Cancer Therapy: Preclinical Accelerated Tumor Growth Mediated by Sublytic Levels of Antibody-InducedComplement Activation Is Associatedwith Activation of the PI3K/AKT Survival Pathway

Purpose: We addressed the possibility that low levels of tumor cell-bound antibodies targeting gangliosides might accelerate tumor growth. Experimental Design: To test this hypothesis, we treated mice with a range of monoclonal antibody (mAb) doses against GM2, GD2, GD3, andCD20 after challenge with tumors expressing these antigens and tested the activity of the same mAbs in vitro. We also explored the mechanisms behind the complementmediated tumor growth acceleration that we observed and an approach to overcome it. Results: Serologically detectable levels of IgM-mAb against GM2 are able to delay or prevent tumor growthof highGM2expressing cell lines both in vitro and in a SCIDmousemodel, whereas very low levels of this mAb resulted in slight but consistent acceleration of tumor growth in both settings. Surprisingly, this is not restricted to IgMmAb targeting GM2 but consistent against an IgGmAb targeting GD3 as well. These findings were mirrored by in vitro studies with antibodies against these antigens as well as GD2 and CD20 (with Rituxan), and shown to be complement-dependent in all cases. Complement-mediated accelerated growth of cultured tumor cell lines initiated by low mAb levels was associated with activation of the phosphoinositide 3-kinase (PI3K)/AKT survival pathway and significantly elevated levels of both p-AKT and p-PRAS40. This complement-mediated PI3K activation and accelerated tumor growth in vitro and in vivo are eliminated by PI3K inhibitors NVP-BEZ235 and Wortmannin. These PI3K inhibitors also significantly increased efficacy of high doses of these four mAbs. Conclusion:Our findings suggest that manipulation of the PI3K/AKT pathway and its signaling network can significantly increase the potency of passively administered mAbs and vaccine-induced antibodies targeting a variety of tumor cell surface antigens. Clin Cancer Res; 19(17); 4728–39. 2013 AACR. Introduction A variety ofmonoclonal antibodies (mAb) against cancer antigens have resulted in prolongation of disease-free and overall survival in preclinical studies and in clinical responses when tumors known to be strongly positive for the relevant antigens were targeted. Several of these mAbs have been U.S. Food and Drug Administration (FDA) approved for these purposes. Although not FDA approved, ganglioside-specific mAbs against gangliosides GD2 and GD3 have showed both preclinical efficacy and clinical responses (1–8) in patients with neuroblastoma and melanoma, respectively, in the setting of strongly antigen-positive tumors. On the other hand, randomized trials with a GM2-keyhole limpet hemocyanin (KLH) vaccine that consistently induces IgM and IgG antibodies against GM2 in patients with melanoma have shown either no benefit (9, 10) or an initial decrease in overall survival compared with no treatment controls (11, 12).GM2 is present in essentially all melanomas, but unlike GD3 and GM3, which are the most highly expressed melanoma gangliosides, it is expressed at only low levels in the majority of cases (13, 14) and very few melanoma cell lines can be lysed with complement andmAbs or immune sera against GM2 (15). We hypothesized therefore that: (i) induction of high titers of antibodies against GM2 results in only a low level of cell surface antibodies against GM2 because of the low level GM2 expression on most melanomas and (ii) this might result in sublytic levels of complement activation resulting in inflammation, angiogenesis, and tumor cell activation, as has been described for sublytic levels of cell surface complement activation in other settings (16–18). Authors' Affiliations: Laboratory of Tumor Vaccinology, Melanoma Sarcoma Service, Department of Medicine and Department of Biostatistics, Memorial Sloan-Kettering Cancer Center, New York, New York Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Current address for P.O. Livingston: MabVax Therapeutics Inc., SanDiego, California Corresponding Author: Govind Ragupathi, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065. Phone: 212639-4022; Fax: 646-422-0516; E-mail: [email protected] doi: 10.1158/1078-0432.CCR-13-0088 2013 American Association for Cancer Research. Clinical Cancer Research Clin Cancer Res; 19(17) September 1, 2013 4728 on April 14, 2017. © 2013 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Published OnlineFirst July 5, 2013; DOI: 10.1158/1078-0432.CCR-13-0088