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

Gene therapy of human cancer using genetically engineered lymphocytes is dependent on the identification of highly reactive T-cell receptors (TCRs) with antitumor activity. We immunized transgenic mice and also conducted high-throughput screening of human lymphocytes to generate TCRs highly reactive to melanoma/ melanocyte antigens. Genes encoding these TCRs were engineered into retroviral vectors and used to transduce autologous peripheral lymphocytes administered to 36 patients with metastatic melanoma. Transduced patient lymphocytes were CD45RA ؊ and CD45RO ؉ after ex vivo expansion. After infusion, the persisting cells displayed a CD45RA ؉ and CD45RO ؊ phenotype. Gene-engineered cells persisted at high levels in the blood of all patients 1 month after treatment, responding patients with higher ex vivo antitumor reactivity than nonresponders. Objective cancer regressions were seen in 30% and 19% of patients who received the human or mouse TCR, respectively. However, patients exhibited destruction of normal melanocytes in the skin, eye, and ear, and sometimes required local steroid administration to treat uveitis and hearing loss.

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