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Clinical Trial
. 2015 Mar 15;21(6):1305-12.
doi: 10.1158/1078-0432.CCR-14-1770. Epub 2014 Nov 25.

Phase I trial of cyclophosphamide as an immune modulator for optimizing oncolytic reovirus delivery to solid tumors

Victoria Roulstone  1 , Khurum Khan  1 , Hardev S Pandha  2 , Sarah Rudman  3 , Matt Coffey  4 , George M Gill  4 , Alan A Melcher  5 , Richard Vile  6 , Kevin J Harrington  1 , Johann de Bono  7 , James Spicer  8
Affiliations
Clinical Trial

Phase I trial of cyclophosphamide as an immune modulator for optimizing oncolytic reovirus delivery to solid tumors

Victoria Roulstone et al. Clin Cancer Res. .

Abstract

Purpose: Reovirus is a wild-type oncolytic virus that is ubiquitous in the environment; most patients are therefore preimmune. Therapeutic administration leads to an increase in neutralizing antireovirus antibody (NARA) titer. We hypothesized that if NARA limited reovirus antitumor activity, the effect might be attenuated by coadministration of cyclophosphamide.

Experimental design: In a phase I study, patients with advanced cancer received cyclophosphamide 3 days before intravenous reovirus serotype 3 Dearing (RT3D). The primary objective was to reduce the resulting rise in NARA titer. Cyclophosphamide dose was escalated from 25-1,000 mg/m(2) through nine cohorts; we aimed to define a well-tolerated immunomodulatory dose.

Results: The combination was well tolerated in 36 patients, with grade 3/4 toxicities only seen at or above the maximum tolerated dose of cyclophosphamide, which was 800 mg/m(2) combined with reovirus. Immunosuppressive effect, defined as maintaining NARA titer rise below a predefined threshold, was observed in only one patient. Furthermore, despite expected myelosuppression seen at higher cyclophosphamide doses, no changes in T-cell subsets, including Tregs, occurred with dose escalation. Viable virus was detected in association with peripheral blood mononuclear cells (PBMC) from 14% of patients 10 days after the last RT3D injection, despite high plasma NARA titer, demonstrating a potential mechanism for prolonged evasion of neutralization by reovirus.

Conclusions: Coadministration of cyclophosphamide with reovirus is safe, but does not attenuate host antiviral responses. Alternative immunomodulation approaches should be explored, but association with PBMCs may allow reovirus to persist and evade even high levels of neutralizing antibodies.

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Figures

Figure 1
Figure 1
A, neutralizing antireovirus antibody (NARA) titer, and (B) regulatory T-cells (Tregs), are unchanged before and after exposure to reovirus. A, NARA titer was assessed at baseline, 10 days after patients' last infusion of reovirus in their first cycle of treatment (“C1D15”), and at C2D15. Significant immunomodulation, defined as less than 50-fold induction of NARA titer, was observed in a single patient treated with cyclophosphamide 50mg/m2 (27-fold rise; ▼ cohort 2). B, the proportion of CD4+CD25+CD127lo/neg T cells was measured for each cohort at baseline, C1D15, and C2D15. Horizontal bars represent medians. This was not significantly affected even at the highest cyclophosphamide doses, despite more prominent suppression of total white blood cell count with increasing dose (Supplementary Fig. S1).
Figure 2
Figure 2
Patients carry live reovirus on their PBMCs up to 10 days after the last administration, which can replicate and kill target cells in vitro despite an increased level of circulating neutralizing antibodies. A, PBMCs were assessed directly for presence of reoviral RNA (“neat”) by RT-PCR at C1D15 and C2D15. PBMCs were also incubated on L929 target cells at the dilutions shown to allow for any functional reovirus to be handed off the PBMCs and then replicate in the target cells, before analysis for reoviral RNA by RT-PCR (“amp”). The appearance of a band demonstrates presence of RT3D, which denotes successful transfer from PBMC cells and replication of reovirus. This becomes apparent after treatment in 3 patients tested (0303, 0602, 0603), but is not detected in the amplified samples taken before treatment (“pre”). 2% DMEM or a dilution (1:10) of stock reovirus was incubated on L929 cells as negative and positive controls, respectively (−amp, +amp). B, all other available samples for these patients were analyzed, and all samples were negative for reovirus by amplification assay, except for sample C1D5 in patient 0303. Photomicrographs show cytopathic effect on L929 cells pre- and posttreatment, and MTT viability data after incubation of sample on L929 cells for patient 0303. C, neutralizing antireovirus antbody titers in patients who carried reovirus on their PBMCs. At C1D15 and C2D15 antibody titers increased as much as 729-fold compared with baseline. Error bars show SEM.
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