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 3

Nucleoside prodrug treatment of E. coli PNP–expressing EMT6 cells elicits signaling pathways associated with immunogenic cell death.

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Nucleoside prodrug treatment of E. coli PNP–expressing EMT6 cells elicit...
(A and B) Cells were treated as in Figure 2 with E. coli PNP substrate (MeP-dR; A) or fludarabine (F-araA; B) for 24 hours, then fixed, stained for immunogenic cell death (ICD) markers, and analyzed by flow cytometry. *P < 0.05; ****P < 0.0001, versus parental control, 2-way ANOVA (post hoc Tukey’s comparison). Data shown as the mean ± SEM; n = 6–9 biologic replicates combined from 2–3 independent experiments performed in triplicate. Flow cytometry histograms are shown for each experiment following PNP/nucleoside treatment or treatment with mitoxantrone (1 μg/ml × 24 hours), a known ICD inducer, as positive control. Cells expressing E. coli PNP exhibit basal elevations of HSP70 at the plasma membrane. When EMT6 cells expressed the R25A (inactive) PNP enzyme, ICD response was minimal (Supplemental Figure 1). Ec PNP, wild-type E. coli PNP; MFI, mean fluorescence intensity; CALR, calreticulin.