Cyclophosphamide facilitates antitumor efficacy against subcutaneous tumors following intravenous delivery of reovirus

Abstract

Purpose: The purpose of the present study was to investigate whether it is possible to achieve truly systemic delivery of oncolytic reovirus, in immunocompetent hosts, using cyclophosphamide to overcome some of the barriers to effective intratumoral delivery and replication of i.v. injected virus.

Experimental design: I.v. delivery of reovirus was combined with different regimens of i.p. administered cyclophosphamide in C57Bl/6 mice bearing established s.c. B16 tumors. Intratumoral viral replication, tumor size, and survival were measured along with levels of neutralizing antibody (NAb) in the blood. Finally, differential toxicities of the virus/cyclophosphamide regimens were monitored through viral replication in systemic organs, survival, and cardiac damage.

Results: Repeated i.v. injection of reovirus was poorly effective at seeding intratumoral viral replication/oncolysis. However, by combining i.v. virus with cyclophosphamide, viral titers of between 10(7) and 10(8) plaque-forming units per milligram were recovered from regressing tumors. Doses of cyclophosphamide that ablated NAb were associated with severe toxicities, characterized by viral replication in systemic organs--toxicities that are mirrored by repeated reovirus injections into B-cell knockout mice. Next, we restructured the dosing of cyclophosphamide and i.v. virus such that a dose of 3 mg cyclophosphamide was administered 24 h before reovirus injection, and this schedule was repeated every 6 days. Using this protocol, high levels of intratumoral viral access and replication ( approximately 10(7) plaque-forming units per milligram tumor) were maintained along with systemically protective levels of NAb and only very mild, non-life-threatening toxicity.

Conclusion: NAb to oncolytic viruses play a dual role in the context of systemic viral delivery; on one hand, they hinder repeated administration of virus but on the other, they provide an important safety mechanism by which virus released from vigorous intratumoral replication is neutralized before it can disseminate and cause toxicity. These data support the use of cyclophosphamide to modulate, but not ablate, patient NAb, in development of carefully controlled clinical trials of the systemic administration of oncolytic viruses.

Fig. 1. Reovirus is an effective oncolytic agent against B16 melanoma

A, C57Bl/6 mice (n = 8–10 per group) were injected s.c. with 2 × 10⁵ B16 tumor cells on day 1. At days 10, 12, and 14, tumors were injected directly with 5 × 10⁸ pfu of reovirus or heat-inactivated virus (HI Virus) or PBS. Survival of mice is shown with time after tumor seeding (mice were euthanized when tumors reached a size of 1.0 cm in the longest diameter). Heat-inactivated virus was indistinguishable from PBS treatment. Data are representative of multiple experiments. B, systemic viral therapy: s.c. B16 tumors were established by injecting 2 × 10⁵ B16 cells s.c., or lung metastatic tumors were established by injecting 10⁵ B16 cells i.v. in C57Bl/6 mice (n = 7–10 per group). On days 7, 13, and 19, mice were injected i.v. with 5 × 10⁸ pfu of reovirus, heat-inactivated reovirus, or PBS. C, survival of mice in the lung metastatic model is shown after treatment of tumor-bearing mice with i.v. reovirus as shown in B (P = 0.0014 reovirus compared with PBS). D, survival of mice bearing a s.c. B16 tumor and treated with i.v. reovirus or PBS is shown with time after tumor seeding (P = 0.054 reovirus compared with PBS). In both C and D, heat-inactivated virus was indistinguishable from PBS treatment.

Fig. 2. High-dose cyclophosphamide enhances the antitumor efficacy of systemically delivered reovirus

A, the was combined with either an i.p. single dose of cyclophosphamide (Cy) onday 6 (Low Dose ;150 mg/kg = 3 mgpermouse), or with three i.p. doses of 3 mg permouse on days 6, 7, and 8 (High Dose) to treat either s.c. or lung metastatic tumors established as described in Fig.1B. B, mice bearing established lung metastases were treated with either PBS or the high-dose cyclophosphamide (CPA) regimen along with three injections i.v. of either PBS or reovirus (days 7, 13, and 19) at doses of5 ×10⁵, 5 ×10⁶, 5 ×10⁷, or 5 ×10⁸ pfu as shown (n = 8 per group). Survival of mice with time is shown. C toH, mice (n = 8) bearing established s.c. B16 tumors were treated with PBS, low-dose cyclophosphamide, or high-dose cyclophosphamide along with three i.v. injections of either PBS or 5 × 10⁸ pfu of reovirus as indicated. Tumor size was measured with time after tumor seeding as shown. I, the cumulative data of C to H, also displayed with a mean measurement of tumor size and SD. Tumor growth curves of each group are terminated on the day of the first death in that group. *, significant differences exist between control groups (PBS or reovirus alone) and both low- and high-dose cyclophosphamide at day 11 (P = 0.02) and between PBS and high-dose cyclophosphamide from days 13 to 18; significant differences exist between the PBS group and reovirus + low-dose cyclophosphamide from day 13 (P = 0.035) onward until day 18 (when the first animals needed to be euthanized); tumor size in the high-dose cyclophosphamide + reovirus is significantly different from all other groups at days 11and 13.

Fig. 3. Cyclophosphamide significantly alters the distribution of virus in vivo after i.v. reovirus delivery

C57Bl/6 mice bearing 6-d established s.c. B16 tumors were treated with (A) three i.v. injections of 5 × 108 pfu of reovirus (as described in Fig.1B); (B) with low-dose cyclophosphamide + three i.v. injections of 5 × 108 pfu of reovirus (as described in Fig. 2A); or (C) with high-dose cyclophosphamide + three i.v. injections of5 ×10⁸ pfu of reovirus (Fig. 2A). Upon euthanasia due to either tumor size (A and B) or systemic toxicity (C), tumor (Tu) and organs [lungs (Lu), blood (Bl), liver (Li), spleen (Sp), intestines (In), brain (Br), heart (He), and bone marrow (Bm)]were harvested, weighed, and lysed by freeze thawing. Virus titers recovered were determined on L929 cells and are shown as pfu of reovirus per milligram of tissue. Results represent data from one mouse in each group and are representative of viral titers from two to four mice per group. D, hearts harvested from mice treated as in A (i.v. reovirus) showed normal architecture and no pathologic abnormalities. E, in contrast, hearts harvested from mice treated as in C (high-dose cyclophosphamide + i.v. reovirus) showed widespread and diffuse abnormalities throughout the heart (see text). F, B6.129S2-Igh-6tm1^Cgn/j mice (Jackson 002288), which lack mature B cells and cannot, therefore, make antiviral antibodies, bearing 6-d established s.c. B16 tumors were treated with two i.v. injections of5 ×10⁸ pfu of reovirus at days 7 and13 (see Fig.1B).Two days after the second i.v. virus injection, mice had to be euthanized due to toxicity. Upon euthanasia, tumor and organs (lungs, blood, liver, spleen, intestines, brain, heart, and bone marrow) were harvested, weighed, and lysed by freeze thawing. Virus titers recovered were determined on L929 cells and are shown as pfu of reovirus per milligram of tissue. Results shown represent data from one mouse and are representative of viral titers from four different mice.

Fig. 4. Metronomic dosing of cyclophosphamide and reovirus balances antitumor efficacy and systemic toxicity

A, the timing of cyclophosphamide injections was modified relative to that in Fig. 2A, to precede each of the three injections of reovirus by 24 h. B, s.c. tumor bearing C57Bl/6 mice (8–10 per group) were treated according to the metronomic dosing regimen of A with three injections of PBS (days 6, 12, and 18) + 5 × 10⁸ pfu of reovirus (days 7, 13, and 19); or with similar regimens of PBS/PBS; cyclophosphamide/PBS; or with cyclophosphamide/reovirus (as shown). Survival of mice in each group is shown with time with euthanasia being required only due to tumor size rather than treatment-associated toxicity. C to F, the treatment regimen of B above was repeated in additional groups of mice and systemic toxicity was assessed through changes of body weight as shown. G, s.c. tumor-bearing C57Bl/6 mice were treated according to the metronomic dosing regimen of A with three injections of cyclophosphamide (days 6, 12, and 18)+ 5 × 10⁸ pfu of reovirus (days 7, 13, and 19) 50 d after tumor seeding, tumor and organs (lungs, liver, spleen, intestines, brain, heart, and kidney) were harvested, weighed, and lysed by freeze thawing. Virus titers recovered were determined on L929 cells and are shown as pfu of reovirus per milligram of tissue. H and I, hearts harvested from mice treated as in G with metronomic cyclophosphamide alone (I) showed normal pathology; those harvested from mice treated with metronomic cyclophosphamide + i.v. reovirus (H) showed predominantly normal pathology but with sporadic and isolated areas of myocarditis.