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 4

Combining immune checkpoint inhibition with PNP/F-araAMP augments ICI activity against triple-negative breast cancer.

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Combining immune checkpoint inhibition with PNP/F-araAMP augments ICI ac...
(A) F-araAMP was administered i.p. at a dose of 167 mg/kg on days 1–3 after tumors reached a size of 100–200 mm3. (B–E) Mice were implanted with bilateral tumors expressing PNP (in the left flank, B and D) or no PNP (right flank, C and E). F-araAMP was administered (by intent) at subcurative dosing (90 mg/kg i.p. on days 1, 2, and 3). (B and C) Anti–CTLA-4 was given i.p. on days 1, 4, and 7 (5 mg/kg on day 1 and 2.5 mg/kg on days 4 and 7). Anti-PDL1 (D and E) was dosed i.p. biweekly for 2 weeks (days 1, 4, 8, and 11). E. coli PNP activity in the EMT6-transduced line was 6,000–7,000 units, with zero activity in parental tumors not expressing PNP. Statistical analysis of tumor volume was performed on day 21 for B and C and days 15 and 18 for D and E. The ICI cohort was found to have significantly larger tumor volumes compared with combined (ICI + F-araAMP) treatment for experiments in B and C (P < 0.05) and D (P < 0.025). In E, because animals were removed/euthanized owing to rapid tumor growth, an alternative analysis for tumors that doubled in size by day 11 was performed, showing that ICI + F-araAMP differed from ICI alone (P < 0.05, 2-tailed t test with Bonferroni’s correction) (n = 5–6 animals per condition). Animals tolerated interventions with minimal weight loss. Data are shown as the mean ± SEM.