proto-oncogene protein belonging to the VAV family,
wherein the therapeutic agent comprises dasatinib or a prodrug thereof or a pharmacologically acceptable salt thereof or a hydrate or solvate thereof as an active ingredient.
 
2. The therapeutic agent according to claim 1, wherein the tumor is a tumor identified to show enhanced phosphorylation of a proto-oncogene protein belonging to the VAV family.
 
3. The therapeutic agent according to claim 1 or 2, wherein the protein is the VAV1 protein.
 
4. A therapeutic agent for a tumor carrying a gene mutation or gene fusion in the RHOA gene and/or in a proto-oncogene belonging to the VAV family,
wherein the therapeutic agent comprises dasatinib or a prodrug thereof or a pharmacologically acceptable salt thereof or a hydrate or solvate thereof as an active ingredient.
 
5. The therapeutic agent according to any one of claims 1 to 4, wherein the tumor is at least one selected from the group consisting of T-cell lymphoma, gastric cancer, pancreatic cancer, skin tumor, colorectal cancer, uterine cancer and nervous system tumor.
 
6. The therapeutic agent according to any one of claims 1 to 5, wherein the tumor is T-cell lymphoma.
 
7. The therapeutic agent according to any one of claims 1 to 6, wherein the T-cell lymphoma is angioimmunoblastic T-cell lymphoma or peripheral T-cell lymphoma, not otherwise specified.
 
8. A pharmaceutical composition for the treatment of a tumor identified by phosphorylation of a proto-oncogene protein belonging to the VAV family, which comprises dasatinib or a prodrug thereof or a pharmacologically acceptable salt thereof or a hydrate or solvate thereof.
 
9. The use of dasatinib or a prodrug thereof or a pharmacologically acceptable salt thereof or a hydrate or solvate thereof for the manufacture of a pharmaceutical agent for the treatment of a tumor identified by phosphorylation of a proto-oncogene protein belonging to the VAV family.
 
10. A therapeutic method for a tumor identified by phosphorylation of a proto-oncogene protein belonging to the VAV family, which comprises administering a subject with dasatinib or a prodrug thereof or a pharmacologically acceptable salt thereof or a hydrate or solvate thereof.
 
11. A therapeutic kit for a tumor identified by phosphorylation of a proto-oncogene protein belonging to the VAV family, which comprises dasatinib or a prodrug thereof or a pharmacologically acceptable salt thereof or a hydrate or solvate thereof.
 
12. A method for testing the efficacy of dasatinib or a prodrug thereof or a pharmacologically acceptable salt thereof or a hydrate or solvate thereof in a patient who is a subject to be administered therewith, wherein the efficacy is indicated by the level of phosphorylation of a proto-oncogene protein belonging to the VAV family in an analyte taken from the patient.
 
13. A method for testing the efficacy of dasatinib or a prodrug thereof or a pharmacologically acceptable salt thereof or a hydrate or solvate thereof in a patient who is a subject to be administered therewith, wherein the efficacy is indicated by the presence or absence of a gene mutation or gene fusion in the RHOA gene in an analyte taken from the patient.
 
14. A method for testing the efficacy of dasatinib or a prodrug thereof or a pharmacologically acceptable salt thereof or a hydrate or solvate thereof in a patient who is a subject to be administered therewith, wherein the efficacy is indicated by the presence or absence of a gene mutation or gene fusion in the VAV1 gene in an analyte taken from the patient.
 
15. A method for testing the efficacy of dasatinib or a prodrug thereof or a pharmacologically acceptable salt thereof or a hydrate or solvate thereof in a patient who is a subject to be administered therewith, wherein the efficacy is indicated by the presence or absence of a gene mutation or gene fusion in the VAV2 gene in an analyte taken from the patient.
 
16. A method for testing the efficacy of dasatinib or a prodrug thereof or a pharmacologically acceptable salt thereof or a hydrate or solvate thereof in a patient who is a subject to be administered therewith, wherein the efficacy is indicated by the presence or absence of a gene mutation or gene fusion in the VAV3 gene in an analyte taken from the patient.
 




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REFERENCES CITED IN THE DESCRIPTION



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Patent documents cited in the description




Non-patent literature cited in the description