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. 2023 Jul 20:30:103-116.
doi: 10.1016/j.omto.2023.07.005. eCollection 2023 Sep 21.

Oncolytic virotherapy with intratumoral injection of vaccinia virus TG6002 and 5-fluorocytosine administration in dogs with malignant tumors

Jérémy Béguin  1   2   3 , Eve Laloy  2   4 , Sandrine Cochin  1 , Murielle Gantzer  1 , Isabelle Farine  1 , Christelle Pichon  1 , Baptiste Moreau  1 , Johann Foloppe  1 , Jean-Marc Balloul  1 , Christelle Machon  5 , Jérôme Guitton  5 , Dominique Tierny  6 , Bernard Klonjkowski  2 , Eric Quéméneur  1 , Christelle Maurey  3 , Philippe Erbs  1
Affiliations

Oncolytic virotherapy with intratumoral injection of vaccinia virus TG6002 and 5-fluorocytosine administration in dogs with malignant tumors

Jérémy Béguin et al. Mol Ther Oncolytics. .

Abstract

TG6002 is an oncolytic vaccinia virus expressing FCU1 protein, which converts 5-fluorocytosine into 5-fluorouracil. The study objectives were to assess tolerance, viral replication, 5-fluorouracil synthesis, and tumor microenvironment modifications to treatment in dogs with spontaneous malignant tumors. Thirteen dogs received one to three weekly intratumoral injections of TG6002 and 5-fluorocytosine. The viral genome was assessed in blood and tumor biopsies by qPCR. 5-Fluorouracil concentrations were measured in serum and tumor biopsies by liquid chromatography or high-resolution mass spectrometry. Histological and immunohistochemical analyses were performed. The viral genome was detected in blood (7/13) and tumor biopsies (4/11). Viral replication was suspected in 6/13 dogs. The median intratumoral concentration of 5-fluorouracil was 314 pg/mg. 5-Fluorouracil was not detected in the blood. An increase in necrosis (6/9) and a downregulation of intratumoral regulatory T lymphocytes (6/6) were observed. Viral replication, 5-fluorouracil synthesis, and tumor microenvironment changes were more frequently observed with higher TG6002 doses. This study confirmed the replicative properties, targeted chemotherapy synthesis, and reversion of the immunosuppressive tumor microenvironment in dogs with spontaneous malignant tumors treated with TG6002 and 5-fluorocytosine.

Keywords: antineoplastic protocols; immunomodulation; oncolytic virotherapy; oncolytic viruses; translational medical research.

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Conflict of interest statement

J.B., S.C., M.G., I.F., C.P., B.M., J.F., J.-M.B., E.Q., and P.E. were employees of Transgene SA when the work was performed. Transgene SA is a publicly traded French biopharmaceutical company, with Institut Mérieux as the major shareholder. The authors declare no other competing interests. J.B. is a recipient of an Industrial Training Convention for Research (CIFRE) doctoral fellowship (2017/0266).

Figures

None
Graphical abstract
Figure 1
Figure 1
Viral genome measured by qPCR in the blood of dogs after TG6002 injections (A) Dog 1. (B) Dog 2. (C) Dog 7. (D) Dog 9. (E) Dog 10. (F) Dog 11. (G) Dog 12. qPCR analyses were performed at each time represented on the x axis. (♢) Increase in viral genome copy number, (✦) persistence of viral genome more than five half-lives. The results are presented as the mean of triplicate experiments ±SD.
Figure 2
Figure 2
Histological and immunohistochemical analyses following treatment with TG6002 and 5-FC (A) Histological microphotograph of uninfected mammary sarcoma (dog 4). (B and C) Histological microphotographs of mammary sarcoma (dog 4) 7 days after TG6002 administration; the yellow dashed line demarcates an area of necrosis (B) on the right side of the line and (C) on the left side of the line. (D) Evolution of necrosis before and after treatment. (E) CD3 immunohistochemistry microphotographs of mammary sarcoma (dog 4). (F) CD3 immunohistochemistry microphotographs of dog 4, 7 days after TG6002 administration; note the increase in CD3 expression. (G) CD8 immunohistochemistry microphotographs of high-grade soft tissue sarcoma (dog 11). (H) CD8 immunohistochemistry microphotographs of dog 11, 38 days after administrations of TG6002; note the increase in CD8 expression. (I) Evolution of the proportion of CD3+ T cells before and after treatment. (J) Evolution of the proportion of CD8+ T cells before and after treatment. (A, B, and C) Hematoxylin-eosin-saffron staining. (E, F, G, and H) Cellular DNA was stained in blue with DAPI. (E and F) CD3+ lymphocytes were stained in red/purple. (G and H) CD8+ lymphocytes were stained in green. Scale bars, 100 μm.
Figure 3
Figure 3
Evolution of intratumoral FOXP3+ regulatory T lymphocytes following treatment with TG6002 and 5-FC (A) FOXP3 immunohistochemistry microphotographs of esophageal sarcoma (dog 7) before treatment. (B) FOXP3 immunohistochemistry microphotographs of dog 7, 38 days after treatment; note the decrease in FOXP3 expression. (C) FOXP3 immunohistochemistry microphotographs of urothelial carcinoma (dog 13) before treatment. (D) FOXP3 immunohistochemistry microphotographs of dog 13, 38 days after treatment, note the decrease in FOXP3 expression. (E) Evolution of the proportion of FOXP3+ regulatory T cells before and after treatment assessed by immunohistochemistry. (A–D) Cellular DNA was stained in blue with DAPI, FOXP3+ cells were stained in red/purple. Scale bars, 100 μm.
Figure 4
Figure 4
Immune responses of dogs after multiple intratumoral injections of TG6002 (A) Anti-VACV antibody titers after several injections of TG6002. (B) Neutralizing antibody titers after several injections of TG6002. (C) Anti-VACV antibody titers for dog 12 after the second session of treatment. (D) Neutralizing antibody titers for dog 12 after the second session of treatment.
Figure 5
Figure 5
Tumor size variation between day 0 and day 38 Progressive disease is defined by an increase in size over 20% (red line) and partial response is defined by a decrease in size over 30% (green line).
Figure 6
Figure 6
Study chart and sample collection BID, twice daily.
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