Through the adoptive transfer of lymphocytes after host immunodepletion, it is possible to mediate objective cancer regression in human patients with metastatic melanoma. However, the generation of tumor-specific T cells in this mode of immunotherapy is often limiting. Here we report the ability to specifically confer tumor recognition by autologous lymphocytes from peripheral blood by using a retrovirus that encodes a T cell receptor. Adoptive transfer of these transduced cells in 15 patients resulted in durable engraftment at levels exceeding 10% of peripheral blood lymphocytes for at least 2 months after the infusion. We observed high sustained levels of circulating, engineered cells at 1 year after infusion in two patients who both demonstrated objective regression of metastatic melanoma lesions. This study suggests the therapeutic potential of genetically engineered cells for the biologic therapy of cancer.In the past two decades, fundamental advances in immunology have introduced opportunities for the development of cellular-based therapies for the treatment of cancer (1,2). After ex vivo expansion, transfer, and clonal repopulation in patients who have received lymphodepleting conditioning, autologous tumor-infiltrating lymphocytes (TILs) have been found to mediate objective cancer regression in a measurable proportion of patients with metastatic melanoma (3-5). A limitation of this approach is the requirement that patients have preexisting tumorreactive cells that can be expanded ex vivo. In addition, in many cancer patients, especially those with cancers other than melanoma, it is difficult to identify these tumor-reactive lymphocytes. To overcome this limitation, we set out to develop an approach to cancer immunotherapy based on the genetic modification of normal peripheral blood lymphocytes (PBLs).Tumor-associated antigens (TAAs) are recognized by the T cell receptor (TCR) on the T lymphocyte surface, which is composed of the TCR alpha and beta chains (6). The genes encoding the TCR that are specific for a variety of TAA have now been cloned, including the TCR-recognizing MART-1 and gp100 melanoma/melanocyte differentiation antigens, the NY-ESO-1 cancer-testis antigen that is present on many common epithelial cancers, and an epitope from the p53 molecule, which is expressed on the surface of approximately 50% of cancers of common epithelial origin (7-12). In each case, these antigens were detected by the TCR when they were presented as peptides by molecules encoded by the major histocompatibility complex protein human lymphocyte antigen (HLA)-A2. In vitro transcribed

We report here the adoptive transfer, to patients with metastatic melanoma, of highly selected tumorreactive T cells directed against overexpressed self-derived differentiation antigens after a nonmyeloablative conditioning regimen. This approach resulted in the persistent clonal repopulation of T cells in those cancer patients, with the transferred cells proliferating in vivo, displaying functional activity, and trafficking to tumor sites. This led to regression of the patients' metastatic melanoma as well as to the onset of autoimmune melanocyte destruction. This approach presents new possibilities for the treatment of patients with cancer as well as patients with human immunodeficiency virus-related acquired immunodeficiency syndrome and other infectious diseases.Immunotherapy of patients with cancer requires the in vivo generation of large numbers of highly reactive antitumor lymphocytes that are not restrained by normal tolerance mechanisms and are capable of sustaining immunity against solid tumors. Immunization of melanoma patients with cancer antigens can increase the number of circulating CD8 + cytotoxic T lymphocyte precursor cells (pCTLs), but to date this has not correlated with clinical tumor regression, suggesting a defect in function or activation of the pCTLs (1).Adoptive cell transfer therapies provide the opportunity to overcome tolerogenic mechanisms by enabling the selection and activation of highly reactive T cell subpopulations and by manipulation of the host environment into which the T cells are introduced. However, prior clinical trials, including the transfer of highly active antitumor T cell clones, failed to demonstrate engraftment and persistence of the transferred cells (2-5). Lymphodepletion can have a marked effect on the efficacy of T cell transfer therapy in murine models (6-9) and may depend on the destruction of regulatory cells, disruption of homeostatic T cell regulation, or abrogation of other normal tolerogenic mechanisms.To determine whether prior lymphodepletion might improve the persistence and function of adoptively transferred cells, 13 HLA-A2 + patients with metastatic melanoma received immunodepleting chemotherapy with cyclophosphamide and fludarabine for 7 days before the

PurposeWe 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 MethodsThirty-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/m 2 ). 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. ResultsEighteen (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. ConclusionLymphodepleting 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