Toca 511 gene transfer and treatment with the prodrug, 5-fluorocytosine, promotes durable antitumor immunity in a mouse glioma model

Leah A Mitchell, Fernando Lopez Espinoza, Daniel Mendoza, Yuki Kato, Akihito Inagaki, Kei Hiraoka, Noriyuki Kasahara, Harry E Gruber, Douglas J Jolly, Joan M Robbins, Leah A Mitchell, Fernando Lopez Espinoza, Daniel Mendoza, Yuki Kato, Akihito Inagaki, Kei Hiraoka, Noriyuki Kasahara, Harry E Gruber, Douglas J Jolly, Joan M Robbins

Abstract

Background: Toca 511 (vocimagene amiretrorepvec) is a retroviral replicating vector encoding an optimized yeast cytosine deaminase (CD). Tumor-selective expression of CD converts the prodrug, 5-fluorocytosine (5-FC), into the active chemotherapeutic, 5-fluorouracil (5-FU). This therapeutic approach is being tested in a randomized phase II/III trial in recurrent glioblastoma and anaplastic astrocytoma (NCT0241416). The aim of this study was to identify the immune cell subsets contributing to antitumor immune responses following treatment with 5-FC in Toca 511-expressing gliomas in a syngeneic mouse model.

Methods: Flow cytometry was utilized to monitor and characterize the immune cell infiltrate in subcutaneous Tu-2449 gliomas in B6C3F1 mice treated with Toca 511 and 5-FC.

Results: Tumor-bearing animals treated with Toca 511 and 5-FC display alterations in immune cell populations within the tumor that result in antitumor immune protection. Attenuated immune subsets were exclusive to immunosuppressive cells of myeloid origin. Depletion of immunosuppressive cells temporally preceded a second event which included expansion of T cells which were polarized away from Th2 and Th17 in the CD4+ T cell compartment with concomitant expansion of interferon gamma-expressing CD8+ T cells. Immune alterations correlated with clearance of Tu-2449 subcutaneous tumors and T cell-dependent protection from future tumor challenge.

Conclusions: Treatment with Toca 511 and 5-FC has a concentrated effect at the site of the tumor which causes direct tumor cell death and alterations in immune cell infiltrate, resulting in a tumor microenvironment that is more permissive to establishment of a T cell mediated antitumor immune response.

Keywords: 5-fluorouracil; cytosine deaminase; immunotherapy; myeloid-derived suppressor cell; retroviral gene transfer.

© The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.

Figures

Fig. 1
Fig. 1
Toca 511 and 5-FC treatment concentrates 5-FU at the site of the tumor and reduces tumor burden; however, changes in immune cell subsets in the tumor 3 days after treatment initiation are minimal. (A) Tumor burden expressed as tumor volume (mm3) over time (days post tumor implant). 5-FC treatment cycles are shown below the graph and collection dates are indicated by black arrows and labeled as days post 5-FC start (n = 5). (B) 5-FC and 5-FU were assessed in plasma and tumor 1 hour after the last administration of 5-FC (n = 4). (C) Pan T cells (CD3+), CD4+, CD8+, or Tregs were analyzed as a percentage of total live lymphocytes in the tumor 3 days post 5-FC treatment initiation (n = 5). (D) 41BB, CD40L, and PD1 expressions on CD4+ T cells were analyzed 3 days after 5-FC treatment initiation. These data were repeated with similar results in at least one additional experiment. *Statistical significance was defined as P < .05.
Fig. 2
Fig. 2
Toca 511 and 5-FC treatment reduces immunosuppressive myeloid cells in the tumor 6 and 9 days after 5-FC treatment initiation. (A) TAMs, MDSCs, and monocytes were analyzed in the tumor 6 days after the initiation of 5-FC and expressed as a percentage of total live lymphocytes in the tumor. (B) CD4+, CD8+, or Tregs were analyzed as a percentage of total live splenocytes 6 days post 5-FC treatment initiation. (C) MDSCs and monocytes were analyzed in the spleen 6 days after the initiation of 5-FC and expressed as a percentage of total live splenocytes. (D) TAMs and MDSCs were analyzed in the tumor 9 days after the initiation of 5-FC and expressed as a percentage of total live lymphocytes in the tumor. These data, n = 5, were repeated with similar results in at least one additional experiment.
Fig. 3
Fig. 3
Toca 511 and 5-FC treatment reduces Th2 and Th17 cells in the tumor 6 and 9 days after 5-FC treatment initiation while enhancing expression of TNFα at day 6 and TNFα and IFNγ at day 9. (A) Intracellular staining for IL-4 and IL-17 was conducted and expressed as a percentage of CD4+ T cells in the tumor 6 days after initiation of treatment with 5-FC. (B) Six days after 5-FC treatment initiation, intracellular staining for TNFα was conducted and expressed as a percentage of CD3−, or non–T cells in the tumor. (C) Intracellular staining for IL-4 and IL-17 was conducted and expressed as a percentage of CD4+ T cells in the tumor 9 days after initiation of treatment with 5-FC. (D) Nine days after 5-FC treatment initiation, intracellular staining for TNFα and IFNγ was conducted and expressed as a percentage of CD3−, or non–T cells in the tumor. (E) Intracellular staining for IFNγ was conducted and expressed as a percentage of CD8+ T cells in the tumor 9 days after initiation of treatment with 5-FC. These data, n = 5, were repeated with similar results in at least one additional experiment.
Fig. 4
Fig. 4
Toca 511 and 5-FC treatment continues to reduce immunosuppressive myeloid cells while increasing T cells in the tumor 14 days after treatment initiation. (A) Total lymphocytes were analyzed as a percentage of total cells in the tumor (data correspond to italicized axis on the left), CD4+, CD8+, or Tregs were analyzed as a percentage of total live lymphocytes in the tumor (data correspond to non-italicized axis on the treatment initiation). (B) Tregs were analyzed as a percentage of total CD4+ T cells in the tumor (right) 14 days post 5-FC treatment initiation. (C) TAMs and MDSCs were analyzed in the tumor 14 days after the initiation of 5-FC and expressed as a percentage of total live lymphocytes in the tumor. (D) Fourteen days after 5-FC treatment initiation, B cells were analyzed as a percentage of total live lymphocytes in the tumor. (E) Mice which had cleared tumors through treatment with Toca 511 and 5-FC and were tumor free for 9 weeks were rechallenged with Tu-2449 subcutaneous tumors and tumor burden was assessed over time (n = 5). Age-matched naïve controls were also challenged (n = 10). (F) Animals which had previously cleared Tu-2449 intracranial tumors through treatment with Toca 511 and 5-FC (termed “immunized”) were used for ACT into recipient mice bearing intracranial Tu-2449 tumors. Before transfer, immunized splenocytes were fractionated. Animals received either unfractionated splenocytes from immunized (black) or naïve mice (orange) or purified T cells from immunized spleens (green) or immunized splenocytes depleted of T cells (blue). These data were repeated with similar results in at least one additional experiment. *Statistical significance was defined as P < .05.
Fig. 5
Fig. 5
Graphic representation of immune cell subset changes over time after initiation of 5-FC treatment.

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