RESUMO
Transfer of autologous tumor infiltrating lymphocytes (TIL) to patients with refractory melanoma has shown clinical efficacy in a number of trials. However, extending the clinical benefit to patients with other cancers poses a challenge. Inefficient costimulation in the tumor microenvironment can lead to T cell anergy and exhaustion resulting in poor anti-tumor activity. Here, we describe a chimeric costimulatory antigen receptor (CoStAR) comprised of FRα-specific scFv linked to CD28 and CD40 intracellular signaling domains. CoStAR signaling alone does not activate T cells, while the combination of TCR and CoStAR signaling enhances T cell activity resulting in less differentiated T cells, and augmentation of T cell effector functions, including cytokine secretion and cytotoxicity. CoStAR activity resulted in superior T cell proliferation, even in the absence of exogenous IL-2. Using an in vivo transplantable tumor model, CoStAR was shown to improve T cell survival after transfer, enhanced control of tumor growth, and improved host survival. CoStAR could be reliably engineered into TIL from multiple tumor indications and augmented TIL activity against autologous tumor targets both in vitro and in vivo. CoStAR thus represents a general approach to improving TIL therapy with synthetic costimulation.
Assuntos
Melanoma , Receptores de Antígenos Quiméricos , Humanos , Linfócitos T , Antígenos CD28 , Linfócitos do Interstício Tumoral , Receptor 1 de Folato , Receptores de Antígenos Quiméricos/genética , Antígenos CD40 , Microambiente TumoralRESUMO
BACKGROUND: It remains challenging to characterize the functional attributes of chimeric antigen receptor (CAR)-engineered T cell product targeting CD19 related to potency and immunotoxicity ex vivo, despite promising in vivo efficacy in patients with B cell malignancies. METHODS: We employed a single-cell, 16-plex cytokine microfluidics device and new analysis techniques to evaluate the functional profile of CD19 CAR-T cells upon antigen-specific stimulation. CAR-T cells were manufactured from human PBMCs transfected with the lentivirus encoding the CD19-BB-z transgene and expanded with anti-CD3/anti-CD28 coated beads. The enriched CAR-T cells were stimulated with anti-CAR or control IgG beads, stained with anti-CD4 RPE and anti-CD8 Alexa Fluor 647 antibodies, and incubated for 16 h in a single-cell barcode chip (SCBC). Each SCBC contains ~12,000 microchambers, covered with a glass slide that was pre-patterned with a complete copy of a 16-plex antibody array. Protein secretions from single CAR-T cells were captured and subsequently analyzed using proprietary software and new visualization methods. RESULTS: We demonstrate a new method for single-cell profiling of CD19 CAR-T pre-infusion products prepared from 4 healthy donors. CAR-T single cells exhibited a marked heterogeneity of cytokine secretions and polyfunctional (2+ cytokine) subsets specific to anti-CAR bead stimulation. The breadth of responses includes anti-tumor effector (Granzyme B, IFN-γ, MIP-1α, TNF-α), stimulatory (GM-CSF, IL-2, IL-8), regulatory (IL-4, IL-13, IL-22), and inflammatory (IL-6, IL-17A) functions. Furthermore, we developed two new bioinformatics tools for more effective polyfunctional subset visualization and comparison between donors. CONCLUSIONS: Single-cell, multiplexed, proteomic profiling of CD19 CAR-T product reveals a diverse landscape of immune effector response of CD19 CAR-T cells to antigen-specific challenge, providing a new platform for capturing CAR-T product data for correlative analysis. Additionally, such high dimensional data requires new visualization methods to further define precise polyfunctional response differences in these products. The presented biomarker capture and analysis system provides a more sensitive and comprehensive functional assessment of CAR-T pre-infusion products and may provide insights into the safety and efficacy of CAR-T cell therapy.
Assuntos
Antígenos CD19/imunologia , Citocinas/imunologia , Feminino , Humanos , Masculino , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
PURPOSE: This pilot feasibility clinical trial evaluated the coadministration of vemurafenib, a small-molecule antagonist of BRAFV600 mutations, and tumor-infiltrating lymphocytes (TIL) for the treatment of metastatic melanoma. EXPERIMENTAL DESIGN: A metastatic tumor was resected for growth of TILs, and patients were treated with vemurafenib for 2 weeks, followed by resection of a second lesion. Patients then received a nonmyeloablative preconditioning regimen, infusion of autologous TILs, and high-dose interleukin-2 administration. Vemurafenib was restarted at the time of TIL infusion and was continued for 2 years or until disease progression. Clinical responses were evaluated by Response Evaluation Criteria in Solid Tumors (RECIST) 1.0. Metastases resected prior to and after 2 weeks of vemurafenib were compared using TCRB deep sequencing, immunohistochemistry, proliferation, and recognition of autologous tumor. RESULTS: The treatment was well tolerated and had a safety profile similar to that of TIL or vemurafenib alone. Seven of 11 patients (64%) experienced an objective clinical response, and 2 patients (18%) had a complete response for 3 years (one response is ongoing at 46 months). Proliferation and viability of infusion bag TILs and peripheral blood T cells were inhibited in vitro by research-grade vemurafenib (PLX4032) when approaching the maximum serum concentration of vemurafenib. TCRB repertoire (clonotypes numbers, clonality, and frequency) did not significantly change between pre- and post-vemurafenib lesions. Recognition of autologous tumor by T cells was similar between TILs grown from pre- and post-vemurafenib metastases. CONCLUSIONS: Coadministration of vemurafenib and TILs was safe and feasible and generated objective clinical responses in this small pilot clinical trial. Clin Cancer Res; 23(2); 351-62. ©2016 AACRSee related commentary by Cogdill et al., p. 327.
Assuntos
Indóis/administração & dosagem , Interleucina-2/administração & dosagem , Melanoma/tratamento farmacológico , Proteínas Proto-Oncogênicas B-raf/genética , Sulfonamidas/administração & dosagem , Adolescente , Adulto , Idoso , Proliferação de Células/efeitos dos fármacos , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Indóis/efeitos adversos , Linfócitos do Interstício Tumoral/patologia , Linfócitos do Interstício Tumoral/transplante , Masculino , Melanoma/genética , Melanoma/patologia , Pessoa de Meia-Idade , Mutação , Metástase Neoplásica , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Sulfonamidas/efeitos adversos , Linfócitos T/patologia , Linfócitos T/transplante , VemurafenibRESUMO
PURPOSE: Adoptive cell transfer, the infusion of large numbers of activated autologous lymphocytes, can mediate objective tumor regression in a majority of patients with metastatic melanoma (52 of 93; 56%). Addition and intensification of total body irradiation (TBI) to the preparative lymphodepleting chemotherapy regimen in sequential trials improved objective partial and complete response (CR) rates. Here, we evaluated the importance of adding TBI to the adoptive transfer of tumor-infiltrating lymphocytes (TIL) in a randomized fashion. PATIENTS AND METHODS: A total of 101 patients with metastatic melanoma, including 76 patients with M1c disease, were randomly assigned to receive nonmyeloablative chemotherapy with or without 1,200 cGy TBI before transfer of tumor-infiltrating lymphcytes. Primary end points were CR rate (as defined by Response Evaluation Criteria in Solid Tumors v1.0) and overall survival (OS). Clinical and laboratory data were analyzed for correlates of response. RESULTS: CR rates were 24% in both groups (12 of 50 v 12 of 51), and OS was also similar (median OS, 38.2 v 36.6 months; hazard ratio, 1.11; 95% CI, 0.65 to 1.91; P = .71). Thrombotic microangiopathy was an adverse event unique to the TBI arm and occurred in 13 of 48 treated patients. With a median potential follow-up of 40.9 months, only one of 24 patients who achieved a CR recurred. CONCLUSION: Adoptive cell transfer can mediate durable complete regressions in 24% of patients with metastatic melanoma, with median survival > 3 years. Results were similar using chemotherapy preparative regimens with or without addition of TBI.
Assuntos
Imunoterapia Adotiva , Depleção Linfocítica , Linfócitos do Interstício Tumoral/imunologia , Melanoma/terapia , Adolescente , Adulto , Idoso , Feminino , Humanos , Masculino , Melanoma/imunologia , Melanoma/mortalidade , Pessoa de Meia-Idade , Metástase Neoplásica , Estudos Prospectivos , Irradiação Corporal TotalRESUMO
PURPOSE: Metastatic cervical cancer is a prototypical chemotherapy-refractory epithelial malignancy for which better treatments are needed. Adoptive T-cell therapy (ACT) is emerging as a promising cancer treatment, but its study in epithelial malignancies has been limited. This study was conducted to determine if ACT could mediate regression of metastatic cervical cancer. PATIENTS AND METHODS: Patients enrolled onto this protocol were diagnosed with metastatic cervical cancer and had previously received platinum-based chemotherapy or chemoradiotherapy. Patients were treated with a single infusion of tumor-infiltrating T cells selected when possible for human papillomavirus (HPV) E6 and E7 reactivity (HPV-TILs). Cell infusion was preceded by lymphocyte-depleting chemotherapy and was followed by administration of aldesleukin. RESULTS: Three of nine patients experienced objective tumor responses (two complete responses and one partial response). The two complete responses were ongoing 22 and 15 months after treatment, respectively. One partial response was 3 months in duration. The HPV reactivity of T cells in the infusion product (as measured by interferon gamma production, enzyme-linked immunospot, and CD137 upregulation assays) correlated positively with clinical response (P = .0238 for all three assays). In addition, the frequency of HPV-reactive T cells in peripheral blood 1 month after treatment was positively associated with clinical response (P = .0238). CONCLUSION: Durable, complete regression of metastatic cervical cancer can occur after a single infusion of HPV-TILs. Exploratory studies suggest a correlation between HPV reactivity of the infusion product and clinical response. Continued investigation of this therapy is warranted.
Assuntos
Alphapapillomavirus , Linfócitos T Citotóxicos , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/terapia , Adulto , Idoso , Feminino , Humanos , Metástase Linfática , Pessoa de Meia-Idade , Indução de RemissãoRESUMO
PURPOSE: Infusion of interleukin-12 (IL12) can mediate antitumor immunity in animal models, yet its systemic administration to patients with cancer results in minimal efficacy and severe toxicity. Here, we evaluated the antitumor activity of adoptively transferred human tumor-infiltrating lymphocytes (TILs) genetically engineered to secrete single-chain IL12 selectively at the tumor site. EXPERIMENTAL DESIGN: Thirty-three patients with metastatic melanoma were treated in a cell dose-escalation trial of autologous TILs transduced with a gene encoding a single-chain IL12 driven by a nuclear factor of the activated T cells promoter (NFAT.IL12). No IL2 was administered. RESULTS: The administration of 0.001 to 0.1 × 10(9) NFAT.IL12-transduced TILs to 17 patients resulted in a single, objective response (5.9%). However, at doses between 0.3 and 3 × 10(9) cells, 10 of 16 patients (63%) exhibited objective clinical responses. The responses tended to be short, and the administered IL12-producing cells rarely persisted at 1 month. Increasing cell doses were associated with high serum levels of IL12 and IFNγ as well as clinical toxicities, including liver dysfunction, high fevers, and sporadic life-threatening hemodynamic instability. CONCLUSIONS: In this first-in-man trial, administration of TILs transduced with an inducible IL12 gene mediated tumor responses in the absence of IL2 administration using cell doses 10- to 100-fold lower than conventional TILs. However, due to toxicities, likely attributable to the secreted IL12, further refinement will be necessary before this approach can be safely used in the treatment of cancer patients.
Assuntos
Interleucina-12/genética , Linfócitos do Interstício Tumoral/fisiologia , Melanoma/terapia , Neoplasias Cutâneas/terapia , Adulto , Idoso , Células Cultivadas , Feminino , Engenharia Genética , Humanos , Imunoterapia , Interleucina-12/biossíntese , Linfócitos do Interstício Tumoral/transplante , Masculino , Melanoma/imunologia , Melanoma/secundário , Pessoa de Meia-Idade , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Ativação Transcricional , Resultado do Tratamento , Adulto JovemRESUMO
PURPOSE: Although adoptive cell therapy can be highly effective for the treatment of patients with melanoma, the application of this approach to the treatment of other solid tumors has been limited. The observation that the cancer germline (CG) antigen NY-ESO-1 is expressed in 70% to 80% and in approximately 25% of patients with synovial cell sarcoma and melanoma, respectively, prompted us to perform this first-in-man clinical trial using the adoptive transfer of autologous peripheral blood mononuclear cells that were retrovirally transduced with an NY-ESO-1-reactive T-cell receptor (TCR) to heavily pretreated patients bearing these metastatic cancers. EXPERIMENTAL DESIGN: HLA-*0201 patients with metastatic synovial cell sarcoma or melanoma refractory to standard treatments and whose cancers expressed NY-ESO-1 received autologous TCR-transduced T cells following a lymphodepleting preparative chemotherapy. Response rates using Response Evaluation Criteria in Solid Tumors (RECIST), as well as immunologic correlates of response, are presented in this report. RESULTS: Eleven of 18 patients with NY-ESO-1(+) synovial cell sarcomas (61%) and 11 of 20 patients with NY-ESO-1(+) melanomas (55%) who received autologous T cells transduced with an NY-ESO-1-reactive TCR demonstrated objective clinical responses. The estimated overall 3- and 5-year survival rates for patients with synovial cell sarcoma were 38% and 14%, respectively, whereas the corresponding estimated survival rates for patients with melanoma were both 33%. CONCLUSIONS: The adoptive transfer of autologous T cells transduced with a retrovirus encoding a TCR against an HLA-A*0201 restricted NY-ESO-1 epitope can be an effective therapy for some patients bearing synovial cell sarcomas and melanomas that are refractory to other treatments.
Assuntos
Antígenos de Neoplasias/imunologia , Proteínas de Membrana/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Adulto , Idoso , Antígenos de Neoplasias/genética , Epitopos de Linfócito T/imunologia , Feminino , Seguimentos , Antígeno HLA-A2/imunologia , Humanos , Imunoterapia Adotiva , Masculino , Melanoma/diagnóstico , Melanoma/genética , Melanoma/imunologia , Melanoma/metabolismo , Melanoma/mortalidade , Melanoma/terapia , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Metástase Neoplásica , Razão de Chances , Fenótipo , Projetos Piloto , Sarcoma Sinovial/diagnóstico , Sarcoma Sinovial/genética , Sarcoma Sinovial/imunologia , Sarcoma Sinovial/metabolismo , Sarcoma Sinovial/mortalidade , Sarcoma Sinovial/terapia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Tomografia Computadorizada por Raios X , Resultado do Tratamento , Adulto JovemRESUMO
PURPOSE: Adoptive transfer of autologous tumor infiltrating lymphocytes (TIL) can mediate durable cancer regression in selected patients with metastatic melanoma. However, the tumor antigens associated with these favorable responses remain unclear. We hypothesized that a clinical strategy involving the iterative adoptive transfer of selected autologous antigen-specific T-cell clones could help systematically define immunologic targets associated with successful cancer therapy, without the interpretative ambiguity of transferring polyclonal populations. Here, we evaluated the clinical efficacy of CD8(+) T-cell clones specific for the melanocyte differentiation antigens (MDA), gp100 and MART-1, respectively. EXPERIMENTAL DESIGN: We conducted two consecutive phase II clinical trials involving the adoptive transfer of highly selected autologous antigen-specific CD8(+) T-cell clones against gp100 and MART-1, respectively. Fifteen patients with HLA-A2(+) treatment-refractory metastatic melanoma received highly avid MDA-specific CD8(+) T-cell clones specific for either gp100 (n = 10) or MART-1 (n = 5) with or without intravenous interleukin-2 (IL2) after a lymphodepleting myeloablative preparative regimen. RESULTS: Of the 15 treated patients, we observed immune-mediated targeting of skin melanocytes in 11 patients (73%) and clonal engraftment in eight patients (53%) after cell transfer. There were only transient minor tumor regressions observed, but no objective tumor responses based on Response Evaluation Criteria in Solid Tumor (RECIST) criteria. CONCLUSIONS: Despite successful clonal repopulation and evidence of in vivo antigen targeting, the poor therapeutic efficacy after the adoptive transfer of autologous MDA-specific T cells raises significant concerns regarding future immunotherapy efforts targeting this class of tumor antigens.
Assuntos
Imunoterapia Adotiva , Antígenos Específicos de Melanoma/imunologia , Melanoma/imunologia , Melanoma/terapia , Linfócitos T Citotóxicos/imunologia , Adulto , Idoso , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Citotoxicidade Imunológica , Dermatite/etiologia , Feminino , Antígeno HLA-A2/imunologia , Humanos , Imunoterapia Adotiva/efeitos adversos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Antígeno MART-1/imunologia , Masculino , Melanoma/patologia , Pessoa de Meia-Idade , Especificidade do Receptor de Antígeno de Linfócitos T/genética , Linfócitos T Citotóxicos/metabolismo , Resultado do Tratamento , Carga Tumoral , Antígeno gp100 de Melanoma/imunologiaRESUMO
PURPOSE: T cells can be genetically modified to express an anti-CD19 chimeric antigen receptor (CAR). We assessed the safety and efficacy of administering autologous anti-CD19 CAR T cells to patients with advanced CD19(+) B-cell malignancies. PATIENTS AND METHODS: We treated 15 patients with advanced B-cell malignancies. Nine patients had diffuse large B-cell lymphoma (DLBCL), two had indolent lymphomas, and four had chronic lymphocytic leukemia. Patients received a conditioning chemotherapy regimen of cyclophosphamide and fludarabine followed by a single infusion of anti-CD19 CAR T cells. RESULTS: Of 15 patients, eight achieved complete remissions (CRs), four achieved partial remissions, one had stable lymphoma, and two were not evaluable for response. CRs were obtained by four of seven evaluable patients with chemotherapy-refractory DLBCL; three of these four CRs are ongoing, with durations ranging from 9 to 22 months. Acute toxicities including fever, hypotension, delirium, and other neurologic toxicities occurred in some patients after infusion of anti-CD19 CAR T cells; these toxicities resolved within 3 weeks after cell infusion. One patient died suddenly as a result of an unknown cause 16 days after cell infusion. CAR T cells were detected in the blood of patients at peak levels, ranging from nine to 777 CAR-positive T cells/µL. CONCLUSION: This is the first report to our knowledge of successful treatment of DLBCL with anti-CD19 CAR T cells. These results demonstrate the feasibility and effectiveness of treating chemotherapy-refractory B-cell malignancies with anti-CD19 CAR T cells. The numerous remissions obtained provide strong support for further development of this approach.
Assuntos
Antígenos CD19/imunologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Imunoterapia Adotiva/métodos , Linfoma de Células B/terapia , Linfoma Difuso de Grandes Células B/terapia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/transplante , Adulto , Idoso , Terapia Combinada , Ciclofosfamida/administração & dosagem , Feminino , Humanos , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/imunologia , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/imunologia , Masculino , Pessoa de Meia-Idade , Linfócitos T/imunologia , Condicionamento Pré-Transplante/métodos , Vidarabina/administração & dosagem , Vidarabina/análogos & derivadosRESUMO
PURPOSE: Cancer immunotherapy with adoptive transfer of tumor-infiltrating lymphocytes (TIL) represents an effective treatment for patients with metastatic melanoma, with the objective regressions in up to 72% of patients in three clinical trials. However, the antigen targets recognized by these effective TILs remain largely unclear. EXPERIMENTAL DESIGN: Melanoma patients 2359 and 2591 both experienced durable complete regressions of metastases ongoing beyond five years following adoptive TIL transfer. Two conventional screening approaches were carried out to identify the antigens recognized by these clinically effective TILs. In addition, a novel approach was developed in this study to identify mutated T-cell antigens by screening a tandem minigene library, which comprised nonsynonymous mutation sequences identified by whole-exome sequencing of autologous tumors. RESULTS: Screening of an autologous melanoma cDNA library using a conventional approach led to the identification of previously undescribed nonmutated targets recognized by TIL 2359 or TIL 2591. In contrast, screening of tandem minigene libraries encoding tumor-specific mutations resulted in the identification of mutated kinesin family member 2C (KIF2C) antigen as a target of TIL 2359, and mutated DNA polymerase alpha subunit B (POLA2) antigen as a target of TIL 2591. Both KIF2C and POLA2 have been found to play important roles in cell proliferation. CONCLUSIONS: These findings suggest that the minigene screening approach can facilitate the antigen repertoire analysis of tumor reactive T cells, and lead to the development of new adoptive cell therapies with purified T cells that recognize candidate-mutated antigens derived from genes essential for the carcinogenesis.
Assuntos
Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Mutação , Neoplasias/genética , Neoplasias/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Alelos , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/química , Autoantígenos/imunologia , Linhagem Celular , Progressão da Doença , Mapeamento de Epitopos , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Biblioteca Gênica , Antígenos de Histocompatibilidade/genética , Antígenos de Histocompatibilidade/imunologia , Humanos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Neoplasias/patologia , Peptídeos/química , Peptídeos/imunologia , Ligação Proteica/imunologiaRESUMO
Limited evidence exists that humans mount a mutation-specific T cell response to epithelial cancers. We used a whole-exomic-sequencing-based approach to demonstrate that tumor-infiltrating lymphocytes (TIL) from a patient with metastatic cholangiocarcinoma contained CD4+ T helper 1 (T(H)1) cells recognizing a mutation in erbb2 interacting protein (ERBB2IP) expressed by the cancer. After adoptive transfer of TIL containing about 25% mutation-specific polyfunctional T(H)1 cells, the patient achieved a decrease in target lesions with prolonged stabilization of disease. Upon disease progression, the patient was retreated with a >95% pure population of mutation-reactive T(H)1 cells and again experienced tumor regression. These results provide evidence that a CD4+ T cell response against a mutated antigen can be harnessed to mediate regression of a metastatic epithelial cancer.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Transferência Adotiva/métodos , Neoplasias dos Ductos Biliares/terapia , Ductos Biliares Intra-Hepáticos , Linfócitos T CD4-Positivos/imunologia , Colangiocarcinoma/terapia , Linfócitos do Interstício Tumoral/transplante , Células Th1/transplante , Adulto , Neoplasias dos Ductos Biliares/genética , Colangiocarcinoma/genética , Ensaios Clínicos Fase II como Assunto , Exoma , Feminino , Humanos , Mutação , Receptor ErbB-2/metabolismoRESUMO
Adoptive transfer of tumor-infiltrating lymphocytes (TILs) can mediate regression of metastatic melanoma; however, TILs are a heterogeneous population, and there are no effective markers to specifically identify and select the repertoire of tumor-reactive and mutation-specific CD8⺠lymphocytes. The lack of biomarkers limits the ability to study these cells and develop strategies to enhance clinical efficacy and extend this therapy to other malignancies. Here, we evaluated unique phenotypic traits of CD8⺠TILs and TCR ß chain (TCRß) clonotypic frequency in melanoma tumors to identify patient-specific repertoires of tumor-reactive CD8⺠lymphocytes. In all 6 tumors studied, expression of the inhibitory receptors programmed cell death 1 (PD-1; also known as CD279), lymphocyte-activation gene 3 (LAG-3; also known as CD223), and T cell immunoglobulin and mucin domain 3 (TIM-3) on CD8⺠TILs identified the autologous tumor-reactive repertoire, including mutated neoantigen-specific CD8⺠lymphocytes, whereas only a fraction of the tumor-reactive population expressed the costimulatory receptor 4-1BB (also known as CD137). TCRß deep sequencing revealed oligoclonal expansion of specific TCRß clonotypes in CD8âºPD-1⺠compared with CD8âºPD-1- TIL populations. Furthermore, the most highly expanded TCRß clonotypes in the CD8⺠and the CD8âºPD-1⺠populations recognized the autologous tumor and included clonotypes targeting mutated antigens. Thus, in addition to the well-documented negative regulatory role of PD-1 in T cells, our findings demonstrate that PD-1 expression on CD8⺠TILs also accurately identifies the repertoire of clonally expanded tumor-reactive cells and reveal a dual importance of PD-1 expression in the tumor microenvironment.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Melanoma/imunologia , Receptor de Morte Celular Programada 1/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Microambiente Tumoral/imunologia , Transferência Adotiva , Antígenos CD/genética , Antígenos CD/imunologia , Linfócitos T CD8-Positivos/patologia , Linhagem Celular Tumoral , Feminino , Receptor Celular 2 do Vírus da Hepatite A , Humanos , Masculino , Melanoma/genética , Melanoma/patologia , Melanoma/terapia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Receptor de Morte Celular Programada 1/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Microambiente Tumoral/genética , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Proteína do Gene 3 de Ativação de LinfócitosRESUMO
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.
Assuntos
Antígenos CD19 , Transfusão de Linfócitos , Linfoma de Células B/terapia , Receptores de Antígenos de Linfócitos T/biossíntese , Transplante de Células-Tronco , Linfócitos T/metabolismo , Linfócitos T/transplante , Adulto , Idoso , Aloenxertos , Feminino , Humanos , Linfoma de Células B/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes de Fusão/biossíntese , Síndrome de Lise Tumoral/etiologia , Síndrome de Lise Tumoral/terapiaRESUMO
Adoptive cell transfer of tumor-infiltrating lymphocytes (TILs) can mediate cancer regression in patients with metastatic melanoma, but whether this approach can be applied to common epithelial malignancies remains unclear. In this study, we compared the phenotype and function of TILs derived from liver and lung metastases from patients with gastrointestinal (GI) cancers (n = 14) or melanoma (n = 42). Fewer CD3(+) T cells were found to infiltrate GI compared with melanoma metastases, but the proportions of CD8(+) cells, T cell differentiation stage, and expression of costimulatory molecules were similar for both tumor types. Clinical-scale expansion up to ~50 × 10(9) T cells on average was obtained for all patients with GI cancer and melanoma. From GI tumors, however, TIL outgrowth in high-dose IL-2 yielded 22 ± 1.4% CD3(+)CD8(+) cells compared with 63 ± 2.4% from melanoma (p < 0.001). IFN-γ ELISA demonstrated MHC class I-mediated reactivity of TIL against autologous tumor in 5 of 7 GI cancer patients tested (9% of 188 distinct TIL cultures) and in 9 of 10 melanoma patients (43% of 246 distinct TIL cultures). In these assays, MHC class I-mediated up-regulation of CD137 (4-1BB) expression on CD8(+) cells suggested that 0-3% of TILs expanded from GI cancer metastases were tumor-reactive. This study implies that the main challenge to the development of TIL adoptive cell transfer for metastatic GI cancers may not be the in vitro expansion of bulk TILs, but the ability to select and enrich for tumor-reactive T cells.
Assuntos
Neoplasias Gastrointestinais/imunologia , Imunoterapia Adotiva , Linfócitos do Interstício Tumoral/imunologia , Melanoma/imunologia , Linfócitos T/imunologia , Feminino , Citometria de Fluxo , Neoplasias Gastrointestinais/patologia , Humanos , Imuno-Histoquímica , Linfócitos do Interstício Tumoral/citologia , Masculino , Melanoma/patologia , Pessoa de Meia-Idade , Metástase Neoplásica/imunologia , Metástase Neoplásica/patologia , Fenótipo , Linfócitos T/citologiaRESUMO
Adoptive cell therapy with tumor-infiltrating lymphocytes (TILs) represents an effective treatment for patients with metastatic melanoma. However, most of the Ag targets recognized by effective melanoma-reactive TILs remain elusive. In this study, patient 2369 experienced a complete response, including regressions of bulky liver tumor masses, ongoing beyond 7 y following adoptive TIL transfer. The screening of a cDNA library generated from the autologous melanoma cell line resulted in the isolation of a mutated protein phosphatase 1, regulatory (inhibitor) subunit 3B (PPP1R3B) gene product. The mutated PPP1R3B peptide represents the immunodominant epitope recognized by tumor-reactive T cells in TIL 2369. Five years following adoptive transfer, peripheral blood T lymphocytes obtained from patient 2369 recognized the mutated PPP1R3B epitope. These results demonstrate that adoptive T cell therapy targeting a tumor-specific Ag can mediate long-term survival for a patient with metastatic melanoma. This study also provides an impetus to develop personalized immunotherapy targeting tumor-specific, mutated Ags.
Assuntos
Epitopos Imunodominantes/imunologia , Imunoterapia Adotiva/métodos , Linfócitos do Interstício Tumoral/imunologia , Melanoma/imunologia , Fosfoproteínas Fosfatases/imunologia , Proteína Fosfatase 1/imunologia , Sequência de Bases , Ensaios Clínicos como Assunto , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Biblioteca Gênica , Humanos , Ativação Linfocitária/imunologia , Masculino , Melanoma/genética , Dados de Sequência Molecular , Mutação , Fosfoproteínas Fosfatases/genética , Proteína Fosfatase 1/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
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.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Imunoterapia Adotiva/métodos , Linfócitos do Interstício Tumoral/imunologia , Melanoma/terapia , Adolescente , Adulto , Idoso , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/patologia , Linfócitos T CD8-Positivos/transplante , Criança , Feminino , Humanos , Imunoterapia Adotiva/efeitos adversos , Interferon gama/imunologia , Interleucina-2/administração & dosagem , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/patologia , Linfócitos do Interstício Tumoral/transplante , Masculino , Melanoma/imunologia , Pessoa de Meia-Idade , Estudos Prospectivos , Células Tumorais Cultivadas , Adulto JovemRESUMO
Nine cancer patients were treated with adoptive cell therapy using autologous anti-MAGE-A3 T-cell receptors (TCR)-engineered T cells. Five patients experienced clinical regression of their cancers including 2 on-going responders. Beginning 1-2 days postinfusion, 3 patients (#'s 5, 7, and 8) experienced mental status changes, and 2 patients (5 and 8) lapsed into comas and subsequently died. Magnetic resonance imagining analysis of patients 5 and 8 demonstrated periventricular leukomalacia, and examination of their brains at autopsy revealed necrotizing leukoencephalopathy with extensive white matter defects associated with infiltration of CD3(+)/CD8(+) T cells. Patient 7, developed Parkinson-like symptoms, which resolved over 4 weeks and fully recovered. Immunohistochemical staining of patient and normal brain samples demonstrated rare positively staining neurons with an antibody that recognizes multiple MAGE-A family members. The TCR used in this study recognized epitopes in MAGE-A3/A9/A12. Molecular assays of human brain samples using real-time quantitative-polymerase chain reaction, Nanostring quantitation, and deep-sequencing indicated that MAGE-A12 was expressed in human brain (and possibly MAGE-A1, MAGE-A8, and MAGE-A9). This previously unrecognized expression of MAGE-A12 in human brain was possibly the initiating event of a TCR-mediated inflammatory response that resulted in neuronal cell destruction and raises caution for clinical applications targeting MAGE-A family members with highly active immunotherapies.
Assuntos
Antígenos de Neoplasias , Terapia Genética/métodos , Imunoterapia Adotiva , Melanoma/terapia , Proteínas de Neoplasias , Receptores de Antígenos de Linfócitos T , Adulto , Idoso , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/uso terapêutico , Encéfalo/metabolismo , Células Dendríticas/imunologia , Feminino , Técnicas de Transferência de Genes , Humanos , Imunoterapia Adotiva/efeitos adversos , Imunoterapia Adotiva/métodos , Imunoterapia Adotiva/mortalidade , Masculino , Melanoma/imunologia , Antígenos Específicos de Melanoma , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/uso terapêutico , RNA Mensageiro/análise , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/uso terapêutico , Vacinas de Subunidades Antigênicas/imunologia , Adulto JovemRESUMO
Adoptive cell transfer immunotherapy against melanoma is highly effective. However, this therapy has seen limited dissemination, mainly due to the complexity and costs of cell expansion protocols. Two bioreactors have recently been described that simplify and streamline the production of individualized cell therapies. Such bioreactors might increase the number of patients that get access to this promising therapeutic modality.
RESUMO
Adoptive immunotherapy using ex vivo-expanded tumor-reactive lymphocytes can mediate durable cancer regression in selected melanoma patients. Analyses of these trials have associated the in vivo engraftment ability of the transferred cells with their antitumor efficacy. Thus, there is intensive clinical interest in the prospective isolation of tumor-specific T cells that can reliably persist after transfer. Animal studies have suggested that central memory CD8(+) T cells (T(CM)) have divergent capabilities including effector differentiation to target antigen and stem cell-like self-renewal that enable long-term survival after adoptive transfer. We sought to isolate human melanoma-specific T(CM) to define their in vivo fate and function after autologous therapeutic transfer to metastatic patients. To facilitate the high-throughput identification of these rare cells from patients, we report that T(CM) have a defined stoichiometric production of interleukin-2 (IL-2) and interferon-γ (IFN-γ) mRNA after antigen stimulation. Melanoma-specific T cells screened for high relative IL-2 production had a T(CM) phenotype and superior in vitro proliferative capacity compared to cells with low IL-2 production. To investigate in vivo effector function and self-renewal capability, we allowed melanoma-specific T(CM) to undergo in vitro expansion and differentiation into lytic effector clones and then adoptively transferred them back into their hosts. These clones targeted skin melanocytes in all five patients and persisted long term and reacquired parental T(CM) attributes in four patients after transfer. These findings demonstrate the favorable engraftment fitness for human T(CM)-derived clones, but further efforts to improve their antitumor efficacy are still necessary.