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Immunotherapy has revolutionized the treatment of advanced melanoma. Because the pathways mediating resistance to immunotherapy are largely unknown, we conducted transcriptome profiling of preimmunotherapy tumor biopsies from patients with melanoma that received PD-1 blockade or adoptive cell therapy with tumor-infiltrating lymphocytes. We identified two melanoma-intrinsic, mutually exclusive gene programs, which were controlled by IFNγ and MYC, and the association with immunotherapy outcome. MYC-overexpressing melanoma cells exhibited lower IFNγ responsiveness, which was linked with JAK2 downregulation. Luciferase activity assays, under the control of JAK2 promoter, demonstrated reduced activity in MYC-overexpressing cells, which was partly reversible upon mutagenesis of a MYC E-box binding site in the JAK2 promoter. Moreover, silencing of MYC or its cofactor MAX with siRNA increased JAK2 expression and IFNγ responsiveness of melanomas, while concomitantly enhancing the effector functions of T cells coincubated with MYC-overexpressing cells. Thus, we propose that MYC plays a pivotal role in immunotherapy resistance through downregulation of JAK2.

Comparison between proteome and transcriptome profiling. Related to Figure 1.

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Immunotherapy has revolutionized the treatment of advanced melanoma. Because the pathways mediating resistance to immunotherapy are largely unknown, we conducted transcriptome profiling of preimmunotherapy tumor biopsies from patients with melanoma that received PD-1 blockade or adoptive cell therapy with tumor-infiltrating lymphocytes. We identified two melanoma-intrinsic, mutually exclusive gene programs, which were controlled by IFNγ and MYC, and the association with immunotherapy outcome. MYC-overexpressing melanoma cells exhibited lower IFNγ responsiveness, which was linked with JAK2 downregulation. Luciferase activity assays, under the control of JAK2 promoter, demonstrated reduced activity in MYC-overexpressing cells, which was partly reversible upon mutagenesis of a MYC E-box binding site in the JAK2 promoter. Moreover, silencing of MYC or its cofactor MAX with siRNA increased JAK2 expression and IFNγ responsiveness of melanomas, while concomitantly enhancing the effector functions of T cells coincubated with MYC-overexpressing cells. Thus, we propose that MYC plays a pivotal role in immunotherapy resistance through downregulation of JAK2.

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Supplementary Data from Clinical Responses in a Phase II Study Using Adoptive Transfer of Short-term Cultured Tumor Infiltration Lymphocytes in Metastatic Melanoma Patients

PDF file - 60K, the baseline characteristics and response to ipilimumab in 19 patients previously treated with TIL ACT.

PDF file - 74K, the baseline and treatment characteristics of evaluated TIL patients treated with or without ipilimumab before TIL ACT.

CCR Translation for This Article from Adoptive Transfer of Tumor-Infiltrating Lymphocytes in Patients with Metastatic Melanoma: Intent-to-Treat Analysis and Efficacy after Failure to Prior Immunotherapies

PDF file - 112K, the correlation of different variables to one another and their p values.

PDF file - 43K, the objective responses of individual patients to various types of immunotherapies.

Purpose: Adoptive cell therapy with autologous tumor-infiltrating lymphocytes (TIL) has shown promising results in metastatic melanoma patients. Although objective response rates of over 50% have been reported, disadvantages of this approach are the labor-intensive TIL production and a very high drop-out rate of enrolled patients, limiting its widespread applicability.Previous studies showed a clear correlation between short TIL culture periods and clinical response. Therefore, we used a new TIL production technique using unselected, minimally cultured, bulk TIL (Young-TIL). The use of Young-TIL is not restricted to human leukocyte antigen (HLA)-A2 patients.The purpose of this study is to explore the efficacy and toxicity of adoptively transferred Young-TIL following lympho-depleting chemotherapy in metastatic melanoma patients, refractory to interleukin-2 and chemotherapy.Experimental Design: Young-TIL cultures for 90% of the patients were successfully generated, enabling the treatment of most enrolled patients. We report here the results of 20 evaluated patients.Results: Fifty percent of the patients achieved an objective clinical response according to the Response Evaluation Criteria in Solid Tumors, including two ongoing complete remissions (20+, 4+ months) and eight partial responses (progression-free survival: 18+, 13+, 10+, 9, 6+, 4, 3+, and 3 months). All responders are currently alive. Four additional patients showed disease stabilization. Side effects were transient and manageable.Conclusion: We showed that lympho-depleting chemotherapy followed by transfer of short-term cultured TIL can mediate tumor regression in 50% of metastatic melanoma with manageable toxicity. The convincing clinical results combined with the simplification of the process may thus have a major effect on cell therapy of cancer. Clin Cancer Res; 16(9); 2646–55. ©2010 AACR.

Purpose: Adoptive cell transfer (ACT) using autologous tumor-infiltrating lymphocytes (TIL) was reported to yield objective responses in about 50% of metastatic patients with melanoma. Here, we present the intent-to-treat analysis of TIL ACT and analyze parameters predictive to response as well as the impact of other immunotherapies.Experimental Design: Eighty patients with stage IV melanoma were enrolled, of which 57 were treated with unselected/young TIL and high-dose interleukin-2 (IL-2) following nonmyeloablative lymphodepleting conditioning.Results: TIL cultures were established from 72 of 80 enrolled patients. Altogether 23 patients were withdrawn from the study mainly due to clinical deterioration during TIL preparation. The overall response rate and median survival was 29% and 9.8 months for enrolled patients and 40% and 15.2 months for treated patients. Five patients achieved complete and 18 partial remission. All complete responders are on unmaintained remission after a median follow-up of 28 months and the 3-year survival of responding patients was 78%. Multivariate analysis revealed blood lactate-dehydrogenase levels, gender, days of TIL in culture, and the total number of infused CD8+ cells as independent predictive markers for clinical outcome. Thirty-two patients received the CTLA-4-blocking antibody ipilimumab prior or post TIL infusion. Retrospective analysis revealed that nonresponders to ipilimumab or IL-2 based therapy had the same overall response rate to ACT as other patients receiving TIL. No additional toxicities to TIL therapy occurred following ipilimumab treatment.Conclusion: Adoptive transfer of TIL can yield durable and complete responses in patients with refractory melanoma, even when other immunotherapies have failed. Clin Cancer Res; 19(17); 4792–800. ©2013 AACR.

CD28-based CD19 chimaeric antigen receptor-modified (CAR-)Tcells were recently FDA-approved for adult acute lymphoblastic leukaemia (ALL). We report long-term outcome of 37 children and young adults treated with autologous CD19 CAR-T cells. The complete remission rate was 86%, of which 71% were polymerase chain reaction (PCR) minimal residual disease (MRD)-negative, 14% were MRD-negative by flow cytometry, and 14% were PCR MRD-positive. 26 patients proceeded to subsequent haematopoietic stem cell transplant (HSCT). 11 patients had a CD19-postive relapse (eight post HSCT and three without) and one had a CD19-negative relapse. All relapse events occurred within two years from cell therapy. With a median follow-up of three years, the median event-free survival (EFS) is 17 months and the median overall survival (OS) is not reached. The three-year EFS is 41% and OS is 56%. Patients with5% blasts in the bone marrow prior to lymphodepletion had an inferior EFS. All patients with a PCR MRD-positive result at day 28 had relapsed after CAR-T-cell therapy. A prior HSCT did not significantly affect outcome, but a consolidative transplant after achieving remission improved long-term results. Overall, prelymphodepletion disease burden and molecular MRD negativity following CAR-T cells are predictors of long-term outcome following CD19 CAR-T-cell therapy for ALL.

Background: Chimeric antigen receptor (CAR) T cells directed against CD19 (CART19) are effective in B-cell malignancies, but little is known about the molecular factors predicting clinical outcome of CART19 therapy. The increasingly recognized relevance of epigenetic changes in cancer immunology prompted us to determine the impact of the DNA methylation profiles of CART19 cells on the clinical course. Methods: We recruited 114 patients with B-cell malignancies, comprising 77 patients with acute lymphoblastic leukemia and 37 patients with non-Hodgkin lymphoma who were treated with CART19 cells. Using a comprehensive DNA methylation microarray, we determined the epigenomic changes that occur in the patient T cells upon transduction of the CAR vector. The effects of the identified DNA methylation sites on clinical response, cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome, event-free survival, and overall survival were assessed. All statistical tests were 2-sided. Results: We identified 984 genomic sites with differential DNA methylation between CAR-untransduced and CAR-transduced T cells before infusion into the patient. Eighteen of these distinct epigenetic loci were associated with complete response (CR), adjusting by multiple testing. Using the sites linked to CR, an epigenetic signature, referred to hereafter as the EPICART signature, was established in the initial discovery cohort (n = 79), which was associated with CR (Fisher exact test, P < .001) and enhanced event-free survival (hazard ratio [HR] = 0.36; 95% confidence interval [CI] = 0.19 to 0.70; P = .002; log-rank P = .003) and overall survival (HR = 0.45; 95% CI = 0.20 to 0.99; P = .047; log-rank P = .04;). Most important, the EPICART profile maintained its clinical course predictive value in the validation cohort (n = 35), where it was associated with CR (Fisher exact test, P < .001) and enhanced overall survival (HR = 0.31; 95% CI = 0.11 to 0.84; P = .02; log-rank P = .02). Conclusions: We show that the DNA methylation landscape of patient CART19 cells influences the efficacy of the cellular immunotherapy treatment in patients with B-cell malignancy., Supported by CERCA Programme/Generalitat de Catalunya, Health Department PERIS #SLT/002/16/00374, AGAUR-project #2017SGR1080; MCI/AEI/ERDF project #RTI2018-094049-B-I00; ERC EPIPHARM; Cellex Foundation; “la Caixa” Foundation (LCF/PR/GN18/51140001 and LCF/PR/GN18/50310007), RF-2016–02364388, Accelerator Award—Cancer Research UK/AIRC—INCAR Associazione Italiana Ricerca per la Ricerca sul Cancro (AIRC) Project 5 × 1000 no. 9962, AIRC IG 2018 id. 21724, AIRC MFAG id. 21769 and id. 20450; MIUR (Grant PRIN 2017); and RCR-2019–23669115.

Tisagenlecleucel (tisa-cel) is an anti-CD19 chimeric antigen receptor (CAR) T-cell therapy approved for patients with relapsed/refractory large B-cell lymphoma. Outcomes of patients with out-of-commercial specification (OOS) CAR T products are not well characterized. We therefore assessed 37 adult patients who underwent leukapheresis for tisa-cel therapy in a single center. In nine (24%) patients, manufactured tisa-cel was considered OOS. Three of them (33%) received tisa-cel after institutional review board approval; 2/9 (22%) did not receive tisa-cel due to disease progression; and 4/9 (44%) received academic point-of-care (POC) CAR T-cell as salvage therapy, at a median of 35 days following OOS notification. Three of those four patients achieved a complete response. In univariate analysis, risk factors for OOS were ≥ 4 prior therapies or previous bendamustine exposure. In conclusion, we report high OOS incidence of 24% in real-life setting. Forty-four percent of those patients received POC CAR T-cell as salvage therapy.

Adoptive cell transfer (ACT) using autologous tumor infiltrating lymphocytes (TILs) was previously shown to yield clinical response in metastatic melanoma patients as an advanced line. Unfortunately, there is no reliable marker for predicting who will benefit from the treatment. We analyzed TIL samples from the infusion bags used for treatment of 57 metastatic melanoma patients and compared their microRNA profiles. The discovery cohort included six responding patients and seven patients with progressive disease, as defined by RECIST1.1. High throughput analysis with NanoString nCounter demonstrated significantly higher levels of miR-34a-5p and miR-22-3p among TIL from non-responders. These results were validated in TIL infusion bag samples from an independent cohort of 44 patients, using qRT-PCR of the individual microRNAs. Using classification trees, a data-driven predictive model for response was built, based on the level of expression of these microRNAs. Patients that achieved stable disease were classified with responders, setting apart the patients with progressive disease. Moreover, the expression levels of miR-34a-5p in the infused TIL created distinct survival groups, which strongly supports its role as a potential biomarker for TIL-ACT therapy. Indeed, when tested against autologous melanoma cells, miRLow TIL cultures exhibited significantly higher cytotoxic activity than miRHigh TIL cultures, and expressed features of terminally exhausted effectors. Finally, overexpression of miR-34a-5p or miR-22-3p in TIL inhibited their cytotoxic ability in vitro. Overall, we show that a two-microRNA signature correlates with failure of TIL-ACT therapy and survival in melanoma patients.

BackgroundAdoptive transfer of tumor-infiltrating lymphocytes (TILs) or blood T cells genetically redirected by an antitumor TCR or CAR induces a strong antitumor response in a proportion of patients with cancer; however, the therapeutic efficacy is often limited by rapid decline in T cell functions. Coadministering supportive cytokines frequently provokes systemic side effects preventing their broad clinical application. We recently showed that cytokines can be anchored to the cell membrane in a functional fashion and that cytokine receptor signaling can synergize with TLR4 and CD40 signaling. Here, we aimed at augmenting T cell activation by simultaneous signaling through the cytokine receptor, toll-like receptor and TNF-type receptor using IL-18, TLR4 and CD40 as prototypes.MethodsGenes were expressed on electroporation of in vitro-transcribed mRNA in CD4+and CD8+T cells from healthy donors redirected against melanoma cells with an anti-melanotransferrin CAR and in TILs derived from melanoma patients. Functional assays included the activation of signaling pathways, expression of activation and differentiation markers, cytokine secretion and killing of melanoma target cells.ResultsTo provide IL-18 costimulation to T cells in-cis while avoiding systemic effects, we genetically anchored IL-18 to the T cell membrane, either alone (memIL-18) or fused with constitutively active (ca)TLR4 and caCD40 signaling domains arranged in tandem, creating a synthetic ‘all-in-one’ memIL-18-TLR4-CD40 receptor. MemIL-18-TLR4-CD40, but not memIL-18, triggered strong NF-κB activation in cells lacking the IL-18 receptor, attesting to functionality of the TLR-CD40 moiety. While the membrane-anchored cytokine was found to act mainly in-cis, some T cell activation in-trans was also observed. The electroporated T cells exhibited spontaneous T-bet upregulation and IFN-γ and TNF-α secretion. Melanoma-induced activation of CAR-T cells and TILs as manifested by cytokine secretion and cytolytic activity was substantially augmented by both constructs, with memIL-18-TLR4-CD40 exerting stronger effects than memIL-18 alone.ConclusionsLinking membrane anchored IL-18 with caTLR4 and caCD40 signaling in one hybrid transmembrane protein provides simultaneous activation of three T cell costimulatory pathways through one genetically engineered membrane molecule, strongly amplifying T cell functions for adoptive T cell therapy of cancer.

Adoptive transfer of tumor-infiltrating lymphocytes (TILs) or gene-modified T cells expressing antitumor TCRs or chimeric antigen receptors often yields a high rate of clinical response in several types of cancer. New approaches for enhancing the functional properties of antitumor T cells could improve the clinical outcome of these treatments. To this end, we created 3 classes of genes, each designed to operate autonomously upon expression in T cells. We recently reported on the enhancing effects of constitutively active toll-like receptor 4 (caTLR4), membrane (mem) interleukin-2, memIL-12, and memIL-15, and self-oligomerizing, constitutively active CD40 (caCD40). Here, we evaluated their combined effects on peripheral blood CD8 T cells and different antimelanoma TIL cultures following mRNA electroporation. Expression in CD8 T cells induced transient production of interferon-γ and prolonged and robust upregulation of CD25, CD69, 4-1BB, and OX40. The adjuvants enhanced cytolytic activity of TILs and production of interferon-γ and TNF-α in the presence of autologous, but not mismatched, melanoma for at least 3 days after electroporation. Expression of the 3 adjuvants in young TILs from different patients markedly increased the expression of CD25, OX40, 4-1BB, CD127, and CD28 and exhibited cooperative and, at times, synergistic effects. Furthermore, predefined mixtures of mRNA encoding these adjuvants markedly enhanced the specific antitumor response of selected TILs and killing of autologous melanoma cells by young TILs. Our findings suggest that combinations of these new genetic adjuvants can substantially improve the functional properties of antitumor T cells, offering a new tool of unique versatility in adoptive cell therapy.

BackgroundAdoptive cell therapy with T cells genetically engineered to express a chimeric antigen receptor (CAR-T) or tumor-infiltrating T lymphocytes (TIL) demonstrates impressive clinical results in patients with cancer. Lymphodepleting preconditioning prior to cell infusion is an integral part of all adoptive T cell therapies. However, to date, there is no standardization and no data comparing different non-myeloablative (NMA) regimens.MethodsIn this study, we compared NMA therapies with different doses of cyclophosphamide or total body irradiation (TBI) in combination with fludarabine and evaluated bone marrow suppression and recovery, cytokine serum levels, clinical response and adverse events.ResultsWe demonstrate that a cumulative dose of 120 mg/kg cyclophosphamide and 125 mg/m2 fludarabine (120Cy/125Flu) and 60Cy/125Flu preconditioning were equally efficient in achieving deep lymphopenia and neutropenia in patients with metastatic melanoma, whereas absolute lymphocyte counts (ALCs) and absolute neutrophil counts were significantly higher following 200 cGyTBI/75Flu-induced NMA. Thrombocytopenia was most profound in 120Cy/125Flu patients. 30Cy/75Flu-induced preconditioning in patients with acute lymphoblastic leukemia resulted in a minor ALC decrease, had no impact on platelet counts and did not yield deep neutropenia. Following cell infusion, 120Cy/125Flu patients with objective tumor response had significantly higher ALC and significant lower inflammatory indexes, such as neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR). Receiver-operating characteristics curve analysis 7 days after cell infusion was performed to determine the cut-offs, which distinguish between responding and non-responding patients in the 120Cy/125Flu cohort. NLR≤1.79 and PLR≤32.7 were associated with clinical response and overall survival. Cytokine serum levels did not associate with clinical response in patients with TIL. Patients in the 120Cy/125Flu cohort developed significantly more acute NMA-related adverse events, including thrombocytopenia, febrile neutropenia and cardiotoxicity, and stayed significantly longer in hospital compared with the 60Cy/125Flu and TBI/75Flu cohorts.ConclusionsBone marrow depletion and recovery were equally affected by 120Cy/125Flu and 60Cy/125Flu preconditioning; however, toxicity and consequently duration of hospitalization were significantly lower in the 60Cy/125Flu cohort. Patients in the 30Cy/75Flu and TBI/75Flu groups rarely developed NMA-induced adverse events; however, both regimens were not efficient in achieving deep bone marrow suppression. Among the regimens, 60Cy/125Flu preconditioning seems to achieve maximum effect with minimum toxicity.

Targeted drug therapy in melanoma patients carrying the BRAF V600E mutation provides temporary remission, often followed by relapse due to acquired drug resistance. Here we propose a functional approach to circumvent drug resistance by applying a personalized prescreening platform that maps points of vulnerability in each tumor, prior to drug treatment. This platform applies siRNAs targeting 81 apoptosis, autophagy and programmed necrosis genes in patient tumor cell cultures, identifying genes whose targeting maximizes cell killing by short-term BRAF inhibition. Melanoma tumors displayed large heterogeneity in the number and identities of soft-spots, providing different tumor-specific functional death signatures. The soft-spots were targeted by replacing functional siRNAs with small compound inhibitors for long-term treatment in combination with vemurafenib. This strategy reduced the number of drug-tolerant persister cells surviving treatment, and most importantly, the number of drug-resistant foci. Thus, prescreening melanoma tumors for soft-spots within the cell death network may enhance targeted drug therapy before resistance emerges, thereby reducing the odds of developing drug-resistant mutations, and preventing tumor relapse.

BackgroundCD19 chimeric antigen receptor T (CAR-T) cells demonstrate remarkable remission rates in pediatric and adult patients with refractory or relapsed (r/r) acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma (NHL). In 2016, we initiated a clinical trial with in-house produced CD19 CAR-T cells with a CD28 co-stimulatory domain. We analyzed, for the first time, differences in production features and phenotype between ALL and NHL patients.MethodsNon-cryopreserved CAR-T cells were produced from patients’ peripheral blood mononuclear cells within 9 to 10 days. 93 patients with r/r ALL and NHL were enrolled under the same study. CAR-T cells of ALL and NHL patients were produced simultaneously, allowing the head-to-head comparison.ResultsAll patients were heavily pretreated. Three patients dropped out from the study due to clinical deterioration (n=2) or production failure (n=1). Cells of ALL patients (n=37) expanded significantly better and contained more CAR-T cells than of NHL patients (n=53). Young age had a positive impact on the proliferation capacity. The infusion products from ALL patients contained significantly more naïve CAR-T cells and a significantly higher expression of the chemokine receptor CXCR3. PD-1, LAG-3, TIM-3, and CD28 were equally expressed. 100% of ALL patients and 94% of NHL patients received the target dose of 1×10e6 CAR-T/kg. The overall response rate was 84% (30/36) in ALL and 62% (32/52) in NHL. We further compared CAR-T cell infusion products to tumor infiltrating lymphocytes (TIL), another common type of T cell therapy, mainly clinically effective in solid tumors. CAR-T cells contained significantly more naïve T cells and central memory T cells and significantly less CCR5 compared to TIL infusion products.ConclusionsThe in-house production of CAR-T cells is highly efficient and fast. Clinical response rate is high. CAR-T cells can be successfully produced for 99% of patients in just 9 to 10 days. Cells derived from ALL patients demonstrate a higher proliferation rate and contain higher frequencies of CAR-T cells and naïve T cells than of NHL patients. In addition, understanding the differences between CAR-T and TIL infusion products, may provide an angle to develop CAR-T cells for the treatment of solid tumors in the future.Trial registration numberClinicalTrials.gov; CAR-T:NCT02772198, First posted: May 13, 2016; TIL:NCT00287131, First posted: February 6, 2006.

Adoptive cell transfer (ACT) using autologous tumor infiltrating lymphocytes (TILs) was previously shown to yield clinical response in metastatic melanoma patients as an advanced line. Unfortunately, there is no reliable marker for predicting who will benefit from the treatment. We analyzed TIL samples from the infusion bags used for treatment of 57 metastatic melanoma patients and compared their microRNA profiles. The discovery cohort included six responding patients and seven patients with progressive disease, as defined by RECIST1.1. High throughput analysis with NanoString nCounter demonstrated significantly higher levels of miR-34a-5p and miR-22-3p among TIL from non-responders. These results were validated in TIL infusion bag samples from an independent cohort of 44 patients, using qRT-PCR of the individual microRNAs. Using classification trees, a data-driven predictive model for response was built, based on the level of expression of these microRNAs. Patients that achieved stable disease were classified with responders, setting apart the patients with progressive disease. Moreover, the expression levels of miR-34a-5p in the infused TIL created distinct survival groups, which strongly supports its role as a potential biomarker for TIL-ACT therapy. Indeed, when tested against autologous melanoma cells, miR

Omics analyses often result in dozens to hundreds of potential targets, requiring validation for their biological relevance. Current high-throughput functional investigation methods are frequently labor-intensive, expensive, and display low reproducibility. The Immune Co-Culture Cell Microarray (ICCM) is a formalin-fixed paraffin-embedded cell block microarray based on co-cultures of patient-derived tumor-infiltrating lymphocytes and their autologous melanoma cells. Each ICCM slide represents the same experiment and can be stained using standard immunohistochemistry and immunofluorescence techniques. Functional dynamics assessment of both proteins and microRNAs using ICCM stained slides demonstrated similar findings to flow cytometry assays and to previously published patient-derived biopsy reports.