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New treatments are needed for B-cell malignancies persisting after allogeneic hematopoietic stem cell transplantation (alloHSCT). We conducted a clinical trial of allogeneic T cells genetically modified to express a chimeric antigen receptor (CAR) targeting the B-cell antigen CD19. T cells for genetic modification were obtained from each patient's alloHSCT donor. All patients had malignancy that persisted after alloHSCT and standard donor lymphocyte infusions (DLIs). Patients did not receive chemotherapy prior to the CAR T-cell infusions and were not lymphocyte depleted at the time of the infusions. The 10 treated patients received a single infusion of allogeneic anti-CD19-CAR T cells. Three patients had regressions of their malignancies. One patient with chronic lymphocytic leukemia (CLL) obtained an ongoing complete remission after treatment with allogeneic anti-CD19-CAR T cells, another CLL patient had tumor lysis syndrome as his leukemia dramatically regressed, and a patient with mantle cell lymphoma obtained an ongoing partial remission. None of the 10 patients developed graft-versus-host disease (GVHD). Toxicities included transient hypotension and fever. We detected cells containing the anti-CD19-CAR gene in the blood of 8 of 10 patients. These results show for the first time that donor-derived allogeneic anti-CD19-CAR T cells can cause regression of B-cell malignancies resistant to standard DLIs without causing GVHD.

CD4(+)FoxP3(+) regulatory T cells (Tregs) have been shown to suppress T cell-mediated host immune responses against self- and nonself-antigens; however, the impact of CD4(+) Tregs on human antitumor immune responses and their influence on cancer treatment are unknown. In the present study, we explored the factors that influence CD4(+) Treg reconstitution in patients receiving adoptive immunotherapy following conditioning regimens designed to enhance T-cell function and evaluated potential associations between CD4(+) Treg levels and clinical responses to therapy. The analysis of 4 trials employing nonmyeloablative chemotherapy with or without total body irradiation (TBI) before adoptive T-cell transfer revealed that the percentage and number of reconstituting CD4(+)FoxP3(+) Tregs observed in the peripheral blood was higher in nonresponders than in responders. The addition of TBI resulted in a further depletion of CD4(+) Tregs, and the degree of depletion was dependent on the TBI dose. The number of administered doses of IL-2 was found to be positively associated with peripheral Treg reconstitution. These observations provide strong evidence that endogenous CD4(+) Tregs have a negative impact on cancer therapy, and suggest that strategies reducing Treg levels may provide clinical benefit to cancer patients. All 5 clinical trials are registered at www.clinicaltrials.gov as NCT00001832, NCT00096382, NCT00335127, NCT00509496, and NCT00513604.

We conducted a clinical trial to assess adoptive transfer of T cells genetically modified to express an anti-CD19 chimeric Ag receptor (CAR). Our clinical protocol consisted of chemotherapy followed by an infusion of anti-CD19-CAR-transduced T cells and a course of IL-2. Six of the 8 patients treated on our protocol obtained remissions of their advanced, progressive B-cell malignancies. Four of the 8 patients treated on the protocol had long-term depletion of normal polyclonal CD19(+) B-lineage cells. Cells containing the anti-CD19 CAR gene were detected in the blood of all patients. Four of the 8 treated patients had prominent elevations in serum levels of the inflammatory cytokines IFNγ and TNF. The severity of acute toxicities experienced by the patients correlated with serum IFNγ and TNF levels. The infused anti-CD19-CAR-transduced T cells were a possible source of these inflammatory cytokines because we demonstrated peripheral blood T cells that produced TNF and IFNγ ex vivo in a CD19-specific manner after anti-CD19-CAR-transduced T-cell infusions. Anti-CD19-CAR-transduced T cells have great promise to improve the treatment of B-cell malignancies because of a potent ability to eradicate CD19(+) cells in vivo; however, reversible cytokine-associated toxicities occurred after CAR-transduced T-cell infusions.

Adoptive transfer of genetically modified T cells is an attractive approach for generating antitumor immune responses. We treated a patient with advanced follicular lymphoma by administering a preparative chemotherapy regimen followed by autologous T cells genetically engineered to express a chimeric antigen receptor (CAR) that recognized the B-cell antigen CD19. The patient's lymphoma underwent a dramatic regression, and B-cell precursors were selectively eliminated from the patient's bone marrow after infusion of anti-CD19-CAR-transduced T cells. Blood B cells were absent for at least 39 weeks after anti-CD19-CAR-transduced T-cell infusion despite prompt recovery of other blood cell counts. Consistent with eradication of B-lineage cells, serum immunoglobulins decreased to very low levels after treatment. The prolonged and selective elimination of B-lineage cells could not be attributed to the chemotherapy that the patient received and indicated antigen-specific eradication of B-lineage cells. Adoptive transfer of anti-CD19-CAR-expressing T cells is a promising new approach for treating B-cell malignancies. This study is registered at www.clinicaltrials.gov as #NCT00924326.

Tumor antigen-specific T cells are found within melanomas, yet tumors continue to grow. Although the tumor microenvironment is thought to influence the suppression of tumor-reactive T cells, the underlying mechanisms for this T-cell dysfunction are not clear. Here, we report that the majority of tumor infiltrating T lymphocytes (TIL), including MART-1/Melan-A melanoma antigen-specific CD8 T cells, predominantly expressed PD-1, in contrast to T cells in normal tissues and peripheral blood T lymphocytes (PBL). PD-1(+) TIL expressed CTLA-4 and Ki-67, markers that were not expressed by PD-1(-) TIL and T cells in the normal tissues and PBL. Moreover, PD-1(+) TIL were primarily HLA-DR(+) and CD127(-), in contrast to PD-1(-) TIL. Effector cytokine production by PD-1(+) TIL was impaired compared with PD-1(-) TIL and PBL. Collectively, the phenotypic and functional characterizations of TIL revealed a significantly higher frequency and level of PD-1 expression on TIL compared with normal tissue T-cell infiltrates and PBL, and PD-1 expression correlated with an exhausted phenotype and impaired effector function. These findings suggest that the tumor microenvironment can lead to up-regulation of PD-1 on tumor-reactive T cells and contribute to impaired antitumor immune responses.

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. Thus, T cells expressing highly reactive TCRs mediate cancer regression in humans and target rare cognate-antigen-containing cells throughout the body, a finding with important implications for the gene therapy of cancer. This trial was registered at www.ClinicalTrials.gov as NCI-07-C-0174 and NCI-07-C-0175.

Adoptive cell therapy (ACT) for metastatic melanoma involves the ex vivo expansion and reinfusion of tumor infiltrating lymphocytes (TIL) obtained from resected specimens. With an overall objective response rate of 56%, this T-cell immunotherapy provides an appealing alternative to other therapies, including conventional therapies with lower response rates. However, there are significant regulatory and logistical concerns associated with the ex vivo activation and large-scale expansion of these cells. The best current practice uses a rapid expansion protocol (REP) consisting of an ex vivo process that occurs in tissue culture flasks (T-flasks) and gas-permeable bags, utilizes OKT3 (anti-CD3 monoclonal antibody), recombinant human interleukin-2, and irradiated peripheral blood mononuclear cells to initiate rapid lymphocyte growth. A major limitation to the widespread delivery of therapy to large numbers of melanoma patients is the open system in which a REP is initiated. To address this problem, we have investigated the initiation, expansion and harvest at clinical scale of TIL in a closed-system continuous perfusion bioreactor. Each cell product met all safety criteria for patient treatment and by head-to-head comparison had a similar potency and phenotype as cells grown in control T-flasks and gas-permeable bags. However, the currently available bioreactor cassettes were limited in the total cell numbers that could be generated. This bioreactor may simplify the process of the rapid expansion of TIL under stringent regulatory conditions thereby enabling other institutions to pursue this form of ACT.

Objective: To describe the ophthalmic and systemic autoimmune findings after successful adoptive cell transfer of ex vivo expanded, autologous tumor-reactive tumor-infiltrating lymphocytes (TIL) for metastatic melanoma., Design: Retrospective, interventional case report., Participant: A 35-year-old man who underwent immunotherapy for metastatic melanoma with adoptive cell transfer of tumor-reactive TIL., Methods: A 35-year-old man with metastatic melanoma was treated with TIL plus interleukin-2 (IL-2) therapy after a lymphodepleting regimen of cyclophosphamide and fludarabine for metastatic melanoma, which led to a complete and durable remission. Bilateral panuveitis, hearing loss, vitiligo, poliosis, and alopecia developed in the patient, requiring local ophthalmic immunosuppressive therapy. The clinical course, diagnostic testing, and therapeutic interventions over a 2-year period are reviewed., Main Outcome Measures: Visual acuity, anterior chamber and vitreous inflammation, optical coherence tomography findings, serial electro-oculograms (EOGs), microperimetry (MP-1) testing, flow cytometric analysis of cells derived from the aqueous humor, and aqueous humor cytokine profiles were evaluated., Results: After melanoma immunotherapy, complete tumor regression was achieved at 5 months after treatment with a durable, ongoing, complete remission at 24 months. Early in the treatment course, a high fever, a diffuse rash, hearing loss, and bilateral anterior uveitis developed acutely in the patient. Late autoimmune sequelae included the development of alopecia, vitiligo, poliosis, and bilateral panuveitis with diffuse retinal pigment epithelium (RPE) hypopigmentation, reminiscent of Vogt-Koyanagi-Harada (VKH) syndrome. Bilateral cystoid macular edema also developed that was responsive to acetazolamide. Serial EOGs showed alterations in RPE standing potentials in dark conditions, and MP-1 testing revealed diminished foveal and perifoveal sensitivity. An aqueous humor aspirate revealed a high concentration of melanoma tumor antigen-reactive T cells compared with that of peripheral blood samples, as well as a proinflammatory aqueous cytokine profile. At the time of cataract surgery 22 months after immunotherapy, a repeat aqueous humor sample showed the disappearance of the previously seen melanoma differentiation antigen-reactive lymphocytes, but the proinflammatory cytokine profile persisted., Conclusions: Ocular and systemic autoimmune sequelae resembling VKH may develop after successful melanoma immunotherapy. This report provides insight into the pathogenesis of VKH disease. The patient's clinical course illustrates the fine balance between tumor-specific immunity and loss of self-tolerance., Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Adoptive cell therapy (ACT) is the best available treatment for patients with metastatic melanoma. In a recent series of three consecutive clinical trials using increasing lymphodepletion before infusion of autologous tumor infiltrating lymphocytes (TIL), objective response rates between 49% and 72% were seen. Persistence of infused cells in the circulation at one month was highly correlated with anti-tumor response as was the mean telomere length of the cells infused and the number of CD8+ CD27+ cells infused. Responses occur at all sites and appear to be durable with many patients in ongoing response beyond three years. In the most recent trial of 25 patients receiving maximum lymphodepletion, seven of the 25 patients (28%) achieved a complete response. Of the 12 patients in the three trials who achieved a complete response all but one are ongoing between 18 and 75 months. We recently demonstrated that ACT using autologous lymphocytes genetically modified to express anti-tumor T cell receptors can mediate tumor regression and this approach is now being applied to patients with common epithelial cancers.

In humans, the pathways of memory T-cell differentiation remain poorly defined. Recently, adoptive cell transfer (ACT) of tumor-reactive T lymphocytes to metastatic melanoma patients after nonmyeloablative chemotherapy has resulted in persistence of functional, tumor-reactive lymphocytes, regression of disease, and induction of melanocyte-directed autoimmunity in some responding patients. In the current study, longitudinal phenotypic analysis was performed on melanoma antigen-specific CD8+ T cells during their transition from in vitro cultured effector cells to long-term persistent memory cells following ACT to 6 responding patients. Tumor-reactive T cells used for therapy were generally late-stage effector cells with a CD27Lo CD28Lo CD45RA- CD62 ligand- (CD62L-) CC chemokine receptor 7- (CCR7-) interleukin-7 receptor alphaLo (IL-7RalphaLo) phenotype. After transfer, rapid up-regulation and continued expression of IL-7Ralpha in vivo suggested an important role for IL-7R in immediate and long-term T-cell survival. Although the tumor antigen-specific T-cell population contracted between 1 and 4 weeks after transfer, stable numbers of CD27+)CD28+ tumor-reactive T cells were maintained, demonstrating their contribution to the development of long-term, melanoma-reactive memory CD8+ T cells in vivo. At 2 months after transfer, melanoma-reactive T cells persisted at high levels and displayed an effector memory phenotype, including a CD27+ CD28+ CD62L- CCR7- profile, which may explain in part their ability to mediate tumor destruction.