Broadening activity of checkpoint blockade agents by intratumoral nucleoside cleavage
Regina Rab, Jeong S. Hong, Brendan L.C. Kinney, Nicole C. Schmitt, William B. Parker, Adrianna Westbrook, Kelsey B. Bennion, Mandy L. Ford, Douglas H. Weitzel, Paula L. Miliani de Marval, Eric J. Sorscher, Annette Ehrhardt
Regina Rab, Jeong S. Hong, Brendan L.C. Kinney, Nicole C. Schmitt, William B. Parker, Adrianna Westbrook, Kelsey B. Bennion, Mandy L. Ford, Douglas H. Weitzel, Paula L. Miliani de Marval, Eric J. Sorscher, Annette Ehrhardt
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Research Article Genetics Oncology

Broadening activity of checkpoint blockade agents by intratumoral nucleoside cleavage

Abstract

We investigated whether destroying malignant cells and the associated tumor microenvironment (TME) by focal gene therapy would broaden immune checkpoint inhibitor (ICI) effectiveness. We show that ICI antitumor activity against syngeneic (murine) triple-negative breast cancer (TNBC) was augmented when a therapeutic transgene (purine nucleoside phosphorylase, referred to here as E. coli PNP) was used to cleave fludarabine (2-fluoro-arabinofuranosyl adenine) to the anticancer purine base, 2-fluoroadenine (F-Ade). We also established strong repression of anatomically distant, non-PNP-expressing tumors being treated by the same strategy. TNBC cytoreduction was associated with decreased intratumoral PD1+ Tregs, increased granzyme B+ NK cells, elevated MKI67+ T8 cells, and rapid immune clearance. Because F-Ade works by a mechanism that destroys quiescent neoplastic and supporting cells in the microenvironment, and since resistance to ICIs depends upon an intact TME, tumor killing by this approach offers a means to sensitize refractory malignancies to immune ablation and points to broad applicability against numerous cancer subtypes.

Authors

Regina Rab, Jeong S. Hong, Brendan L.C. Kinney, Nicole C. Schmitt, William B. Parker, Adrianna Westbrook, Kelsey B. Bennion, Mandy L. Ford, Douglas H. Weitzel, Paula L. Miliani de Marval, Eric J. Sorscher, Annette Ehrhardt

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Figure 2

Characterization of EMT6 triple-negative breast cancer–expressing E. coli PNP.

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Characterization of EMT6 triple-negative breast cancer–expressing E. col...
(A) Nucleoside cleavage activity of cell lysates (nmol prodrug converted to purine base per mg cell extract per hour; see Methods). n = 6 biologic replicates per condition. Data are shown as the mean ± SEM. ****P < 0.0006, 1-way ANOVA with Bonferroni’s correction for multiple comparisons. (B and C) In vitro cell killing of murine triple-negative breast cancer (TNBC) cells following cleavage of nucleoside by E. coli PNP. Cells were grown to 70%–80% confluency and treated with (B) 100 μM MeP-dR (F-AraA homologue used for in vitro analyses) or (C) 7 μM F-araA. Representative experiments are shown, with n = 9 biological replicates per condition from 3 independent experiments. Cells were stained with 0.1% crystal violet. Complete ablation of tumor cells in culture was observed specifically in the presence of functional E. coli PNP. Parental, TNBC without transduction; Vector Alone, TNBC transduced with empty lentiviral vector (no PNP expression); EcPNP wt, stable transduction with wild-type PNP; EcPNP R25A, TNBC transduced with E. coli PNP encoding an active site mutant with diminished activity. The HPLC assay did not report very low-level prodrug conversion responsible for barely detectable cytotoxicity shown on far right of B.