Human monoclonal antibody CP751871, combined with rapamycin is effective treatment for childhood sarcoma xenografts

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Background: Under growth factor deficient conditions, rapamycin a specific inhibitor of mTORC1 signaling, induces apoptosis selectively in sarcoma cells with dysfunctional p53. Apoptosis is blocked uniquely by insulin like growth factors-1 and -2. Consequently, we have evaluated the combination of rapamycin and an antibody that prevents signaling through the IGF-1 receptor (IGF-1R).
 Methods: The sensitivity of rhabdomyosarcoma (RMS) and Ewing sarcoma (EWS) cell lines to CP-751871 was determined by Alamar Blue staining after 4 days treatment. The ability of CP-751871 to block IGF-1-mediated rescue from rapamycin treatment was determined by ApoAlert assay. Secreted levels of IGF-1 and IGF-2 were determined by ELISA assay. Levels of IGF-1R, Akt, S6 and their phosphorylated forms were determined by western blot. In vivo testing was performed against subcutaneous sarcomas in CB17/scid^-/- mice. CP-751871 was administered twice weekly (0.5 mg/mouse) for 4 weeks and rapamycin was administered i.p. daily for 5 days for 12 consecutive weeks.
 Results: Maximum growth inhibition (range for RMS 39.2-52.0%; range for EWS: 5.5-64%) for all cell lines was obtained at 1 μg/ml. CP-751871 induced only minor decrease in phospho-Akt, and mTORC1 signaling. Rapamycin induced phosphorylation of both Akt (S⁴⁷³) and IGF-IR (Y¹¹⁴⁶) in RMS cells that were both inhibited by CP-751871. Both rapamycin and CP-751871 significantly increased IGF-1 but not IGF-2 secreted in medium by EWS cells. CP-751871 effectively blocked IGF-1-mediated rescue from rapamycin-induced apoptosis in vitro. In vivo, CP-751871 markedly downregulated IGF-1R levels and pAkt (>70%) within 24 hr in 9/11 childhood sarcoma xenografts. We evaluated the antitumor activity of CP-751871, rapamycin and the combination against 6 in vivo models that are IGF-driven, and harbor dysfunctional (mutated) p53: EW-5, ES-2, ES-8 (Ewing sarcoma) and OS-1, OS-9 (osteosarcoma), and Rh18 (RMS; HDM2 amplification). Pharmacodynamics (PD) studies are ongoing. IGF-1R was completely downregulated at day 7 of combination treatment, whereas it was still detected in CP-751871 treated tumors. At day 7, p-Akt levels had returned to control levels in some combination treated tumors, whereas it was suppressed in tumors from mice receiving CP-751871 alone. The combination treatment was additive in 5 of 6 models, and induced complete regressions in 4 lines (EW-5, OS-1, OS-9, Rh18) that were maintained at week 12.
 Conclusions: CP-751871 significantly retarded the proliferation of most sarcoma cell lines. In vivo, CP751871 retarded tumor growth, as did rapamycin. In 4 IGF-driven xenograft models the combination induced complete regressions of established tumors. Our studies are being extended to additional sarcoma models. PD studies may potentially identify biomarkers of responsiveness to this combination. Supported by CA23099 and CA77776. (2593 characters)