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Cancer remains among the most common causes of death globally, and more effective therapies are urgently needed. Personalized biotherapies customized to individual patients, including adoptive therapies such as chimeric antigen receptor (CAR) T cells, have shown significant clinical benefit even in heavily pretreated patients. However, the commercial manufacture of these cellular therapies at centralized facilities requires the transport of cryopreserved product under complex logistic conditions that increase cost, carbon footprint, and the vein-to-vein time (i.e., time from apheresis to infusion of adoptive cells). An alternate paradigm is to produce these cellular therapies locally at the academic centers where patients are treated. In this editorial, we highlight the key advantages of such point-of-care manufacture of cell therapies, which include a significantly reduced cost and a shorter time to final product, and summarize the technological, safety, and regulatory challenges of this approach.

Background The extracellular matrix (ECM) is a major component of tumors. Several recent findings link ECM composition with outcome in cancer patients treated with immune checkpoint inhibitors (ICIs). The aim here was to explore circulating ECM- and tissue-derived metabolites to enable clustering of patients with metastatic melanoma (MM) into putative endotypes and evaluate patient characteristics and outcome when treated with Ipilimumab accordingly. Methods Serum was collected from MM patients prior to treatment with Ipilimumab (n = 64). Wards hierarchical clustering of patients was based on 15 ECM- and tissue-derived metabolites measured in serum by ELISA. Identified clusters (endotypes) were compared to clinical characteristics and evaluated for associations with disease control rate (DCR), progression free survival (PFS) and overall survival (OS). Results Three putative endotypes (cluster A, B, C) were identified including 14, 30 and 20 patients, respectively. Overall a stepwise increase in ECM metabolite median levels were detected from C-B-A with the largest absolute change seen from B-A. There was no difference between A, B, and C according to age, gender, lactate dehydrogenase levels, number of metastasized organs and whether patients were treated previously. At follow-up, the DCR was 0%, 60% and 45% in A, B, and C, respectively. Likewise, patients with endotype A had a median PFS/OS time of 69/85 days versus 174/520 and 165/589 days for endotype B and C. In support, endotype A predicted for poor survival outcomes (PFSAvsB+C:HR=3.9, 95%CI:2.0-7.6, p = 0.0001; OSAvsB+C:HR:2.5, 95%CI:1.2-4.9, p = 0.0108). Conclusions Hierarchical clustering of MM patients based on 15 ECM- and tissue-derived metabolites measured in a liquid biopsy identifies 3 putative endotypes. One endotype (A) seems to reflect patients with an overall high and differentiated ECM turnover profile. These patients experience poor outcome when treated with Ipilimumab. If validated, this supports a link between ECM remodeling and outcome in cancer patients treated with ICIs. Legal entity responsible for the study Nordic Bioscience. Funding Has not received any funding. Disclosure N. Willumsen: Full / Part-time employment: Nordic Bioscience. C.L. Bager: Full / Part-time employment: Nordic Bioscience. C. Jensen: Full / Part-time employment: Nordic Bioscience. M.A. Karsdal: Leadership role, Shareholder / Stockholder / Stock options, Full / Part-time employment: Nordic Bioscience. H. Schmidt: Advisory / Consultancy, Travel / Accommodation / Expenses: BMS; Advisory / Consultancy: Incyte; Advisory / Consultancy: Novartis; Research grant / Funding (institution): MSD; Travel / Accommodation / Expenses: Roche. I.M. Svane: Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Novartis; Honoraria (self), Advisory / Consultancy: Pierre Faber; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: MSD; Honoraria (self), Advisory / Consultancy: Incyte; Honoraria (self): Roche-Genentech; Travel / Accommodation / Expenses: Pfizer. All other authors have declared no conflicts of interest.

Background CD137 (4-1BB) is widely considered a comprehensive marker of T cell activation, and it is largely used in cancer immunology to determine the proportion of tumor-reactive T cells. However, CD137 detection does not provide specific information regarding the functions of activated T cells. Hence, in this study we tried to determine whether the simultaneous detection of CD137 plus common antitumor immune functions (type 1 functions such as production of cytokines TNFα and IFN-γ, or upregulation of CD107a) was feasible and provided advantages over using both methods separately. Methods The influence of common protein transport inhibitors used to allow cytokine detection, such as Brefeldin A (BFA) or Monensin (MN), on surface and intracellular expression of CD137 was tested in vitro. Activation of tumor infiltrating lymphocytes (TILs) was achieved with unspecific stimuli (PMA and Ionomycin) or naturally presented tumor-antigens (autologous tumor cells). TIL activation was defined as the proportion of CD4+ or CD8+ TILs staining for either CD137 (CD137+ TILs) and/or at least one of TNFα, IFNγ and CD107a (Type 1 function+ TILs) via flow cytometry. Results Our results showed that MN had minimal impact on the ability to detect surface CD137, whereas BFA had a much larger effect regardless of the stimulus employed. Nonetheless, robust results could be obtained when type 1 functions detection was combined with intracellular staining of CD137 in the presence of both BFA and MN. Using this method, we identified three tumor-antigen specific T cell subpopulations, characterized by combined expression of CD137 and type 1 functions or CD137 without type 1 functions and vice-versa. The combined method allowed the identification of a much larger proportion of tumor-reactive T cells, over either of the two methods used alone. Indeed, CD137 alone identified 46% of the total tumor-reactive CD8+ TILs, while 86% of the total tumor-reactive CD8+ TILs were positive for type 1 functions. Conclusion Combined detection of CD137 and common type 1 functions represents a more comprehensive method to identify and characterize the total repertoire of tumor-antigen specific, activated T lymphocytes, regardless of the antigen recognized. Legal entity responsible for the study Herlev and Gentofte Hospital. Funding Research grant A11806 and A12535, The Danish Cancer Society; Research grant R233-2016-3728 and R286-2018-991 Lundbeck Foundation; Clinician-Scientist Grant from the Herlev and Gentofte Hospital Research Council. Disclosure All authors have declared no conflicts of interest.

Background The majority of neoantigens arise from unique mutations which are not shared between individual patients. Thus, neoantigen-directed immunotherapy is a fully personalized treatment. Novel technical advances in next-generation sequencing and artificial intelligence (AI) allow fast and systematic prediction of cancer neoantigens for each individual patient. In this study, the proprietary AI platform PIONEERTM is used for fast and accurate identification of tumor-derived neoantigens to be included in an immunotherapy (NeoPepVac), tailored to each individual patient. NeoPepVac immunotherapy, consisting of 5-15 predicted neoantigens as synthetic peptides, is formulated with a novel cationic liposomal adjuvant (CAF09b) to strengthen CD8+ T-cell immunity towards cancer. Immune checkpoint inhibitors, targeting PD-1 or PD-L1, are administered before, during and after neoantigen-targeted immunotherapy to augment the activity of induced immune responses. Methods The study is designed as an open phase I/IIa trial. Patients with either unresectable or metastatic melanoma, non-small cell lung cancer (NSCLC) or bladder (urothelial) cancer who meet the criteria for treatment with anti-PD-1 or anti-PD-L1 are included. The NeoPepVac immunotherapy is given every second week, for a total of 6 immunizations. Blood samples are collected before, during and after NeoPepVac treatment to monitor the induction of a neoantigen-specific immune response and immune-related changes. Results Four patients have been included and treated with personalized NeoPepVac immunotherapy. So far, only CTCAE Grade 1 mild flu-like symptoms have been observed as NeoPepVac-related AEs at the first dose level (500 mg total peptide). At least 50% of the neoantigens are generating a specific T-cell response as determined by IFN-γ ELISPOT analyses of patient PBMCs. Conclusion So far the vaccine is well tolerated and safe. Neoantigen prediction and immunotherapy manufacturing is feasible within 6 weeks of patient enrolment using the PIONEERTM platform. Preliminary analyses indicate induction of NeoPepVac-specific immune responses. Clinical trial identification EudraCT: 2018-002892-16. Legal entity responsible for the study Inge Marie Svane. Funding Innovations fonden. Disclosure J.V. Kringelum: Full / Part-time employment, PhD, Director, Genomic Immuno-Oncology: Evaxion. T. Bogenrieder: Full / Part-time employment, CMO at Evaxion: Evaxion. B. Rono: Full / Part-time employment, PhD, Director, Cancer Vaccines: Evaxion. A.B. Sorensen: Full / Part-time employment, Project Manager, Personalized Immuno-oncology: Evaxion. N.V. Petersen: Full / Part-time employment: Evaxion. L.V. Andreasen: Leadership role: SSI. D. Christensen: Leadership role, Full / Part-time employment: SSI. P.L. Andersen: Leadership role, Full / Part-time employment: SSI. All other authors have declared no conflicts of interest.

Background The study of crosstalks between cancer cells and T cells in the tumour microenvironment (TME) can inform on tumour immune escape and help in predicting responses to immunotherapy. By reproducing in vitro how tumour infiltrating lymphocytes (TILs) interact with cancer cells, novel methodologies may allow a thorough characterization of the transcriptomic changes induced in tumours undergoing a T cell attack (TuTACK). Methods Thirteen pairs of highly tumour-reactive TILs and autologous cancer cell lines, covering four distinct tumour types, were selected and co-cultured to induce a cytostatic effect in tumour cultures. The global TuTACK transcriptome was characterized, and a TuTACK gene signature was extracted to be tested for prediction of response to anti-PD-1/anti-PD-L1. Results We showed that an autologous T cell attack induced large transcriptomic changes in tumours, that were independent of IFN-g signaling. Transcriptomic changes were tumour-type independent and allowed the identification of a gene-set (TuTACK gene-set) containing 256 genes covering 55 defined biological processes, that included several potential actionable adaptive immune resistance pathways. We extracted a 14-gene signature (TuTACK signature) that allowed a novel scoring of the TME. The TuTACK score correlated well to the estimated immunological activity in the TME (measured by the T cell-inflamed gene expression profile or GEP), and was predictive of response to anti-PD-1/PD-L1 therapy across 26 tumour types (Spearman R = 0.54, Pearson R = 0.57) outperforming the T cell-inflamed GEP score (Spearman R = 0.25, Pearson R = 0.34) and with a comparable predictive power to the tumour mutational burden (TMB; Spearman R = 0.65, Pearson R = 0.56). The TuTACK score was only moderately correlated to the TMB (Spearman R = 0.36; Pearson R = 0.53), indicating that these biomarkers may be independent. Conclusion TuTACK measured the effects of an immune response rather than its activity and it represents an innovative method to identify immunologically hot tumours. Our findings suggested that TuTACK may allow better patient selection in immunotherapy clinical trials and provide hints to improve the treatment of “hot” tumours resistant to current immunotherapies. Legal entity responsible for the study The authors. Funding The Danish Cancer Society (grant number R148-A9862); The Lundbeck Foundation (grant number R233-2016-3728); The Capital Region of Denmark Research Foundation (grant number R146-A5693); The National Research, Development and Innovation Fund of Hungary (FIEK_16-1-2016-0005). Disclosure All authors have declared no conflicts of interest.

Aim: Recent clinical trial results suggest programmed cell death ligand-1 (PD-L1) expression may predict response to anti-PD-1 therapy. There are conflicting data regarding PD-L1 expression as a prognostic factor among patients with melanoma treated with standard of care (SOC) therapies. Methods: We evaluated the relationship between PD-L1 expression and overall survival (OS) in 223 melanoma patients diagnosed in Denmark between 2001 and 2010. Tumor PD-L1 expression was measured using a prototype immunohistochemistry assay with the 22C3 antibody, and PD-L1 positivity was defined as staining in ≥1% of cells. Analyses of PD-L1 expression with survival outcomes were performed using Cox proportional hazards models, Kaplan-Meier methods, and the log-rank test, adjusting for age, gender, stage, and performance status. Results: Median (range) age was 59 years (19-90), and 36% of patients were female. At diagnosis, 48% had stage I-IIIA disease and 45% had stage IIIB-IV disease (6% had missing stage information). 88% of the tumor samples were collected within 6 months of diagnosis. 212 patients received surgery as initial therapy. Among the 167 patients who received chemotherapy or immunotherapy, 17% received 1 regimen, 43% received 2 regimens, and 40% received 3+ regimens. The first-line regimen, excluding adjuvant therapy, was interleukin-2 or interferon (49%), temozolomide (24%), ipilumumab (19%), vemurafenib (3%), or other (5%). 49% of patients were PD-L1 positive. There was no significant association between PD-L1 expression and OS from time of diagnosis, with an adjusted hazard ratio (AHR) of 0.71 for the PD-L1–positive group versus the PD-L1–negative group (95% confidence interval [CI], 0.42-1.21; n = 108) among stage I-IIIA patients, and 1.12 (95% CI, 0.66-1.88; n = 101) among stage IIIB-IV patients. There was also no association between PD-L1 expression and OS among ipilimumab-treated patients (n = 136), with a 1-yr survival rate of 45% (95% CI, 32%-57%) in the PD-L1–positive group and 47% (33%-60%) in the PD-L1–negative group (AHR, 0.93; 95% CI, 0.60-145). Conclusions: Our results do not suggest that PD-L1 is a strong prognostic factor among patients with melanoma treated with SOC therapies, including immunotherapy. Disclosure: T. Steiniche: Corporate-sponsored research–Merck Sharp & Dohme; Z. Wang: Full-time employment–Merck & Co., Inc.; M. Dolled-Filhart: Full-time employment–Merck & Co., Inc.; K. Emancipator: Full-time employment–Merck & Co., Inc.; R. Weiner: Full-time employment–Merck & Co., Inc.; M. Busch-Sorensen: Full-time employment–Merck & Co., Inc.; W. Zhou: Merck Research Laboratories–Full-time employment with stock ownership.; All other authors have declared no conflicts of interest.

Aim: Adoptive cell therapy (ACT) with tumor infiltrating lymphocytes (TILs) achieved impressive clinical results in several single institution phase I/II clinical trials performed outside Europe, and holds the promise to enter the mainstream of standard melanoma care in the near future. However, although transient, the toxicities associated with high-dose interleukin-2 (IL-2) classically administered together with TILs are severe and recent results have questioned its use. To further scrutinize IL-2 dosing, we have carried out a phase II trial using TILs after classical lymphodepletion but followed by an attenuated regimen of IL-2. Methods: A total of 25 patients with progressive metastatic melanoma, PS ≤1, age Results: The trial is fully recruited. Data indicate that the lower dose of IL-2 considerably decreased the toxicity of the treatment, and imaging evaluations of the first 20 patients evaluated showed two complete responses (29 + , 15+ months) and seven partial responses (28 + , 12, 19 + , 12 and 11 + , 8, 5+ months) with most responses still ongoing. Clinical responses were associated with high numbers of tumor reactive T-cells infused. Importantly, in most responding patients we observed induction and durable persistence of anti-melanoma T-cell responses in the peripheral blood. Updated results will be presented at the 2014 ESMO conference. Conclusions: As the first European institution we show that TIL-based ACT is reliable, logistically feasible to administer and clinically effective in metastatic melanoma. Importantly, a high response rate including long-lasting complete responses can be induced after treatment with TILs followed by an attenuated regimen of IL-2, which considerably reduced the occurrence of severe side effects. Effective TIL treatment is associated with induction and long-term persistence in the blood of T cells producing in vitro anticancer responses. Disclosure: All authors have declared no conflicts of interest.

During the last decade the field of cancer therapy has been reformed by the development of new and effective treatment modalities with a major focus on immune therapeutic strategies. Beside the successful immune check point antibodies different forms of adoptive cellular immunotherapies have been exploited in the past years. Among these adoptive cell therapy (ACT) based on the infusion of ex vivo expanded tumor infiltrating lymphocytes (TILs) has been the most applied and successful. TIL based ACT is a personalized treatment defined as the infusion of T cells harvested from autologous fresh autologous tumor tissues after ex vivo activation and extensive expansion. The final cell infusionproduct consist of a polyclonal T cell population comprising a highly variable amount and diversity of tumor specific T-cells. The expanded TILs are infused i.v. after one week of intensive lymphodepleting chemotherapy and followed by treatment with high i.v. doses of IL-2. TIL based ACT has yet only been tested in smaller phase I/II studies but these studies consistently confirm an impressive clinical response rate of up to 50% in metastatic melanoma - response rates able to compete with the immune check point antibodies. These impressive results justify the need for a pivotal phase III trial documenting the efficacy of this type of T-cell based Advanced Therapy Medicinal Products (ATMP) in order to pave the way for regulatory approval and implementation of TIL therapy as a new treatment standard in oncology practice. The complexity and severe acute toxicity associated with this treatment is likely to significantly limit the dissemination of TIL therapy to cancer centers outside highly specialized units. Much of the toxicity is associated with high-dose IL-2 and recent data have questioned the need for this high dose regimen. Thus, the role of IL-2 dosing requires further clarification. Other approaches might contribute to the improved efficacy of TIL therapy. These strategies could specifically aim at increasing the anti-tumor activity of T cell products, or at improving host conditioning for a better in vivo TIL survival and tumor targeting by immune sensitization of tumors. Furthermore, initiatives to extend TIL therapy to other diagnoses are ongoing. Disclosure: I.M. Svane: limited research grant has been recieved from BMS and Roche.