Mark E. Dudley

Purpose: Adoptive cell therapy (ACT) with autologous tumor-infiltrating lymphocytes (TILs) and high-dose interleukin-2 (IL-2) administered to lymphodepleted patients with melanoma can cause durable tumor regressions. The optimal TIL product for ACT is unknown.Patients and Methods: Patients with metastatic melanoma were prospectively assigned to receive unselected young TILs versus CD8⁺-enriched TILs. All patients received lymphodepleting chemotherapy and high-dose IL-2 therapy and were assessed for response, toxicity, survival, and immunologic end points.Results: Thirty-four patients received unselected young TILs with a median of 8.0% CD4⁺lymphocytes, and 35 patients received CD8⁺-enriched TILs with a median of 0.3% CD4⁺lymphocytes. One month after TIL infusion, patients who received CD8⁺-enriched TILs had significantly fewer CD4⁺peripheral blood lymphocytes (P = .01). Twelve patients responded to therapy with unselected young TILs (according to Response Evaluation Criteria in Solid Tumors [RECIST]), and seven patients responded to CD8⁺-enriched TILs (35% v 20%; not significant). Retrospective studies showed a significant association between response to treatment and interferon gamma secretion by the infused TILs in response to autologous tumor (P = .04), and in the subgroup of patients who received TILs from subcutaneous tumors, eight of 15 patients receiving unselected young TILs responded but none of eight patients receiving CD8⁺-enriched TILs responded.Conclusion: A randomized selection design trial was feasible for improving individualized TIL therapy. Since the evidence indicates that CD8⁺-enriched TILs are not more potent therapeutically and they are more laborious to prepare, future studies should focus on unselected young TILs.

Background: Adoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TIL) in patients with metastatic melanoma has been reported to have a 56% overall response rate with 20% complete responders. To increase the availability of this promising therapy in patients with advanced melanoma, a minimally invasive approach to procure tumor for TIL generation is warranted. Methods: A feasibility study was performed to determine the safety and efficacy of laparoscopic liver resection to generate TIL for ACT. Retrospective review of a prospectively maintained database identified 22 patients with advanced melanoma and visceral metastasis (AJCC Stage M1c) who underwent laparoscopic liver resection between 1 October 2005 and 31 July 2011. The indication for resection in all patients was to receive postoperative ACT with TIL. Results: Twenty patients (91%) underwent resection utilizing a closed laparoscopic technique, one required hand-assistance and another required conversion to open resection. Median intraoperative blood loss was 100 mL with most cases performed without a Pringle maneuver. Median hospital stay was 3 days. Three (14%) patients experienced a complication from resection with no mortality. TIL were generated from 18 of 22 (82%) patients. Twelve of 15 (80%) TIL tested were found to have in vitro tumor reactivity. Eleven patients (50%) received the intended ACT. Two patients were rendered no evidence of disease after surgical resection, with one undergoing delayed ACT with generated TIL after relapse. Objective tumor response was seen in 5 of 11 patients (45%) who received TIL, with one patient experiencing an ongoing complete response (32+ months). Conclusions: Laparoscopic liver resection can be performed with minimal morbidity and serve as an effective means to procure tumor to generate therapeutic TIL for ACT to patients with metastatic melanoma.

A patient with metastatic melanoma who had progressive disease after prior surgical resections, high dose interleukin-2, and anti-cytotoxic T lymphocyte antigen-4 antibody received sequential treatments with autologous tumor infiltrating lymphocytes that recognized the gp100 melanocyte differentiation antigen. Although no clinical response was seen when cells were administered alone, an objective clinical response to therapy was seen with tumor infiltrating lymphocytes administered together with a highly immunogenic fowlpox vaccine expressing a gp100: 209-217 (210M) epitope. Persistence of the transferred antigen-specific lymphocytes in the peripheral blood was observed only after adoptive cell therapy plus administration of vaccine. Cell proliferation in vitro was further stimulated by additional vaccine and interleukin-2. The patient has an ongoing partial response at 10 months after the last treatment.

Purpose: The two approved treatments for patients with metastatic melanoma, interleukin (IL)-2 and dacarbazine, mediate objective response rates of 12% to 15%. We previously reported that adoptive cell therapy (ACT) with autologous antitumor lymphocytes in lymphodepleted hosts mediated objective responses in 51% of 35 patients. Here, we update that study and evaluate the safety and efficacy of two increased-intensity myeloablative lymphodepleting regimens. Patients and Methods: We performed two additional sequential trials of ACT with autologous tumor-infiltrating lymphocytes (TIL) in patients with metastatic melanoma. Increasing intensity of host preparative lymphodepletion consisting of cyclophosphamide and fludarabine with either 2 (25 patients) or 12 Gy (25 patients) of total-body irradiation (TBI) was administered before cell transfer. Objective response rates by Response Evaluation Criteria in Solid Tumors (RECIST) and survival were evaluated. Immunologic correlates of effective treatment were studied. Results: Although nonmyeloablative chemotherapy alone showed an objective response rate of 49%, when 2 or 12 Gy of TBI was added, the response rates were 52% and 72% respectively. Responses were seen in all visceral sites including brain. There was one treatment-related death in the 93 patients. Host lymphodepletion was associated with increased serum levels of the lymphocyte homeostatic cytokines IL-7 and IL-15. Objective responses were correlated with the telomere length of the transferred cells. Conclusion: Host lymphodepletion followed by autologous TIL transfer and IL-2 results in objective response rates of 50% to 70% in patients with metastatic melanoma refractory to standard therapies.

Adoptive cell therapy (ACT) with tumor-reactive lymphocytes in patients with refractory melanoma can result in tumor regression and prolonged survival. Generating tumor-reactive lymphocyte cultures is technically difficult and resource intensive; these limitations have restricted the widespread application of ACT. Tumor-infiltrating lymphocytes (TIL) from melanoma contain tumor antigen-reactive cells. The “standard” method for producing TIL cultures for clinical administration requires extended in vitro expansion in interleukin-2, then identification of tumor-reactive cells by immunologic assays. We show here that limitations in reagents and methods during screening underrepresent the actual reactivity of TIL cultures. Furthermore, the extended culture times necessitated by the screening assays resulted in telomere shortening and reduced expression of CD27 and CD28 in the TIL cultures, properties that our prior studies showed are correlated with in vivo persistence and clinical response. We have thus developed an alternative “young” TIL method that demonstrated superior in vitro attributes compared with standard TIL. This approach uses the entire resected tumor to rapidly expand TIL for administration without in vitro testing for tumor recognition. Our observations suggest that younger TIL can have an undetermined but high level of antigen reactivity, and other advantageous attributes such as long telomeres and high levels of CD27 and CD28. We suggest that minimally cultured, unselected lymphocytes represent an alternative strategy for generating TIL cultures suitable for use in ACT that, if effective in vivo, may facilitate the widespread application of this approach to a broader population of patients with melanoma.

3032 Background: T cell adoptive immunotherapy can mediate objective cancer regression in patients with invasive melanoma. To extend T cell based therapies to patients with common epithelial cancers, the tumor associated antigen (TAA) p53 has been extensively studied in animal models and humans. We investigated tumor recognition in vitro and in vivo by normal lymphocytes gene modified to express an anti-p53 T cell receptor (TCR). Methods: A MSGV1 retroviral construct encoding an HLA-A2.1 restricted p53:264–272 specific α/β TCR (p53:264 TCR), derived from HLA-A2.1 transgenic mice, was used to optimize transduction (Td) of activated peripheral blood lymphocytes (PBL). A panel of well-characterized tumor cell lines with known HLA-A type and p53 gene sequences including the NCI-60 lines were used to test the recognition by p53:264 TCR Td PBL. Results: After Td optimization, expression of the p53:264 TCR was routinely achieved in > 80% of T cells. A panel of tumor cell lines including breast, colon, lung, renal, liver, ovarian, melanoma, central nervous system, and hematopoietic cancers were recognized by p53:264 TCR Td T cells irrespective of the level of p53 expression determined by FACS or western blot. Although this casts doubt on p53 as an ideal TAA based on the concept that p53 overexpression creates a therapeutic window for immune discrimination of tumor from normal cells, with few exceptions a panel of normal cells was ignored by p53:264 TCR Td PBL. We developed a method to expand p53:264 TCR Td PBL up to 1000 fold in vitro and a pilot clinical trial was conducted in metastatic cancer patients with breast, melanoma, esophageal, bile duct, small bowel, and parotid gland tumors. Eleven patients received lymphodepleting conditioning chemotherapy, p53:264 TCR Td PBL, and high dose interleukin-2, and no toxicity attributable to the Td PBL was observed. One patient achieved a partial response in all sites of disease including lung nodules and a hilar lymph node. Conclusions: Lymphocytes transduced with genes encoding anti-p53 TCR can recognize a wide array of cancer cell lines but recognition does not correlate with p53 gene expression. Adoptive immunotherapy clinical studies are underway using these gene-modified cells. No significant financial relationships to disclose.

Adoptive-cell-transfer (ACT) therapy for patients with cancer relies on the ex vivo generation of highly active, tumour-specific lymphocytes, and their administration in large numbers to the autologous host. Preclinical models have identified characteristics of lymphocyte cultures that are required for successful ACT therapy. The most important characteristic is the presence of high affinity, tumour-antigen-specific CD8⁺ T cells. There is generally a direct correlation between treatment efficacy and the number of transferred, tumour-specific cells. Preclinical models have also identified ways to manipulate the host immune environment that increase ACT therapeutic efficacy. These include immunosuppression before cell administration and concurrent interleukin 2 administration with the transferred T cells. ACT therapy directed at viral antigens has been effective for elimination of Epstein–Barr virus (EBV)-induced post-transplant lymphoproliferative disease. EBV-specific lymphocyte cultures suitable for ACT therapy were generated by repetitive in vitro stimulation using EBV-transformed lymphoblastoid lines. Lymphocyte cultures that were selected for reactivity against melanoma antigens, including melanocyte-differentiation antigens, mediated cancer regression in some patients with metastatic melanoma. Melanoma-reactive cultures that were suitable for ACT therapy were generated from tumour-infiltrating lymphocytes that were rapidly expanded with anti-CD3 antibody. The generation of tumour-antigen-specific lymphocyte cultures is evolving rapidly, spurred on by the identification of tumour antigens and the T-cell receptors that recognize them. Further improvements to ACT therapy will depend on a deeper understanding of basic immunological processes, including the role of CD4⁺ T cells in the antitumour inflammatory response, the ability of lymphocytes to persist in vivo and travel to tumours, and the mechanisms of ACT augmentation by previous host immunosuppression.

The generation of T lymphocytes with specific reactivity against tumor antigens is a prerequisite for effective adoptive transfer therapies. Melanoma-specific lymphocyte cultures can be established from tumor infiltrating lymphocytes (TILs) by in vitro culture in high levels of IL-2. We have optimized methods for generating melanoma-reactive TIL cultures from small resected tumor specimens. We report a retrospective analysis of 860 attempted TIL cultures from 90 sequential melanoma biopsy specimens from 62 HLA-A2⁺ patients. Multiple independent TIL derived from a single tumor often exhibited substantial functional and phenotypic variation. Tumor specific activity was detected in TIL from 29 (81%) of 36 patients screened. TIL cultures selected for high activity were generally capable of large numerical expansion using a single round of a rapid expansion protocol. Limited clonal T-cell populations in an oligoclonal TIL culture could confer specific tumor recognition in these highly selected, highly expanded TIL cultures. These methods were efficient at generating TILs suitable for adoptive transfer therapy.