Intratumoral recombinant GM-CSF-encoding virus as gene therapy in patients with cutaneous melanoma Michael J. Mastrangelo, 1 Henry C. Maguire Jr., 1 Laurence C. Eisenlohr, 2 Carol E. Laughlin, 2 Claude E. Monken, 1 Peter A. McCue, 3 Albert J. Kovatich, 3 and Edmund C. Lattime 1,2 Departments of 1 Medicine, 2 Microbiology and Immunology, and 3 Pathology, Anatomy, and Cell Biology, Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pennsylvania 19107. Seven immunocompetent, revaccinated patients with surgically incurable cutaneous melanoma underwent treatment of dermal and/or subcutaneous metastases with twice-weekly intratumoral injections of escalating doses (10 4 -2  10 7 plaque-forming units (PFU)/lesion; 10 4 -8  10 7 PFU/session) of a vaccinia/GM-CSF recombinant virus for 6 weeks. Patients with stable or responding disease were maintained on treatment until tumor resolution or progression. Systemic toxicity was infrequent, dose-related, and limited to mild flu-like symptoms that resolved within 24 hours. Local inflammation, at times with pustule formation, was consistently seen with doses of 10 7 PFU/lesion. Chronically treated lesions showed a dense infiltration, with CD4 + and CD8 + lymphocytes, histiocytes, and eosinophils. All seven patients developed an antivaccinia humoral immune response 14 –21 days following revaccination. Despite the presence of these antivaccinia antibodies, the reporter gene was expressed, as judged by the development of anti--galactosidase antibodies in all patients. Passenger cytokine gene function was evidenced by the presence of virally encoded GM-CSF mRNA at injection sites both early (weeks 1 and 5) and late (week 31) in the course of treatment. Eosinophilia at treatment sites indicated that physiologically significant levels of functional cytokine were generated. However, there were no changes in the total number of peripheral white blood cells or in the numbers or percentages of polymorphonuclear leukocytes, monocytes, or eosinophils. GM-CSF was not detected in the sera. The two patients with the largest tumor burdens failed to respond even at treatment sites. Three patients had mixed responses, with regression of treated and untreated dermal metastases and progression of disease elsewhere. One patient had a partial response, with regression of injected and uninjected regional dermal metastases. Residual melanoma was excised, rendering the patient disease free. One patient with only dermal metastases confined to the scalp achieved a complete remission. Sequential administration of escalating doses of a GM-CSF recombinant vaccinia virus is safe, effective at maintaining passenger gene function, and can induce tumor regression. Key words: Vaccinia; recombinant GM-CSF; intratumoral; gene therapy, melanoma. T he demonstration by Prehn and Main 1 of function- ally specific tumor-associated antigens (Ags) that could be targeted to effect tumor regression provided a scientific foundation for the pursuit of active specific immunization as a cancer treatment. Early clinical trials of active specific tumor immunotherapy used whole tumor cells as immunogens almost exclusively. Autolo- gous tumor cells display by definition not one but all of the potential Ag epitopes expressed by the host tumor, both tumor rejection-related and tumor rejection-irrel- evant. The historical development of tumor cell-based vaccines has been reviewed by Mastrangelo et al. 2 When used alone, cell-based vaccines demonstrated little abil- ity to induce tumor regression or to prolong disease-free survival. The addition of adjuvants (bacillus Calmette- Gue ´rin, Corynebacterium parvum, and Detox®, Ribi Immuno Chem Research, Hamilton, Mont), haptens (keyhole limpet hemocyanin, and dinitrophenyl), and immunomodulators (cyclophosphamide) enhanced effi- cacy; still, only a minority of patients benefited. Advances in basic immunology led to an appreciation of the cascade of molecular events integral to the development of an immune response. Requisite is an array of cytokines that provide the costimulatory signals important for T-lymphocyte activation. We and others hypothesized that enrichment of the cytokine milieu at the vaccination site would enhance the acquisition of immunity. Reasoning that higher and perhaps more effective cytokine levels could be sustained at the immu- nization site by local production rather than by local or systemic injection, tumor cells or fibroblasts genetically engineered to produce the molecule(s) of interest were included in vaccine preparations. The demonstration by Golumbek et al 3 that immunization with tumor cells engineered to secrete Interleukin(IL)-4 could induce Received June 26, 1998; accepted November 1, 1998. Address correspondence and reprint requests to Dr. Michael J. Mas- trangelo, Division of Medical Oncology, Jefferson Medical College, Suite 1024, Curtis Building, 1015 Walnut Street, Philadelphia, PA 19107-5099. © 1999 Stockton Press 0929-1903/99/$12.00/+0 Cancer Gene Therapy, Vol 6, No 5, 1998: pp 409–422 409