Gene network-based and ensemble modeling-based selection of tumor-associated antigens with a predicted low risk of tissue damage for targeted immunotherapy.

BACKGROUND: Tumor-associated antigens and their derived peptides constitute an opportunity to design off-the-shelf mainline or adjuvant anti-cancer immunotherapies for a broad array of patients. A performant and rational antigen selection pipeline would lay the foundation for immunotherapy trials with the potential to enhance treatment, tremendously benefiting patients suffering from rare, understudied cancers. METHODS: We present an experimentally validated, data-driven computational pipeline that selects and ranks antigens in a multipronged approach. In addition to minimizing the risk of immune-related adverse events by selecting antigens based on their expression profile in tumor biopsies and healthy tissues, we incorporated a network analysis-derived antigen indispensability index based on computational modeling results, and candidate immunogenicity predictions from a machine learning ensemble model relying on peptide physicochemical characteristics. RESULTS: In a model study of uveal melanoma, Human Leukocyte Antigen (HLA) docking simulations and experimental quantification of the peptide-major histocompatibility complex binding affinities confirmed that our approach discriminates between high-binding and low-binding affinity peptides with a performance similar to that of established methodologies. Blinded validation experiments with autologous T-cells yielded peptide stimulation-induced interferon-γ secretion and cytotoxic activity despite high interdonor variability. Dissecting the score contribution of the tested antigens revealed that peptides with the potential to induce cytotoxicity but unsuitable due to potential tissue damage or instability of expression were properly discarded by the computational pipeline. CONCLUSIONS: In this study, we demonstrate the feasibility of the de novo computational selection of antigens with the capacity to induce an anti-tumor immune response and a predicted low risk of tissue damage. On translation to the clinic, our pipeline supports fast turn-around validation, for example, for adoptive T-cell transfer preparations, in both generalized and personalized antigen-directed immunotherapy settings.

The future of affordable cancer immunotherapy.

The treatment of cancer was revolutionized within the last two decades by utilizing the mechanism of the immune system against malignant tissue in so-called cancer immunotherapy. Two main developments boosted cancer immunotherapy: 1) the use of checkpoint inhibitors, which are characterized by a relatively high response rate mainly in solid tumors; however, at the cost of serious side effects, and 2) the use of chimeric antigen receptor (CAR)-T cells, which were shown to be very efficient in the treatment of hematologic malignancies, but failed to show high clinical effectiveness in solid tumors until now. In addition, active immunization against individual tumors is emerging, and the first products have reached clinical approval. These new treatment options are very cost-intensive and are not financially compensated by health insurance in many countries. Hence, strategies must be developed to make cancer immunotherapy affordable and to improve the cost-benefit ratio. In this review, we discuss the following strategies: 1) to leverage the antigenicity of "cold tumors" with affordable reagents, 2) to use microbiome-based products as markers or therapeutics, 3) to apply measures that make adoptive cell therapy (ACT) cheaper, e.g., the use of off-the-shelf products, 4) to use immunotherapies that offer cheaper platforms, such as RNA- or peptide-based vaccines and vaccines that use shared or common antigens instead of highly personal antigens, 5) to use a small set of predictive biomarkers instead of the "sequence everything" approach, and 6) to explore affordable immunohistochemistry markers that may direct individual therapies.

Autologous regulatory T-cell transfer in refractory ulcerative colitis with concomitant primary sclerosing cholangitis.

OBJECTIVE: Ulcerative colitis (UC) is a chronic, debilitating immune-mediated disease driven by disturbed mucosal homeostasis, with an excess of intestinal effector T cells and an insufficient expansion of mucosal regulatory T cells (Tregs). We here report on the successful adoptive transfer of autologous, ex vivo expanded Tregs in a patient with refractory UC and associated primary sclerosing cholangitis (PSC), for which effective therapy is currently not available. DESIGN: The patient received a single infusion of 1×106 autologous, ex vivo expanded, polyclonal Tregs per kilogram of body weight, and the clinical, biochemical, endoscopic and histological responses were assessed 4 and 12 weeks after adoptive Treg transfer. RESULTS: The patient showed clinical, biochemical, endoscopic and histological signs of response until week 12 after adoptive Treg transfer, which was associated with an enrichment of intestinal CD3+/FoxP3+ and CD3+/IL-10+ T cells and increased mucosal transforming growth factor beta and amphiregulin levels. Moreover, there was marked improvement of PSC with reduction of liver enzymes. This pronounced effect lasted for 4 weeks before values started to increase again. CONCLUSION: These findings suggest that adoptive Treg therapy might be effective in refractory UC and might open new avenues for clinical trials in PSC. TRIAL REGISTRATION NUMBER: NCT04691232.

A One-Armed Phase I Dose Escalation Trial Design: Personalized Vaccination with IKKß-Matured, RNA-Loaded Dendritic Cells for Metastatic Uveal Melanoma.

Uveal melanoma (UM) is an orphan disease with a mortality of 80% within one year upon the development of metastatic disease. UM does hardly respond to chemotherapy and kinase inhibitors and is largely resistant to checkpoint inhibition. Hence, further therapy approaches are urgently needed. To improve clinical outcome, we designed a trial employing the 3rd generation personalized IKKß-matured RNA-transfected dendritic cell (DC) vaccine which primes T cells and in addition activates NK cells. This ongoing phase I trial [NCT04335890 (www.clinicaltrials.gov), Eudract: 2018-004390-28 (www.clinicaltrialsregister.eu)] investigates patients with treatment-naive metastatic UM. Monocytes are isolated by leukapheresis, differentiated to immature DCs, matured with a cytokine cocktail, and activated via the NF-κB pathway by electroporation with RNA encoding a constitutively active mutant of IKKß. Three types of antigen-RNA are co-electroporated: i) amplified mRNA of the tumor representing the whole transcriptome, ii) RNA encoding driver mutations identified by exome sequencing, and iii) overexpressed non-mutated tumor antigens detected by transcriptome sequencing. This highly personalized DC vaccine is applied by 9 intravenous infusions in a staggered schedule over one year. Parallel to the vaccination, standard therapy, usually an immune checkpoint blockade (ICB) as mono (anti-PD-1) or combined (anti-CTLA4 and anti-PD-1) regimen is initiated. The coordinated vaccine-induced immune response encompassing tumor-specific T cells and innate NK cells should synergize with ICB, perhaps resulting in measurable clinical responses in this resistant tumor entity. Primary outcome measures of this trial are safety, tolerability and toxicity; secondary outcome measures comprise overall survival and induction of antigen-specific T cells.

Assessment of sorafenib induced changes in tumor perfusion of uveal melanoma metastases with dynamic contrast-enhanced ultrasound (DCE-US).

PURPOSE: Dynamic contrast-enhanced ultrasound (DCE-US) was used to monitor early response to sorafenib therapy in patients with liver metastases from uveal melanoma. METHODS: In total, 21 patients with liver metastases were recruited within a prospective trial and underwent daily sorafenib therapy. DCE-US of a target lesion was performed before initiation of treatment, on day 15 and 56. Two independent blinded investigators performed software analysis for DCE-US parameters and inter-observer-correlation was calculated. Response to treatment was evaluated on day 56. DCE-US parameters were correlated with clinical response and RECIST1.1 criteria. RESULTS: Inter-observer-correlation (r) of DCE-US parameters [time-to-peak (TTP), mean-transit-time (MTT), peak intensity (PI), regional blood volume (RBV), regional blood flow (RBF)] at baseline, day 15, and day 56 was highly significant (r-range 0.73-0.97, all p < 0.001). Out of 17 evaluable patients, 12 patients survived day 56 (clinical responders, cRE), whereas, five patients died before day 56 and were classified as non-responders (cNR). TTP values significantly increased in the cRE group 15 days after initiation of treatment for investigator 1 (p = 0.034) and at day 56 for both investigators (p = 0.028/0.028). MTT had increased significantly in the cRE group on day 56 (p = 0.037/0.022). In the cNR group changes for TTP and MTT remained insignificant. Thus, increase of the DCE-US parameters TTP and MTT are associated with response to treatment and prognosis. CONCLUSION: An increase of TTP and MTT at frequent intervals could serve as a surrogate marker for early response evaluation to anti-angiogenic treatment of metastatic uveal melanoma.

Safety and tolerability of a single infusion of autologous ex vivo expanded regulatory T cells in adults with ulcerative colitis (ER-TREG 01): protocol of a phase 1, open-label, fast-track dose-escalation clinical trial.

INTRODUCTION: Accumulating evidence suggests that the adoptive transfer of ex vivo expanded regulatory T cells (Treg) may overcome colitogenic immune responses in patients with inflammatory bowel diseases. The objective of the ER-TREG 01 trial is to assess safety and tolerability of a single infusion of autologous ex vivo expanded Treg in adults with ulcerative colitis. METHODS AND ANALYSIS: The study is designed as a single-arm, fast-track dose-escalation trial. The study will include 10 patients with ulcerative colitis. The study intervention consists of (1) a baseline visit; (2) a second visit that includes a leukapheresis to generate the investigational medicinal product, (3) a third visit to infuse the investigational medicinal product and (4) five subsequent follow-up visits within the next 26 weeks to assess safety and tolerability. Patients will intravenously receive a single dose of 0.5×106, 1×106, 2×106, 5×106 or 10×106 autologous Treg/kg body weight. The primary objective is to define the maximum tolerable dose of a single infusion of autologous ex vivo expanded Treg. Secondary objectives include the evaluation of safety of one single infusion of autologous ex vivo expanded Treg, efficacy assessment and accompanying immunomonitoring to measure Treg function in the peripheral blood and intestinal mucosa. ETHICS AND DISSEMINATION: The study protocol was approved by the Ethics Committee of the Friedrich-Alexander University Erlangen-Nürnberg, Erlangen, Germany (number 417_19 Az). In addition, the study was approved by the Paul-Ehrlich Institute, Federal Institute for Vaccines and Biomedicines, Langen, Germany (number 3652/01). The study is funded by the German Research Foundation (DFG, KFO 257 project 08 and SFB/TransRegio 241 project C04). The trial will be conducted in compliance with this study protocol, the Declaration of Helsinki, Good Clinical Practice and Good Manufacturing Practice. The results will be published in peer-reviewed scientific journals and disseminated in scientific conferences and media. TRIAL REGISTRATION NUMBER: NCT04691232.

BRAF and MEK Inhibitors Affect Dendritic-Cell Maturation and T-Cell Stimulation.

BRAF and MEK inhibitor (BRAFi/MEKi) combinations are currently the standard treatment for patients with BRAFV600 mutant metastatic melanoma. Since the RAS/RAF/MEK/ERK-pathway is crucial for the function of different immune cells, we postulated an effect on their function and thus interference with anti-tumor immunity. Therefore, we examined the influence of BRAFi/MEKi, either as single agent or in combination, on the maturation of monocyte-derived dendritic cells (moDCs) and their interaction with T cells. DCs matured in the presence of vemurafenib or vemurafenib/cobimetinib altered their cytokine secretion and surface marker expression profile. Upon the antigen-specific stimulation of CD8+ and CD4+ T cells with these DCs or with T2.A1 cells in the presence of BRAFi/MEKi, we detected a lower expression of activation markers on and a lower cytokine secretion by these T cells. However, treatment with any of the inhibitors alone or in combination did not change the avidity of CD8+ T cells in peptide titration assays with T2.A1 cells. T-helper cell/DC interaction is a bi-directional process that normally results in DC activation. Vemurafenib and vemurafenib/cobimetinib completely abolished the helper T-cell-mediated upregulation of CD70, CD80, and CD86 but not CD25 on the DCs. The combination of dabrafenib/trametinib affected DC maturation and activation as well as T-cell activation less than combined vemurafenib/cobimetinib did. Hence, for a potential combination with immunotherapy, our data indicate the superiority of dabrafenib/trametinib treatment.

Plasma-derived extracellular vesicles discriminate type-1 allergy subjects from non-allergic controls.

BACKGROUND: Allergies are on the rise globally, with an enormous impact on affected individuals' quality of life as well as health care resources. They cause a wide range of symptoms, from slightly inconvenient to potentially fatal immune reactions. While allergies have been described and classified phenomenologically, there is an unmet need for easily accessible biomarkers to stratify the severity of clinical symptoms. Furthermore, biomarkers marking the success of specific immunotherapy are urgently needed. OBJECTIVES: Plasma extracellular vesicles (pEV) play a role in coordinating the immune response and may be useful future biomarkers. A pilot study on differences in pEV content was carried out between patients with type I allergy, suffering from rhinoconjunctivitis with or without asthma, and voluntary non-allergic donors. METHODS: We examined pEV from 38 individuals (22 patients with allergies and 16 controls) for 38 chemokines, cytokines, and soluble factors using high-throughput data mining approaches. RESULTS: Patients with allergies had a distinct biomarker pattern, with 7 upregulated (TNF-alpha, IL-4, IL-5, IL-6, IL-17F, CCL2, and CCL17) and 3 downregulated immune mediators (IL-11, IL-27, and CCL20) in pEV compared to controls. This reduced set of 10 factors was able to discriminate controls and allergic patients better than the total array. CONCLUSIONS: The content of pEV showed potential as a target for biomarker research in allergies. Plasma EV, which are readily measurable via blood test, may come to play an important role in allergy diagnosis. In this proof-of-principle study, it could be shown that pEV's discriminate patients with allergies from controls. Further studies investigating whether the content of pEVs may predict the severity of allergic symptoms or even the induction of tolerance to allergens are needed.

A Chimeric IL-15/IL-15Rα Molecule Expressed on NFκB-Activated Dendritic Cells Supports Their Capability to Activate Natural Killer Cells.

Natural killer (NK) cells, members of the innate immune system, play an important role in the rejection of HLA class I negative tumor cells. Hence, a therapeutic vaccine, which can activate NK cells in addition to cells of the adaptive immune system might induce a more comprehensive cellular response, which could lead to increased tumor elimination. Dendritic cells (DCs) are capable of activating and expanding NK cells, especially when the NFκB pathway is activated in the DCs thereby leading to the secretion of the cytokine IL-12. Another prominent NK cell activator is IL-15, which can be bound by the IL-15 receptor alpha-chain (IL-15Rα) to be transpresented to the NK cells. However, monocyte-derived DCs do neither secrete IL-15, nor express the IL-15Rα. Hence, we designed a chimeric protein consisting of IL-15 and the IL-15Rα. Upon mRNA electroporation, the fusion protein was detectable on the surface of the DCs, and increased the potential of NFκB-activated, IL-12-producing DC to activate NK cells in an autologous cell culture system with ex vivo-generated cells from healthy donors. These data show that a chimeric IL-15/IL-15Rα molecule can be expressed by monocyte-derived DCs, is trafficked to the cell surface, and is functional regarding the activation of NK cells. These data represent an initial proof-of-concept for an additional possibility of further improving cellular DC-based immunotherapies of cancer.

Cryopreservation impairs 3-D migration and cytotoxicity of natural killer cells.

Natural killer (NK) cells are important effector cells in the immune response to cancer. Clinical trials on adoptively transferred NK cells in patients with solid tumors, however, have thus far been unsuccessful. As NK cells need to pass stringent safety evaluation tests before clinical use, the cells are cryopreserved to bridge the necessary evaluation time. Standard degranulation and chromium release cytotoxicity assays confirm the ability of cryopreserved NK cells to kill target cells. Here, we report that tumor cells embedded in a 3-dimensional collagen gel, however, are killed by cryopreserved NK cells at a 5.6-fold lower rate compared to fresh NK cells. This difference is mainly caused by a 6-fold decrease in the fraction of motile NK cells after cryopreservation. These findings may explain the persistent failure of NK cell therapy in patients with solid tumors and highlight the crucial role of a 3-D environment for testing NK cell function.

Blood Eosinophilia is an on-Treatment Biomarker in Patients with Solid Tumors Undergoing Dendritic Cell Vaccination with Autologous Tumor-RNA.

BACKGROUND: The approvals of immune checkpoint inhibitors for several cancer types and the rapidly growing recognition that T cell-based immunotherapy significantly improves outcomes for cancer patients led to a re-emergence of cancer vaccines, including dendritic cell (DC)-based immunotherapy. Blood and tissue biomarkers to identify responders and long-term survivors and to optimize cost and cost-effectiveness of treatment are greatly needed. We wanted to investigate whether blood eosinophilia is a predictive biomarker for patients with solid tumors receiving vaccinations with DCs loaded with autologous tumor-RNA. METHODS: In total, 67 patients with metastatic solid tumors, who we treated with autologous monocyte-derived DCs transfected with total tumor mRNA, were serially analyzed for eosinophil counts and survival over the course of up to 14 years. Eosinophilic counts were performed on peripheral blood smears. RESULTS: Up to 87% of the patients treated with DC-based immunotherapy experienced at least once an eosinophilia of ≥ 5% after initiation of therapy; 61 % reached levels of ≥ 10% eosinophils, and 13% of patients showed eosinophil counts of 20% or above. While prevaccination eosinophil levels were not associated with survival, patients with blood eosinophilia at any point after initiation of DC-based immunotherapy showed a trend towards longer survival. There was a statistically significant difference for the patients with eosinophil counts of 20% or more (p = 0.03). In those patients, survival was prolonged to a median of 58 months (range 2-111 months), compared to a median of 20 months (range 0-119 months) in patients with lower eosinophil counts. In 12% of the patients, an immediate increase in eosinophil count of at least 10 percentage points could be detected after the first vaccine, which also appeared to correlate with survival (65 vs. 24 months; p = 0.06). CONCLUSION: Blood eosinophilia appears to be an early, on-therapy biomarker in patients with solid tumors undergoing vaccination with RNA-transfected DC, specifically autologous tumor mRNA-transfected DC vaccines, and it correlates with long-term patient outcome. Eosinophilia should be systematically investigated in future trials.

Therapeutic Cancer Vaccination with Ex Vivo RNA-Transfected Dendritic Cells-An Update.

Over the last two decades, dendritic cell (DC) vaccination has been studied extensively as active immunotherapy in cancer treatment and has been proven safe in all clinical trials both with respect to short and long-term side effects. For antigen-loading of dendritic cells (DCs) one method is to introduce mRNA coding for the desired antigens. To target the whole antigenic repertoire of a tumor, even the total tumor mRNA of a macrodissected biopsy sample can be used. To date, reports have been published on a total of 781 patients suffering from different tumor entities and HIV-infection, who have been treated with DCs loaded with mRNA. The majority of those were melanoma patients, followed by HIV-infected patients, but leukemias, brain tumors, prostate cancer, renal cell carcinomas, pancreatic cancers and several others have also been treated. Next to antigen-loading, mRNA-electroporation allows a purposeful manipulation of the DCs' phenotype and function to enhance their immunogenicity. In this review, we intend to give a comprehensive summary of what has been published regarding clinical testing of ex vivo generated mRNA-transfected DCs, with respect to safety and risk/benefit evaluations, choice of tumor antigens and RNA-source, and the design of better DCs for vaccination by transfection of mRNA-encoded functional proteins.

Merkel Cell Carcinoma of the Head and Neck Compared to Other Anatomical Sites in a Real-World Setting: Importance of Surgical Therapy for Facial Tumors.

Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine skin tumor with a high propensity for nodal involvement, local recurrence, and distant metastases. Up to 50% of MCC arises on head and neck (HN), which may impede oncological treatment due to insufficiently wide excisions and a lower rate of sentinel lymph node detection due to more complicated lymph drainage. Several studies have compared the clinical outcome of HN-MCC with those of non-head and neck (NHN) MCC yielding inconsistent results. This single-center, retrospective analysis compared the clinical outcome of 26 HN-MCC patients with 30 NHN-MCC patients. Overall survival (OS) and disease-free survival (DFS) were calculated with the Kaplan-Meier method assuming proportional hazards. The mean resection margins were 1.6 and 2.0 cm for the HN and NHN cohort, respectively. Local relapses were more frequently observed in patients with HN-MCC (19 vs. 10%). Patients with HN-MCC had a median OS of 4.3 years compared with 7.5 years in patients with NHN-MCC (p = 0.277). The median OS by tumor stage was 11, 3, 2, and 3 years in stage I, II, III, and IV disease, respectively (p = 0.009). The median DFS in HN-MCC was 10 years and not reached in the cohort with NHN-MCC patients (p = 0.939). Our data suggest a trend toward poorer outcomes of HN-MCC compared with NHN-MCC. Patients with MCC on the head and neck carry a higher risk for local relapse, requiring resolute surgical treatment also in facial localizations at early stages.

NF-κB activation triggers NK-cell stimulation by monocyte-derived dendritic cells.

BACKGROUND: In therapeutic cancer vaccination, monocyte-derived dendritic cells (moDCs) efficiently activate specific T-cell responses; however, optimizing the activation of innate immune cells could support and improve the antitumor effects. A major disadvantage of moDCs matured with the standard cytokine cocktail (consisting of IL-1ß, IL-6, TNFα, and PGE2) is their inability to secrete IL-12p70. IL-12 prominently activates natural killer (NK) cells, which are crucial in innate antitumor immunity, as they act as helper cells for the induction of a cytotoxic T lymphocyte (CTL) response and are also able to directly kill the tumor. METHODS: Previously we have shown that triggering the NF-κB pathway in moDCs by transfection of mRNA encoding constitutively active IKKß (caIKKß) led to IL-12p70 secretion and improved the dendritic cells' capability to activate and expand CTLs with a memory-like phenotype. In this study, we examined whether such dendritic cells could activate autologous NK cells. RESULTS: moDCs matured with the standard cytokine cocktail followed by transfection with the caIKKß-RNA were able to activate autologous NK cells, detected by the upregulation of CD54, CD69, and CD25 on the NK cells, their ability to secrete IFNγ, and their high lytic activity. Moreover, the ability of NK-cell activation was not diminished by simultaneous T-cell activation. CONCLUSION: The capacity of caIKKß-DCs to activate both the adaptive and innate immune response indicates an enhanced potential for clinical efficacy.

Automated Good Manufacturing Practice-compliant generation of human monocyte-derived dendritic cells from a complete apheresis product using a hollow-fiber bioreactor system overcomes a major hurdle in the manufacture of dendritic cells for cancer vaccines.

BACKGROUND: Although dendritic cell (DC)-based cancer vaccines represent a promising treatment strategy, its exploration in the clinic is hampered due to the need for Good Manufacturing Practice (GMP) facilities and associated trained staff for the generation of large numbers of DCs. The Quantum bioreactor system offered by Terumo BCT represents a hollow-fiber platform integrating GMP-compliant manufacturing steps in a closed system for automated cultivation of cellular products. In the respective established protocols, the hollow fibers are coated with fibronectin and trypsin is used to harvest the final cell product, which in the case of DCs allows processing of only one tenth of an apheresis product. MATERIALS AND RESULTS: We successfully developed a new protocol that circumvents the need for fibronectin coating and trypsin digestion, and makes the Quantum bioreactor system now suitable for generating large numbers of mature human monocyte-derived DCs (Mo-DCs) by processing a complete apheresis product at once. To achieve that, it needed a step-by-step optimization of DC-differentiation, e.g., the varying of media exchange rates and cytokine concentration until the total yield (% of input CD14+ monocytes), as well as the phenotype and functionality of mature Mo-DCs, became equivalent to those generated by our established standard production of Mo-DCs in cell culture bags. CONCLUSIONS: By using this new protocol for the Food and Drug Administration-approved Quantum system, it is now possible for the first time to process one complete apheresis to automatically generate large numbers of human Mo-DCs, making it much more feasible to exploit the potential of individualized DC-based immunotherapy.

C-reactive protein as an early marker of immune-related adverse events.

PURPOSE: Immune checkpoint inhibitors (ICIs) are effective against a wide variety of cancers. However, they also induce a plethora of unique immune-related adverse events (irAEs). Since for many organ systems symptoms can be unspecific, differential diagnosis with progression of disease or infection may be difficult. C-reactive protein (CRP) has been suggested as a marker for infection. The purpose of this study was to evaluate the diagnostic value of CRP in differentiating infectious causes from autoimmune side effects induced by ICIs. METHODS: In order to investigate the role of CRP in irAEs, we screened our patient data base. Only events with full infectious workup were included. In total 88 events of irAEs in 37 melanoma patients were analyzed. CRP levels before and during irAEs were evaluated. Statistical analyses were conducted using the Chi-square test for categorical variables. RESULTS: At the onset of irAE, CRP rose in 93% of cases to a mean of 52.7 mg/L (CI 35.1-70.3) from 8.4 mg/L at baseline (normal < 5 mg/L) (P < 0.0001). Other causes of CRP elevation including infectious diseases were excluded, and procalcitonin (PCT) levels were normal in 92% of events. Importantly, in 42% of cases CRP elevations preceded clinical symptoms. CONCLUSION: CRP elevation can predict the onset of irAEs in patients treated with ICIs in the absence of infectious disease.

Curatopes Melanoma: A Database of Predicted T-cell Epitopes from Overly Expressed Proteins in Metastatic Cutaneous Melanoma.

Therapeutic anticancer vaccination has been adapted as an immunotherapy in several solid tumors. However, the selection of promising candidates from the total quantity of possible epitopes poses a challenge to clinicians and bioinformaticians alike, and very few epitopes have been tested in experimental or clinical settings to validate their efficacy. Here, we present a comprehensive database of predicted nonmutated peptide epitopes derived from genes that are overly expressed in a group of 32 melanoma biopsies compared with healthy tissues and that were filtered against expression in a curated list of survival-critical tissues. We hypothesize that these "self-tolerant" epitopes have two desirable properties: they do not depend on mutations, being immediately applicable to a large patient collective, and they potentially cause fewer autoimmune reactions. To support epitope selection, we provide an aggregated score of expected therapeutic efficiency as a shortlist mechanism. The database has applications in facilitating epitope selection and trial design and is freely accessible at https://www.curatopes.com. SIGNIFICANCE: A database is presented that predicts and scores antitumor T-cell epitopes, with a focus on tolerability and avoidance of severe autoimmunity, offering a supplementary epitope set for further investigation in immunotherapy.

Clinical-Scale Production of CAR-T Cells for the Treatment of Melanoma Patients by mRNA Transfection of a CSPG4-Specific CAR under Full GMP Compliance.

Chimeric antigen receptor (CAR)-T cells already showed impressive clinical regressions in leukemia and lymphoma. However, the development of CAR-T cells against solid tumors lags behind. Here we present the clinical-scale production of CAR-T cells for the treatment of melanoma under full GMP compliance. In this approach a CAR, specific for chondroitin sulfate proteoglycan 4 (CSPG4) is intentionally transiently expressed by mRNA electroporation for safety reasons. The clinical-scale protocol was optimized for: (i) expansion of T cells, (ii) electroporation efficiency, (iii) viability, (iv) cryopreservation, and (v) potency. Four consistency runs resulted in CAR-T cells in clinically sufficient numbers, i.e., 2.4 × 109 CAR-expressing T cells, starting from 1.77x108 PBMCs, with an average expansion of 13.6x, an electroporation efficiency of 88.0% CAR-positive cells, a survival of 74.1% after electroporation, and a viability of 84% after cryopreservation. Purity was 98.7% CD3+ cells, with 78.1% CD3+/CD8+ T cells and with minor contaminations of 1.2% NK cells and 0.6% B cells. The resulting CAR-T cells were tested for cytolytic activity after cryopreservation and showed antigen-specific and very efficient lysis of tumor cells. Although our work is descriptive rather than investigative in nature, we expect that providing this clinically applicable protocol to generate sufficient numbers of mRNA-transfected CAR-T cells will help in moving the field of adoptive cell therapy of cancer forward.

Enhancing lentiviral transduction to generate melanoma-specific human T cells for cancer immunotherapy.

Introduction of a tumor antigen-specific T cell receptor (TCR) into patient-derived lymphocytes has already exhibited promising results for the treatment of melanoma and other malignancies in clinical trials. However, insufficient or unsuccessful ex vivo manufacturing of engineered T cells due to low expansion and/or transduction rate can still be observed in some patients. Thus, we isolated human CD8+ T cells from healthy donors and equipped them with a gp100-specific TCR using a lentiviral construct in combination with a novel chemical lentiviral transduction enhancer (Lentiboost) to increase the rate of transduced cells. Following experiments to determine the ideal multiplicity of infection (MOI) and to analyze the efficacy of the transduction enhancer using a GFP-encoding lentivirus, we analyzed in the next step the transduction rate, cell count, and functionality of gp100 TCR-transduced T cells, i.e. antigen-specific cytokine secretion and lytic capacity. In order to increase the number of transduced cells, antigen-specific stimulation was performed, either once for 1 week (1st activation) or twice for another week (2nd activation). In general, each cycle of antigen-specific stimulation resulted in expansion of TCR-positive cells, while no further significant increase of transduced cells was observed after 2nd activation. Cytokine production pattern of transduced cells after antigen encounter, however, revealed significant antigen-specific secretion of TNF and IFNγ after the 1st as well as the 2nd activation. Furthermore, TCR T cells, either activated once or twice, showed significant cytotoxicity towards antigen-positive tumor cells. Taken together, these results show that it is feasible to transduce human T cells using a lentiviral construct in combination with this novel lentiviral transduction enhancer, which shows potential in the growing field of cancer immunotherapy.

CSPG4-Specific CAR T Cells for High-Risk Childhood B Cell Precursor Leukemia.

The advent of CD19-specific chimeric antigen receptor (CAR) T cells has proven to be a powerful asset in the arsenal of cancer immunotherapy of acute lymphoblastic leukemia and certain B cell lymphomas. However, a sizable portion of patients treated with CD19-CAR T cells relapse with CD19-negative cancer cells, necessitating the quest for back-up antigens. Chondroitin sulfate proteoglycan 4 (CSPG4) expression has been reported on leukemic blasts bearing the ill-fated MLL 11q23 rearrangement. We aimed at exploring the use of CSPG4-specific CAR T cells against mixed-lineage leukemia (MLL)-rearranged leukemic blasts, using the precursor B cell leukemia cell line KOPN8 (MLL-MLLT1 translocation) as a model. First, we confirmed CSPG4 expression on KOPN8 cells. Bulk T cells electroporated with mRNA encoding a CSPG4-specific CAR upregulated activation markers and secreted the Th1 cytokines TNF and IFNγ in an antigen-specific manner upon co-culture with KOPN8 cells. More importantly, CSPG4-specific CAR T cells evinced specific degranulation towards KOPN8 cells and specifically lysed KOPN8 target cells in chromium lysis experiments. CSPG4 is a well-established CAR target in cutaneous melanoma. Here, we provide proof-of-principle data for the use of CSPG4-specific CAR T cells against MLL-translocated leukemias.