Adoptive cell transfer therapy fo...
Adoptive Cell Transfer Therapy Following Non-Myeloablative but Lymphodepleting Chemotherapy for the Treatment of Patients With Refractory Metastatic Melanoma Mark E. Dudley, John R. Wunderlich, James C. Yang, Richard M. Sherry, Suzanne L. Topalian, Nicholas P. Restifo, Richard E. Royal, Udai Kammula, Don E. White, Sharon A. Mavroukakis, Linda J. Rogers, Gerald J. Gracia, Stephanie A. Jones, David P. Mangiameli, Michelle M. Pelletier, Juan Gea-Banacloche, Michael R. Robinson, David M. Berman, Armando C. Filie, Andrea Abati, and Steven A. Rosenberg From the Surgery Branch Experimental Immunology and Transplantation Branch Laboratory of Pathology, Center for Cancer Research, National Cancer Institute National Eye Institute, National Institutes of Health, Bethesda, MD. Abstract Purpose We investigated the combination of lymphodepleting chemotherapy followed by the adoptive transfer of autologous tumor reactive lymphocytes for the treatment of patients with refractory metastatic melanoma. Patients and Methods Thirty-five patients with metastatic melanoma, all but one with disease refractory to treatment with high-dose interleukin (IL)-2 and many with progressive disease after chemotherapy, underwent lymphodepleting conditioning with two days of cyclophosphamide (60 mg/kg) followed by five days of fludarabine (25 mg/m2). On the day following the final dose of fludarabine, all patients received cell infusion with autologous tumor-reactive, rapidly expanded tumor infiltrating lymphocyte cultures and high-dose IL-2 therapy. Results Eighteen (51%) of 35 treated patients experienced objective clinical responses including three ongoing complete responses and 15 partial responses with a mean duration of 11.5 �� 2.2 months. Sites of regression included metastases to lung, liver, lymph nodes, brain, and cutaneous and subcutaneous tissues. Toxicities of treatment included the expected hematologic toxicities of chemotherapy including neutropenia, thrombocytopenia, and lymphopenia, the transient toxicities of high-dose IL-2 therapy, two patients who developed Pneumocystis pneumonia and one patient who developed an Epstein-Barr virus-related lymphoproliferation. Conclusion Lymphodepleting chemotherapy followed by the transfer of highly avid antitumor lymphocytes can mediate significant tumor regression in heavily pretreated patients with IL-2 refractory metastatic melanoma. Address reprint requests to Steven A. Rosenberg, MD, PhD, Surgery Branch, National Cancer Institute, NIH, CRC 3-3940, 10 Center Dr MSC 1201, Bethesda MD 20892-1202 e-mail: firstname.lastname@example.org.. Authors��� Disclosures of Potential Conflicts of Interest The authors indicated no potential conflicts of interest. NIH Public Access Author Manuscript J Clin Oncol. Author manuscript available in PMC 2006 June 12. Published in final edited form as: J Clin Oncol. 2005 April 1 23(10): 2346���2357. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
INTRODUCTION Adoptive cell transfer (ACT) immunotherapy is based on the ex vivo selection of tumor- reactive lymphocytes, and their activation and numerical expansion before re-infusion to the autologous tumor-bearing host.1 Murine models of ACT have established the ability of this approach to mediate the regression of established cancers and have provided important principles to guide human studies.2-4In murine models, prior host immunosuppression can dramatically improve the antitumor effects of ACT therapy.5,6 Additional improvements can be achieved by the systemic administration of interleukin (IL)-2 or other cytokines to support the transferred cells, and concurrent immunization with inflammatory formulations of the tumor antigen recognized by the transferred cells.7,8 In the National Cancer Institute (NCI Bethesda, MD) Surgery Branch, we have developed improved methods for the generation of human tumor-infiltrating lymphocytes (TILs) with antitumor activity9,10 and have administered these TILs in combination with prior immunosuppression and the administration of IL-2.11 We previously reported that six (47%) of 13 patients with IL-2 refractory metastatic melanoma treated with this combination experienced an objective partial response. Two of the responding patients exhibited a transient lymphocytosis composed primarily of the transferred, tumor-reactive TIL cells. These two patients further demonstrated a clonal repopulation of their immune systems, with more than 70% of their peripheral blood lymphocytes (PBLs) consisting of a single tumor reactive clone for over four months after treatment. In the current study, we extend these results to report on the treatment of a total of 35 patients with refractory metastatic melanoma using selected, rapidly expanded TIL cultures and high- dose IL-2 therapy after nonmyeloablative but lymphodepleting chemotherapy. Eighteen (51%) of the 35 treated patients demonstrated an objective response to treatment, and eight others demonstrated a mixed or minor response. Some patients also exhibited symptoms of autoimmune melanocyte destruction including vitiligo or uveitis. The results and analysis reported here help elucidate many of the principles required for the immune-mediated destruction of established cancers. PATIENTS AND METHODS Patient Treatments and Clinical Assessment Patients older than 18 years with stage IV melanoma who were negative for hepatitis B and C and HIV infection and had a good performance status and a life expectancy of at least 3 months were eligible for treatment on this protocol. All patients signed an institutional review board- approved consent and had melanoma that was histologically confirmed by pathologists at the Clinical Center, National Institutes of Health (Bethesda, MD). All patients had assessable disease (measurable disease on computed tomography scan or by physical exam) refractory to standard treatments including high-dose IL-2 therapy (except patient 31, who did not receive IL-2 before entry into this protocol). Five (14.7%) of the 34 patients who received high-dose IL-2 initially responded to IL-2 therapy alone, but then exhibited progressive disease and were enrolled on this protocol. Granulocyte colony-stimulating factor (G-CSF)-mobilized stem cells were obtained by leukapheresis from all patients and cryopreserved before initiation of chemotherapy in the event that patients did not reconstitute their hematopoetic system. Patients received nonmyeloablative lymphodepleting chemotherapy as previously described12,13 consisting of 2 days of cyclophosphamide (60 mg/kg) followed by 5 days of fludarabine (25 mg/m2). On the day following the final dose of fludarabine, patients received cell infusion with tumor-reactive lymphocytes and high-dose IL-2 therapy consisting of 720,000 IU/kg intravenously every 8 hours to tolerance as previously described.14,15 After enrollment of 15 patients, the protocol was amended through the institutional review process so that all Dudley et al. Page 2 J Clin Oncol. Author manuscript available in PMC 2006 June 12. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
subsequent patients who had MART-1-specific16 or gp100-specific17 TIL received vaccination with 1 mg MART-1:26-35(27L) or gp100:209-217(210M) peptide in incomplete Freund���s adjuvant (IFA) injected subcutaneously. Patients��� hematologic parameters were monitored daily by obtaining complete and differential blood counts, and by flow cytometric analysis of peripheral mononuclear cells. Patient response was assessed using standard radiographic studies and physical examination at four weeks following cell administration and at regular intervals thereafter. A complete response (CR) was defined as the disappearance of all clinical evidence of disease. A partial response (PR) was defined as a 50% or greater decrease in the sum of the products of perpendicular diameters of all measurable lesions for at least one month with no increase in any lesion and no new lesions. Appearance of any new lesion or greater than 25% increase in any lesion following a PR or CR was considered a relapse. A CR or PR was considered to be an objective response, and duration was measured from the initiation of treatment to the time of relapse. Patients with mixed or minor responses or progressive disease were considered nonresponders. Patient Materials and Cell Lines TIL cultures for treatment were generated as previously described.9 This clinical trial was not designed to predict response rates on an intent-to-treat basis. Our prior analysis of sequential melanoma biopsies from HLA-A2+ patients demonstrated that tumor specific activity was detectable in TILs from 29 (81%) of the 36 patients screened.9 In brief, the method for TIL generation involved the initiation of multiple independent cultures in 24 well plates, which were maintained at 0.8-1.5 �� 106 cells/mL. When sufficient TILs were available, each culture was tested by cytokine-release assay for antigen specificity and tumor reactivity. Active TIL cultures were expanded to treatment levels using a rapid expansion protocol (REP) as previously described18,19 with OKT3(anti-CD3) antibody (Ortho Biotech, Bridgewater, NJ) and IL-2 in the presence of irradiated, allogeneic peripheral-blood mononuclear cells from at least three different donors. The rapidly expanded cultures were typically maintained for 13 to 14 days before patient infusion, and were infused into the patient by intravenous administration over 30 minutes. Immunologic Assays TIL activity and phenotype were determined by analysis of cytokine secretion, flourescence activated cell scanning (FACS), and immunocytochemistry as previously described.9,20,21 Statistical Analysis Significance of variation between groups was evaluated using a two-tailed Student t test assuming equal or unequal variance with P ��� .05 considered significant. RESULTS Patient Characteristics Thirty-five patients with assessable metastatic melanoma who had TIL cultures that recognized autologous or HLA-matched tumor cells were eligible for treatment on this protocol. The demographic characteristics of this patient population are shown in Table 1. All but two patients (patients 34 and 35) were HLA-A*0201+. All patients had progressive disease after receiving multiple prior therapies before treatment on this protocol, including surgery and (except patient 31) high-dose IL-2 therapy, and 18 of the patients had progressive disease after receiving chemotherapy. Each patient had one or more metastatic lesions resected for the generation of lymphocyte cultures and autologous tumor cell targets.9 The activity and specificity of three representative Dudley et al. Page 3 J Clin Oncol. Author manuscript available in PMC 2006 June 12. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript