Published OnlineFirst July 26, 2012; DOI: 10.1158/0008-5472.CAN-12-0600 Cancer Therapeutics, Targets, and Chemical Biology Research Systemic Combination Virotherapy for Melanoma with Tumor Antigen-Expressing Vesicular Stomatitis Virus and Adoptive T-Cell Transfer Diana M. Rommelfanger1,2, Phonphimon Wongthida1, Rosa M. Diaz1,3, Karen M. Kaluza1,3, Jill M. Thompson1, Timothy J. Kottke1, and Richard G. Vile1,3 Abstract Oncolytic virotherapy offers the potential to treat tumors both as a single agent and in combination with traditional modalities such as chemotherapy and radiotherapy. Here we describe an effective, fully systemic treatment regimen, which combines virotherapy, acting essentially as an adjuvant immunotherapy, with adoptive cell transfer (ACT). The combination of ACT with systemic administration of a vesicular stomatitis virus (VSV) engineered to express the endogenous melanocyte antigen glycoprotein 100 (gp100) resulted in regression of established melanomas and generation of antitumor immunity. Tumor response was associated with in vivo T-cell persistence and activation as well as treatment-related vitiligo. However, in a proportion of treated mice, initial tumor regressions were followed by recurrences. Therapy was further enhanced by targeting an additional tumor antigen with the VSV-antigen þ ACT combination strategy, leading to sustained response in 100% of mice. Together, our findings suggest that systemic virotherapy combined with antigen-expressing VSV could be used to support and enhance clinical immunotherapy protocols with adoptive T-cell transfer, which are already used in the clinic. Cancer Res; 72(18); 1–12. Ó2012 AACR. Introduction with conditioning of the patient for improved T-cell survival, Oncolytic virotherapy is based on the concept that even persistence, and activation (15, 16). low levels of a replication competent virus introduced into a We and others have shown that adoptive cell transfer (ACT; fi tumor will result in rapid spread through, and lysis of, the of both antigen-speci c T cells and other cell types) can be used tumor, with tumor-selective viral replication being possible to enhance the delivery of oncolytic viruses, especially as a through natural, or engineered, selectivity (1–4). Although method to protect the viruses from neutralization in the – preclinical, as well as several clinical, studies have shown circulation (17 20). There is also considerable potential to highly encouraging results (1, 3, 5–7), virotherapy is still combine virotherapy with ACT as a means to improve the being developed for routine clinical use. Moreover, it is clear immunotherapy component of either, or both, modalities (21, that there is also considerable potential value in the use of 22). In this respect, we, and others, have shown that the negative oncolytic viruses in combination with more conventional strand, enveloped, RNA virus vesicular stomatitis virus (VSV) is in vitro treatment modalities leading to enhancement of efficacy of a potent cytolytic agent against a wide range of tumor – each of the respective agents alone (8–14). In contrast, types (23 25). However, we have observed that, in immuno- adoptive T-cell therapy is more well established in clinical competent, C57BL/6 mice bearing B16ova tumors, therapy use. Despite the technical challenges associated with isola- mediated by intratumoral VSV is primarily mediated by the tion of T cells specific for tumor-associated antigens (TAA) innate immune response to viral infection and does not require – from patients, their subsequent transfer has shown consid- viral replication for tumor control (24, 26 28). On the basis of erable efficacy against several types of cancer, especially these studies, we hypothesized that it would be possible to when used in combination with regimens often associated exploit this potent immunogenicity of VSV as an adjuvant to improve the efficacy of ACT. Thus, we showed that intratu- moral administration of VSV encoding a model TAA (OVA) led Authors' Affiliations: 1Department of Molecular Medicine, 2Department of fi Molecular Pharmacology and Experimental Therapeutics, 3Department of to the activation of adoptively transferred, ova-speci c OT-I T Immunology, Mayo Clinic, Rochester, Minnesota cells. Similarly, when adoptive transfer of Pmel T cells, specific Note: Supplementary data for this article are available at Cancer Research for the endogenous melanocyte differentiation antigen gp100 Online (http://cancerres.aacrjournals.org/). (against which full tolerance is in place in C57BL/6 mice) was Corresponding Author: Richard Vile, Mayo Clinic, 200 First Street SW, combined with intratumoral treatment with VSV-hgp100, we Rochester, MN 55905. Phone: 507-284-9941; Fax: 507-266-2122; E-mail: observed very effective control of the local tumors that were [email protected] directly injected with virus. We also observed, more signifi- doi: 10.1158/0008-5472.CAN-12-0600 cantly, effective treatment of distant disease that was Ó2012 American Association for Cancer Research. not treated directly by virus injection (22), suggesting that www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2012 American Association for Cancer Research. Published OnlineFirst July 26, 2012; DOI: 10.1158/0008-5472.CAN-12-0600 Rommelfanger et al. VSV-hgp100 may act to enhance the antitumor efficacy of Pmel Viral titers were measured by standard plaque assay on BHK- T cells independent of any directly oncolytic, and/or local 21 cells (28, 31). immune stimulating, activities of the virus. In light of these results (22), we proceeded toward our long- Adoptive transfer and tumor treatment term goal of developing a systemic treatment in which no All procedures were approved by the Mayo Foundation tumor has to be directly accessed for viral injections. Here we Institutional Animal Care and Use Committee. To establish show that a combination of intravenous injections of Pmel T subcutaneous tumors, 5  105 B16 tumor cells in 100 mL of PBS cells, followed by VSV-hgp100, resulted in regression of estab- were injected into the flanks of C57BL/6 mice. Following tumor lished B16 tumors and cures in a large proportion of mice. establishment, virus or PBS control (100 mL) was administered However, in some mice, regression was followed by recurrence, intravenously. mimicking a common observation in patients. By targeting 2 Naive T cells were isolated from the spleens and lymph TAAs simultaneously with the combination of VSV-TAA þ nodes of euthanized OT-I and Pmel-1 transgenic mice. Single ACT, we observed regression of tumors in all of the treated cell suspensions were prepared by crushing tissues through a mice with long-term, recurrence-free survival over 100 days. 100 mm filter and red blood cells were removed by incubation in Therefore, our data suggest that systemic virotherapy with ACK buffer (distilled H2O containing 0.15 mol/L NH4Cl, 1.0 antigen-expressing VSV could be used to support, and enhance, mmol/L KHCO3, and 0.1 mmol/L EDTA adjusted to pH 7.2– þ clinically useful protocols of immunotherapy with ACT. 7.4). CD8 T cells were isolated using the MACS CD8a (Ly-2) microbead magnetic cell sorting system (Miltenyi Biotec). For Materials and Methods adoptive transfer experiments, mice were intravenously Cell lines administered naive Pmel, OT-I T cells or a combination of  6 B16(LIF) and B16ova melanoma cells (H2-Kb) (29) were both following tumor establishment (1 10 total cells in 100 grown in Dulbecco's Modified Eagle's Medium (Life Techno- mL PBS). Mice were examined daily for overall health and signs logies) supplemented with 10% (v/v) fetal calf serum (Life of autoimmunity. Tumor sizes were measured 3 times weekly  2  Technologies), and L-glutamine (Life Technologies). B16ova using calipers and volume was calculated as 0.52 width cells were derived from a separate stock of B16 cells transduced length (34). Mice were euthanized when tumor size was  with a cDNA encoding the chicken ovalbumin gene and were approximately 1.0 1.0 cm in 2 perpendicular directions. maintained in 5 mg/mL G418 (Mediatech) to retain the ova Immune cell depletions were carried out by intraperitoneal gene. All cell lines were free of Mycoplasma infection. injections of antibody to CD8 (0.1 mg/mouse; Lyt 2.43; BioX- cell), CD4 (0.1 mg/mouse; GK1.5; BioXcell), Gr1 (0.2 mg/mouse; Mice RB6-8C5; BioXcell), antibody to deplete natural killer (NK) cells þ C57BL/6 mice (Thy 1.2 ) were purchased from The Jackson (25 mL/mouse; anti-asialo-GM-1; Cedarlane), or immunoglob- Laboratory at 6 to 8 weeks of age. The OT-I mouse strain ulin G (IgG) control (0.2 mg/mouse; ChromPure Rat IgG; expresses a transgenic T-cell receptor, Va2, specific for the Jackson Immunoresearch) on days 3, 6, 10 posttumor cell SIINFEKL peptide of ovalbumin in the context of MHC class I, implantation and then weekly until experiment completion. H-2Kb (30). OT-I breeding pairs were obtained as a gift from Fluorescence-activated cell sorting (FACS) analysis confirmed Dr. Larry Pease, Mayo Clinic. Pmel-1 transgenic mice express subset-specific depletions. the Va1/Vb13 T-cell receptor that recognizes amino acids 25 to 33 of gp100 (Pmel-17) presented by H2-Db MHC class I Reverse-transcriptase PCR (21). Pmel-1 breeding colonies were purchased from The Tumors were excised from euthanized mice and dissociated Jackson Laboratory at 6 to 8 weeks of age. to achieve single-cell suspensions. RNA was extracted using the Qiagen RNeasy Kit (Qiagen). cDNA was made from 1 mg total Viruses RNA using the First Strand cDNA Synthesis Kit (Roche). A VSV-GFP and VSV-ova (Indiana serotype) were generated by cDNA equivalent of 1 ng RNA was amplified by PCR with gene- cloning the cDNA for GFP or chicken ovalbumin, respectively, specific primers. Expression of mouse GAPDH (mgapdh) was into the plasmid pVSV-XN2 as described previously (31). used as a positive control for both gp100 and OVA antigen Plasmid pVSV-hgp100 was constructed by PCR amplifying the expression.