Field of the Invention

[0001] The present invention relates to veterinary cancer therapy, and particularly to the mesenchymal stem cells (MSC) expressing cytosine deaminase and uracil phosphoribosyltransferase (CDy::UPRT MSC) and further to the use of CDy::UPRT MSC in combination with prodrug 5-fluorocytosine (5FC) in veterinary cancer therapy for companion animals.

Background of the Invention

[0002] Approximately 1 in 4 dogs and 1 in 6 cats will develop cancer in their lifetimes. Like humans, cancers in animals pose a significant challenge with standard treatment practices. In humans, one novel approach is the use of gene- directed enzyme pro-drug therapy (GDEPT) in cancer treatment. Inherent tumour tropism of mesenchymal stem cells (MSC) serve as a unique platfonn to utilize MSC as cell vehicles to deliver suicide genes specifically to tumors (Li et al. 2015).

[0003] One GDEPT approach employs the combination of two suicide genes, cytosine deaminase (CDy) and uracil phosphoribosyl-transferase (UPRT), and a prodrug 5- fluorocytosine (5FC) to treat cancers. The CDy efficiently converts the prodrug 5- fluorocytosine (5FC) to 5-fluorouracil (5FU), and UPRT, a pyrimidine salvage enzyme, directly converts 5FU to 5-fluorouridine monophosphate (FUMP).

[0004] Bourbeau et al. disclose the construction of adenoviruses expressing the fusion protein CD::UPRT and the test of their anti-tumor activity in combination with 5-FC in glioblastoma cell lines.

[0005] Miyagi et al. disclose the construction of two separate adenovirus vectors expressing the E. coli CDy (AdCA-CD) and E. coli UPRT (AdCA-UPRT) genes and the test of their anti-cancer activity in combination of 5-FC by intratumoral injection of AdCA- Cd and AdCA-UPRT into tumors growing from DU145 cells inoculated into athymic (nude) mice and systemic administration of 5-FC. [0006] Chung-Fayer et al. disclose the construction of a replication-deficient adenovirus containing a CDyxUPRT bifunctional fusion gene (AdCDUPRT) and the test of their anti-cancer activity in combination of 5-FC by intratumoral injection of AdCDUPRT into athymic mice with colon cancer xenografts and intraperitoneal injection of 5-FC.

[0007] Cavarretta et al. discloses the construction of retroviral vector containing

CDxUPRT, the transduction of AT-MSCs (CDy-AT-MSCs), and the test of their anti cancer activity by intravenous injection of CDy-AT-MSCs and intraperitoneal injection of 5-FC.

[0008] Erbs et al. disclose the construction of adenoviruses expressing a bifunctional yeast CDxUPRT fusion gene, and the test of their anti-cancer activity in combination with 5-FC by intratumoral injection of Ad-CD::UPRT and intraperitoneal administration of 5-FC.

[0009] However, as Malekshah et al. summarized, despite the significant progress of different suicide gene therapy protocols in prec!inical studies and early clinical trials, none has reached the clinic due to several shortcomings. These include slow prodiug-drug conversion rate, low transfection/transduction efficiency of the vectors and nonspecific toxicity/immunogenicity related to the delivery systems, plasmid DNA, enzymes and/or prodrugs.

Summary of the Invention

[0010] The present invention provides the use of stem cells expressing cytosine deaminase and uracil phosphoribosyltransferase (CD::UPRT) in combination with a prodrug to treat cancers in companion animals.

[0011] In another embodiment of the use, the cytosine deaminase and uracil phosphoribosyltransferase are from E coli. or yeast.

[0012] In another embodiment of the use, the stem cells are mesenchymal stem cells (MSC).

[0013] In another embodiment of the use, the cytosine deaminase and uracil phosphoribosyltransferase are expressed by inserting encoding sequences into an adenovirus (Ad) vector or an adeno-associated virus (AAV) vector. [0014] In another embodiment of the use, the prodrug is 5-fluorocytosine (5FC).

[0015] The present invention also provides a method for treating veterinary cancers in companion animals. In one embodiment, the method comprises administering stem cells expressing cytosine deaminase and uracil phosphoribosyltransferase (CD::UPRT) in combination with a prodrug.

[0016] In another embodiment of the method, the cytosine deaminase and uracil phosphoribosyltransferase are from E coli. or yeast.

[0017] In another embodiment of the method, the stem cells are mesenchymal stem cells (MSC).

[0018] In another embodiment of the method, the cytosine deaminase and uracil phosphoribosyltransferase are expressed by inserting encoding sequences into an adenovirus (Ad) vector or an adeno-associated virus (AAV) vector.

[0019] In another embodiment of the method, the prodrug is 5-fluorocytosine

(5FC).

[0020] In another embodiment of the method, the stem cells are intratumorally administered.

[0021] In another embodiment of the method, the stem cells are intravenously administered.

[0022] In another embodiment of the method, the prodrug is orally given.

[0023] The objectives and advantages of the invention will become apparent from the following detailed description of preferred embodiments thereof in connection with the accompanying drawings.

Brief Description of the Drawings

[0024] Preferred embodiments according to the present invention will now be described with reference to the Figures, in which like reference numerals denote like elements.

[0025] FIG 1 shows intratumoral injection of the TrafEn stem cells into the solid tumour. Detailed Description of the Invention

[0026] The present invention may be understood more readily by reference to the following detailed description of certain embodiments of the invention.

[0027] Throughout this application, where publications are referenced, the disclosures of these publications are hereby incorporated by reference, in their entireties, into this application in order to more fully describe the state of art to which this invention pertains.

[0028] Referring now to FIG 1 , it shows intratumoral injection of the TrafEn stem cells into the solid tumour.

[0029] CD and UPRT are from E coli. or yeast; the nucleotide and amino acid sequences for CD and UPRT can be obtained from available database e.g. Genbank.

[0030] Expression vectors for CD::UPRT are exemplarily illustrated by e.g. US

20150361449 Al.

[0031] MSCs are from umbilical cord lining. MSCs could be obtained, transfected and characterized by procedures disclosed e.g. in EP1937326 and US 9,085,755.

[0032] The experiments are performed in canines only with different breeds and different cancer types. The Ad-CD::UPRT MSCs were intratumorally administered. Also, the Ad-CD::UPRT MSCs were intravenously administered if intratumoral administration is not possible. 5FC was orally given for ease of administration by owners, allowing for systemic distribution of 5FC. The treated dogs showed reduction in tumor sizes.

[0033] Modification of mesenchymal stem cells (MSC) for human GDEPT clinical trials mainly uses viral over lion-viral gene delivery systems. This is due to the poor efficiency of transfection approaches using liposomes or polymers (0-50 %). TrafEn (US 20150361449 Al), a combination of re-purposed regulatory approved reagents, enabled cationic polymer based modification of human umbilical cord, bone marrow and adipose tissue derived MSC at > 90% efficiency. Previous studies have demonstrated the anticancer efficacy of modified human MSC producing fused yeast cytosine deaminase:: uracil phosphoribosyltransferase (CDy::UPRT) in in vitro studies. CDy::UPRT-MSC exhibited strong cytotoxic effect towards human gastric, breast and glioblastoma cancer cells in culture. [0034] The application of CDy::UPRT and/or derivative (eg. CDy::UPRTGFP) producing MSC for the treatment of solid tumours in veterinary companion animal species is perfonned via the delivery of the TrafEn stem cells to the tumours, followed by the oral administration of prodrug 5FC. The TrafEn stem cells (10 million cells suspended in 1ml PBS) are delivered via direct intratumoural injections via a 30G insulin syringe needle into the solid tumours that are easily accessible. The injections are given over multiple sites on the solid tumour. The patients who have tumours that are inaccessible by the syringe needle are infused with the cells intravenously (10 million cells suspended in 10ml). The intravenous infusion is administered gradually over an hour with the use of a syringe pump. The tumours are measured before and after the therapy to gauge the effectiveness of the treatment.

[0035] This is the first application of the prodrug CDy::UPRT and/or derivative

(eg. CDy::UPRTGFP) producing MSC delivered intratumourally into companion animals. The initial results have seen good reductions of up to 40% in size of the solid tumours.

[0036] While the present invention has been described with reference to particular embodiments, it will be understood that the embodiments are illustrative and that the invention scope is not so limited. Alternative embodiments of the present invention will become apparent to those having ordinary skill in the art to which the present invention pertains. Such alternate embodiments are considered to be encompassed within the scope of the present invention. Accordingly, the scope of the present invention is defined by the appended claims and is supported by the foregoing description.

References list

Bourbeau D, Lavoie G, Nalbantoglu J, Massie B. Suicide gene therapy with an adenovirus expressing the fusion gene CD::UPRT in human glioblastomas: different sensitivities correlate with p53 status. J Gene Med. 2004; 6:1320-32.

Cavarretta IT, Altanerova V, Matuskova M, Kucerova L, Cuhg Z, Altaner C. Adipose tissue-derived mesenchymal stem cells expressing prodrug-converting enzyme inhibit human prostate tumor growth. Mol Ther. 2010; 18:223-31

Chung-Faye GA, Chen MJ, Green NK, Burton A, Anderson D, Mautner V, Searle PF, Ken- DJ. In vivo gene therapy for colon cancer using adenovirus-mediated, transfer of the fusion gene cytosine deaminase and uracil phosphoribosyltransferase. Gene Ther.

2001 ;8(20): 1547-54. Erbs P, Regulier E, Kintz J, Leroy P, Poitevin Y, Exinger F, Jund R, Mehtali M. In vivo cancer gene therapy by adenovirus-mediated transfer of a bifunctional yeast cytosine deaminase/uracil phosphoribosyl transferase fusion gene. Cancer Res. 2000; 60:3813-22.

Li Z, Fan D, and Xiong D. Mesenchymal stem cells as delivery vectors for anti-tumor therapy. Stem Cell Investig. 2015; 2: 6.

Malekshah OM, Chen X, Nomani A, Sarkar S, Hatefi A. Enzyme/Prodrug Systems for Cancer Gene Therapy. Curr Pharmacol Rep. 2016; 2:299-308

Miyagi T, Koshida K, Hori O, Konaka H, Katoh H, Kitagawa Y, Mizokami A, Egawa M, Ogawa S, Hamada H, Na iki M. Gene therapy for prostate cancer using the cytosine deaminase/uracil phosphoribosyltransferase suicide system. J Gene Med. 2003; 5:30-7.