Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) and play a crucial role in antitumor immunity. Consequently, various strategies have been explored to utilize DCs as therapeutic vaccines, leveraging their ability to target tumor cells. A variety of antigen formulations were tested to evaluate their efficacy in vaccination, including purified tumor-associated antigens, synthetic polypeptides, and mRNA encoding tumor-associated antigens or neoantigens. In this protocol, we describe the ex vivo generation of dendritic cells from peripheral blood mononuclear cells and loading with in vitro transcribed mRNA for vaccination purposes under Good and Manufacturing Practices.

Background Ovarian cancer (OC) is the leading cause of death from gynecologic malignancies in the Western world. Contributing factors include a high frequency of late-stage diagnosis, the development of chemoresistance, and the evasion of host immune responses. Currently, debulking surgery and platinum-based chemotherapy are the treatment cornerstones, although recurrence is common. As the clinical efficacy of immune checkpoint blockade is low, new immunotherapeutic strategies are needed. Chimeric antigen receptor (CAR) T cell therapy empowers patients’ own T cells to fight and eradicate cancer, and has been tested against various targets in OC. A promising candidate is the MUC16 ectodomain. This ectodomain remains on the cell surface after cleavage of cancer antigen 125 (CA125), the domain distal from the membrane, which is currently used as a serum biomarker for OC. CA125 itself has not been tested as a possible CAR target. In this study, we examined the suitability of the CA125 as a target for CAR T cell therapy.Methods We tested a series of antibodies raised against the CA125 extracellular repeat domain of MUC16 and adapted them to the CAR format. Comparisons between these candidates, and against an existing CAR targeting the MUC16 ectodomain, identified K101 as having high potency and specificity. The K101CAR was subjected to further biochemical and functional tests, including examination of the effect of soluble CA125 on its activity. Finally, we used cell lines and advanced orthotopic patient-derived xenograft (PDX) models to validate, in vivo, the efficiency of our K101CAR construct.Results We observed a high efficacy of K101CAR T cells against cell lines and patient-derived tumors, in vitro and in vivo. We also demonstrated that K101CAR functionality was not impaired by the soluble antigen. Finally, in direct comparisons, K101CAR, which targets the CA125 extracellular repeat domains, was shown to have similar efficacy to the previously validated 4H11CAR, which targets the MUC16 ectodomain.Conclusions Our in vitro and in vivo results, including PDX studies, demonstrate that the CA125 domain of MUC16 represents an excellent target for treating MUC16-positive malignancies.

We previously demonstrated the antitumor effectiveness of transiently T cell receptor (TCR)-redirected T cells recognizing a frameshift mutation in transforming growth factor beta receptor 2. We here describe a clinical protocol using mRNA TCR-modified T cells to treat a patient with progressive, treatment-resistant metastatic microsatellite instability-high (MSI-H) colorectal cancer. Following 12 escalating doses of autologous T cells electroporated with in-vitro-transcribed Radium-1 TCR mRNA, we assessed T cell cytotoxicity, phenotype, and cytokine production. Tumor markers and growth on computed tomography scans were evaluated and immune cell tumor infiltrate at diagnosis assessed. At diagnosis, tumor-infiltrating CD8+ T cells had minimal expression of exhaustion markers, except for PD-1. Injected Radium-1 T cells were mainly naive and effector memory T cells with low expression of exhaustion markers, except for TIGIT. We confirmed cytotoxicity of transfected Radium-1 T cells against target cells and found key cytokines involved in tumor metastasis, growth, and angiogenesis to fluctuate during treatment. The treatment was well tolerated, and despite his advanced cancer, the patient obtained a stable disease with 6 months survival post-treatment. We conclude that treatment of metastatic MSI-H colorectal cancer with autologous T cells electroporated with Radium-1 TCR mRNA is feasible, safe, and well tolerated and that it warrants further investigation in a phase 1/2 study.

Acute myeloid leukemia (AML) is characterized by the accumulation of immature myeloid cells in the bone marrow and the peripheral blood. Nearly half of the AML patients relapse after standard induction therapy, and new forms of therapy are urgently needed. Chimeric antigen receptor (CAR) T therapy has so far not been successful in AML due to lack of efficacy and safety. Indeed, the most attractive antigen targets are stem cell markers such as CD33 or CD123. We demonstrate that CD37, a mature B cell marker, is expressed in AML samples, and its presence correlates with the European LeukemiaNet (ELN) 2017 risk stratification. We repurpose the anti -lymphoma CD37CAR for the treatment of AML and show that CD37CAR T cells specifically kill AML cells, secrete proinflammatory cytokines, and control cancer progression in vivo . Importantly, CD37CAR T cells display no toxicity toward hematopoietic stem cells. Thus, CD37 is a promising and safe CAR T cell AML target.

Osteosarcoma (OS) remains a dismal malignancy in children and young adults, with poor outcome for metastatic and recurrent disease. Immunotherapies in OS are not as promising as in some other cancer types due to intra-tumor heterogeneity and considerable off-target expression of the potentially targetable proteins. Here we show that chimeric antigen receptor (CAR) T cells could successfully target an isoform of alkaline phosphatase, ALPL-1, which is highly and specifically expressed in primary and metastatic OS. The target recognition element of the second-generation CAR construct is based on two antibodies, previously shown to react against OS. T cells transduced with these CAR constructs mediate efficient and effective cytotoxicity against ALPL-positive cells in in vitro settings and in state-of-the-art in vivo orthotopic models of primary and metastatic OS, without unexpected toxicities against hematopoietic stem cells or healthy tissues. In summary, CAR-T cells targeting ALPL-1 show efficiency and specificity in treating OS in preclinical models, paving the path for clinical translation.

Metastatic castration-resistant prostate cancer (mCRPC) is an immunologically cold disease with dismal outcomes. Cryoablation destroys cancer tissue, releases tumor-associated antigens and creates a pro-inflammatory microenvironment, while dendritic cells (DCs) activate immune responses through processing of antigens. Immunotherapy combinations could enhance the anti-tumor efficacy. This open-label, single-arm, single-center phase I trial determined the safety and tolerability of combining cryoablation and autologous immature DC, without and with checkpoint inhibitors. Immune responses and clinical outcomes were evaluated. Patients with mCRPC, confirmed metastases and intact prostate gland were included. The first participants underwent prostate cryoablation with intratumoral injection of autologous DCs in a 3 + 3 design. In the second part, patients received cryoablation, the highest acceptable DC dose, and checkpoint inhibition with either ipilimumab or pembrolizumab. Sequentially collected information on adverse events, quality of life, blood values and images were analyzed by standard descriptive statistics. Neither dose-limiting toxicities nor adverse events > grade 3 were observed in the 18 participants. Results indicate antitumor activity through altered T cell receptor repertoires, and 33% durable (> 46 weeks) clinical benefit with median 40.7 months overall survival. Post-treatment pain and fatigue were associated with circulating tumor cell (CTC) presence at inclusion, while CTC responses correlated with clinical outcomes. This trial demonstrates that cryoimmunotherapy in mCRPC is safe and well tolerated, also for the highest DC dose (2.0 × 10 8 ) combined with checkpoint inhibitors. Further studies focusing on the biologic indications of antitumor activity and immune system activation could be considered through a phase II trial focusing on treatment responses and immunologic biomarkers.

Intensive induction chemotherapy achieves complete remissions (CR) in >60% of patients with acute myeloid leukemia (AML) but overall survival (OS) is poor for relapsing patients not eligible for allogeneic hematopoietic stem cell transplantation (allo-HSCT). Oral azacytidine may be used as maintenance treatment in AML in first remission, but can be associated with substantial side effects, and less toxic strategies should be explored. Twenty AML patients in first CR (CR1) ineligible for allo-HSCT were treated with FDC101, an autologous RNA-loaded mature dendritic cell (mDC) vaccine expressing two leukemia-associated antigens (LAAs). Each dose consisted of 2.5–5 × 106 mDCs per antigen, given weekly until week 4, at week 6, and then monthly, during the 2-year study period. Patients were followed for safety and long-term survival. Treatment was well tolerated, with mild and transient injection site reactions. Eleven of 20 patients (55%) remained in CR, while 4 of 6 relapsing patients achieved CR2 after salvage therapy and underwent allo-HSCT. OS at five years was 75% (95% CI: 50–89), with 70% of patients ≥60 years of age being long-term survivors. Maintenance therapy with this DC vaccine was well tolerated in AML patients in CR1 and was accompanied by encouraging 5-year long-term survival.

Patients with high‐risk prostate cancer (PC) can experience biochemical relapse (BCR), despite surgery, and develop noncurative disease. The present study aimed to reduce the risk of BCR with a personalized dendritic cell (DC) vaccine, given as adjuvant therapy, after robot‐assisted laparoscopic prostatectomy (RALP).

Background:Adoptive cellular therapy (ACT) with genetically modified T cells aims to redirect T cells against resistant cancers through introduction of a T cell receptor (TCR). The Radium-4 TCR was isolated from a responding patient in a cancer vaccination study and recognizes the enzymatic component of human Telomerase Reverse Transcriptase (hTERT) presented on MHC class II (HLA-DP04). hTERT is a constitutively overexpressed tumor-associated antigen present in most human cancers, including non-small-cell lung cancer (NSCLC), which is the second most common type of cancer worldwide. Treatment alternatives for relapsing NSCLC are limited and survival is poor. To improve patient outcome we designed a TCR-based ACT study targeting hTERT.Methods:T-RAD is a phase I/II study to evaluate the safety and efficacy of Radium-4 mRNA electroporated autologous T cells in the treatment of metastatic NSCLC with no other treatment option. Transient TCR expression is applied for safety considerations. Participants receive two intravenous injections with escalating doses of redirected T cells weekly for 6 consecutive weeks. Primary objectives are safety and tolerability. Secondary objectives include progression-free survival, time to progression, overall survival, patient reported outcomes and overall radiological response.Discussion:Treatment for metastatic NSCLC is scarce and new personalized treatment options are in high demand. hTERT is a tumor target applicable to numerous cancer types. This proof-of-concept study will explore for the first time the safety and efficacy of TCR mRNA electroporated autologous T cells targeting hTERT. The T-RAD study will thus evaluate an attractive candidate for future immunotherapy of solid tumors.

Success of adoptive cell therapy mainly depends on the ability of immune cells to persist and function optimally in the immunosuppressive tumor microenvironment. Although present at the cancer site, immune cells become exhausted and/or inhibited, due to the presence of inhibitory receptors such as PD-L1 on malignant cells. Novel genetic strategies to manipulate the PD1/PD-L1 axis comprise (i) PD-1 reversion where the receptor intracellular domain is replaced with an activating unit, (ii) the use of anti-PD-L1 CAR or (iii) the disruption of the PD-1 gene. We here present an alternative strategy to equip therapeutic cells with a truncated PD-1 (tPD-1) to abrogate PD-1/PD-L1 inhibition. We show that engagement of tPD-1 with PD-L1-positive tumor unleashes NK-92 activity in vitro. Furthermore, this binding was sufficiently strong to induce killing of targets otherwise not recognized by NK-92, thus increasing the range of targets. In vivo treatment with NK-92 tPD-1 cells led to reduced tumor growth and improved survival. Importantly, tPD-1 did not interfere with tumor recognition in PD-L1 negative conditions. Thus, tPD-1 represents a straightforward method for improving antitumor immunity and revealing new targets through PD-L1 positivity.

Patients with acute myeloid leukemia (AML), not eligible for allogeneic hematopoietic stem cell transplantation (AHSCT), have a significant risk of disease relapse and death due to a lack of long-term disease control. To investigate the possibility of relapse prevention after initial chemotherapy, a safety and feasibility, single-center, open label, first in human phase I/II study using dendritic cells (DC) targeting PRAME and WT-1 was recently completed. Mature autologous DCs were rapidly (3-4 days) generated using full-length PRAME and WT-1 antigens and a maturation cocktail with a TLR 7/8 agonist. Intradermal vaccination of 2.5-5x106 WT-1 and 2.5-5x106 PRAME RNA-loaded DCs was performed at week 1, 2, 3, 4, 6 and then monthly for the remainder of a 2-year study period. Patients with WT1-positive AML, with or without PRAME positivity, had to be in morphological remission (+/- hematological recovery) after induction chemotherapy without being eligible for AHSCT. The study consisted of a phase I part (n=6) to assess early safety followed by a phase II part (n=14) which assessed further safety as well as clinical and immunological effects of vaccination. A total of 20 eligible patients (5 female and 15 male, ECOG score of 0) with a median age of 59 years (range 24 to 73) were included into the study. Risk groups based on HOVON/SAKK 102 criteria at screening identified 13 patients as good, 5 as intermediate and 2 as poor risk. All patients were positive for WT-1 and 17 for PRAME prior to chemotherapy. The mean time from first diagnosis to first vaccination was 10.1±3.7 months. Vaccinations were well tolerated, without any withdrawals from the study due to toxicity. No related serious or unexpected adverse events (AEs) were reported and the most common AEs were injection site related, accounting for 28% of all AEs, which were mild and transient in nature (Grade I; CTCAE v4.03). Grade I/II toxicities were experienced by 18/20 patients, whilst 5 (25%) experienced Grade III toxicity unlikely or not related to treatment. The 2-year survival probability from time of first vaccination was 80% (95% CI: 55-92). Importantly, patients at or above the age of 60 years (n=10) also had an 80% survival rate at 2 years (risk groups: 4 good, 4 intermediate, 2 poor). Nine patients relapsed under vaccination, of whom 4 died thereafter (three due to the underlying disease, one due to GvHD after transplantation) within the 2-year study duration, equating to a probability of progression free survival (PFS) of 55% (95% CI: 31-74). The PFS rate for the elderly patients (≥ 60 years) was 50%, while it was 60% for the younger patient group. Most relapses (5/9) appeared early, within 80 days after first vaccination. Out of the 9 relapsed patients, 6 could be successfully transplanted (104 to 380 days after first vaccination; risk groups: 4 good and 2 poor risk) and 4 were still alive at the end of the 2-year observation period. The DC manufacturing process reproducibly yielded a high number of DCs expressing the transfected tumor antigens, WT-1 or PRAME, as well as high surface levels of co-stimulatory molecules. After freeze-thawing, DCs were capable of CCL19-directed migration and, upon stimulation, secreted IL-12, but not IL-10. These phenotypic and functional characteristics indicate efficient DC maturation and activation despite the patients´ previous therapeutic regimens. DC properties were comparable irrespective of the patients' clinical outcome. Immune-monitoring of bulk T cells from vaccinated patients revealed that patients in stable remission had higher levels of HLA-DR-expressing T cells when compared to relapsing patients, both in peripheral blood and bone marrow. Such difference was observed prior to vaccination and throughout the entire treatment period, suggesting that T cell activation may play a role in maintaining remission. Analysis of 23 common AML mutations revealed that 6/9 relapsed patients had at least 1 mutation, with 4 patients having 3-6 mutations. In contrast, only 1 mutation already present at baseline was found in 4/11 patients in remission. Administration of autologous DC transfected with PRAME- and WT1-RNA is feasible, safe and well tolerated. The 2-year 80% survival rate, particularly in the elderly, and the 55% progression free survival warrant further studies to assess the efficacy of this vaccine approach in improving outcomes in patients with AML. Disclosures Raffegerst: Medigene Immunotherapies GmbH: Current Employment, Current equity holder in publicly-traded company. Schnorfeil:Medigene Immunotherapies GmbH: Current Employment. Addo:Medigene Immunotherapies GmbH: Current Employment. Schendel:Medigene AG: Current Employment, Current equity holder in publicly-traded company. Pinkernell:Medigene Immunotherapies GmbH: Current Employment, Current equity holder in publicly-traded company.

Islet isolation procedure destroys islet vasculature and extra cellular matrix molecules, which can negatively affect islet function post transplantation. Developing scaffold that favors islet micro-environment is one solution to preserve islet function post isolation. 3D bioprinting of islets using hydrogel-based bioinks has been reported previously. However, modifying bioinks and presence of supporting cells such as adipose-derived stromal cells (ASCs) can improve survival and reduce cellular stress in islet scaffolds. We designed an implantable multi-layered 3D bioprinted scaffold for human or mouse islets -/+ human ASCs (1.2 ×106 ASCs/scaffold). Cells were embedded in alginate/nanocellulose bioink and printed with INKREDIBLE 3D Bioprinter from CELLINK AB. Micro-CT was used to study the distribution of islets inside scaffolds. In vitro cell viability and function were assessed using FDA/PI staining and glucose stimulated insulin secretion on day 1, 8 and 14 post print. The level of secreted human- and mouse-specific cytokines, IP-10, MCP-1 and GRO-α were measured on day 1, 8 and 14 post print. Islet scaffolds -/+ ASCs were transplanted intraperitoneal (IP) in diabetic immune-compromised mouse model and followed for 60 days post transplantation. We printed 100 islets/scaffold for in vitro and 400 islets/scaffold for in vivo studies. In vitro viability analysis revealed viable islets and ASCs throughout the studies. However, scaffolds with human and mouse islets + ASCs showed improved insulin secretion in response to glucose compared to the islet alone group on day 1, 8 and 14 post print (selected data points for human islets-day 8: islet+ASC, basal insulin 2387 ± 1262, stimulated insulin 4312 ± 606.8, recovery insulin post stimulation 1637 ± 100.1 pmol/L, * p< 0.05 vs stimulated insulin. Islet alone, basal insulin 2358 ± 887.9, stimulated insulin 1149 ± 352.6, recovery insulin post stimulation 999.0 ± 267.7 pmol/L, n=4). This is followed by significant reduction in the level of both human and mouse IP-10, MCP-1 and GRO-α in islet+ASC group compared to the islet alone group on day 1, 8 and 14 post print. IP transplantation of mouse islets -/+ ASCs to diabetic mice normalizes random blood glucose in both groups starting from day 6 post transplantation for islet+ASC group and day 40 for islet alone group. We detected c-peptide in both groups on day 3 and 13 post transplantation. However, at termination of the studies, the level of c-peptide was significantly higher in islet+ASC compared to the islet alone group (islet+ASC 94.01 ± 14.47 vs. islet alone 27.08 ± 18.93, p<0.05). This study presents a successful multi-layered scaffold design for islets and ASCs. Presence of ASCs in the scaffold can create a favorable micro-environment for the islets, resulting in reduced islet loss and improved islet function.

T-cell receptor (TCR) redirected T cells are considered as the next generation of care for the treatment of numerous solid tumors. KRAS mutations are driver neoantigens that are expressed in over 25% of all cancers and are thus regarded as ideal targets for Adoptive Cell Therapy (ACT). We have isolated four KRAS-specific TCRs from a long-term surviving pancreatic cancer patient vaccinated with a mix of mutated KRAS peptides. The sequence of these TCRs could be identified and expressed in primary cells. We demonstrated stable expression of all TCRs as well as target-specific functionality when expressing T cells were co-incubated with target cells presenting KRAS peptides. In addition, these TCRs were all partially co-receptor independent since they were functional in both CD4 and CD8 T cells, thus indicating high affinity. Interestingly, we observed that certain TCRs were able to recognize several KRAS mutations in complex with their cognate Human leukocyte antigen (HLA), suggesting that, here, the point mutations were less important for the HLA binding and TCR recognition, whereas others were single-mutation restricted. Finally, we demonstrated that these peptides were indeed processed and presented, since HLA-matched antigen presenting cells exogenously loaded with KRAS proteins were recognized by TCR-transduced T cells. Taken together, our data demonstrate that KRAS mutations are immunogenic for CD4 T cells and are interesting targets for TCR-based cancer immunotherapy.

Background A Phase I/II dendritic cell (DC) vaccine trial was completed in 20 patients with acute myeloid leukemia (AML) in complete remission or CRi after chemotherapy who were ineligible for hematopoietic stem cell transplantation (NCT02405338). The DC vaccines were designed to delay disease progression by mobilizing natural killer (NK) cells through secretion of IL-12(p70) and activating T cells by stimulation with WT-1 and PRAME, two prominent antigens in AML. DC vaccination was carried out in weeks 1, 2, 3, 4, 6 and monthly thereafter for 2 years. Two questions were prominent at the trial start. First, could mature DCs (mDCs) be efficiently prepared to accommodate the vaccine regimen, including use of separate DC-fractions for each antigen. Second, could suitable quality DC vaccines be generated from patients with myeloid disease, since all had received intensive chemotherapy, impairing hematopoiesis, such that several patients showed extended times for monocyte recovery in peripheral blood before being able to undergo apheresis for production. Methods Immune monitoring tools were used to assess DC vaccines: multi-color flow cytometry for surface and intracellular protein staining, dual-color ELISpot for secretion of IL-10/IL-12, and chemokine-directed trans-well migration. Results Adequate regeneration of monocytes occurred post-chemotherapy in all patients, allowing production of sufficient numbers of cryopreserved vaccine cells (2.5 or 5.0 × 106 mDCs/antigen/ampule) to be completed. In 15/20 patients one batch was sufficient to cover all vaccinations, while 5 patients with lower initial monocyte counts required an additional production.Phenotypic and functional parameters of patient DC vaccines were compared to cells of a healthy control (HC). Patient mDCs expressed CD83, CD40, CD80, CD86 and HLA-DR at frequencies/levels comparable to the HC. Both DC-fractions displayed intracellular protein antigen expression in most cells. Polarized secretion of IL-12(p70) without IL-10 was seen with few exceptions. Furthermore, mDCs displayed chemokine-directed migration. Detection of delayed type hypersensitivity responses post-vaccination at six weeks indicated the DC vaccines were active in vivo in all patients. Conclusions DC vaccine production feasibility was clearly fulfilled and high quality mDCs were generated for every patient. Quantity and quality of DC vaccines did not differ in the patient groups that relapsed or remained in remission, nor in patients who succumbed to disease during the trial. DC vaccines were remarkably consistent, although originating from patients differing in age, AML subtype and receiving varied amounts of standard chemotherapy regimens. Ethics Approval The study was approved by the responsible Norwegian ethics committee, approval number 2014/1677.

The original version of this article unfortunately included a mistake in Fig. 2b where the images of mice in the tumour control group (right), day 30 (bottom) should be removed as the wrong images (duplicate of day 17) were inserted by mistake. At this time point the tumour control mice were no longer alive and the images were replaced by black areas.

Abstract Objectives Innovative post‐remission therapies are needed to eliminate residual AML cells. DC vaccination is a promising strategy to induce anti‐leukaemic immune responses. Methods We conducted a first‐in‐human phase I study using TLR7/8‐matured DCs transfected with RNA encoding the two AML‐associated antigens WT1 and PRAME as well as CMVpp65. AML patients in CR at high risk of relapse were vaccinated 10× over 26 weeks. Results Despite heavy pretreatment, DCs of sufficient number and quality were generated from a single leukapheresis in 11/12 cases, and 10 patients were vaccinated. Administration was safe and resulted in local inflammatory responses with dense T‐cell infiltration. In peripheral blood, increased antigen‐specific CD8+ T cells were seen for WT1 (2/10), PRAME (4/10) and CMVpp65 (9/10). For CMVpp65, increased CD4+ T cells were detected in 4/7 patients, and an antibody response was induced in 3/7 initially seronegative patients. Median OS was not reached after 1057 days; median RFS was 1084 days. A positive correlation was observed between clinical benefit and younger age as well as mounting of antigen‐specific immune responses. Conclusions Administration of TLR7/8‐matured DCs to AML patients in CR at high risk of relapse was feasible and safe and resulted in induction of antigen‐specific immune responses. Clinical benefit appeared to occur more likely in patients <65 and in patients mounting an immune response. Our observations need to be validated in a larger patient cohort. We hypothesise that TLR7/8 DC vaccination strategies should be combined with hypomethylating agents or checkpoint inhibition to augment immune responses. Trial registration The study was registered at https://clinicaltrials.gov on 17 October 2012 (NCT01734304) and at https://www.clinicaltrialsregister.eu (EudraCT‐Number 2010‐022446‐24) on 10 October 2013.

CAR T cells targeting the B-lymphocyte antigen CD19 have led to remarkable clinical results in B-cell leukemia and lymphoma, but eliminate all B-lineage cells, leading to increased susceptibility to severe infections. As malignant B cells will express either immunoglobulin (Ig) light chain κ or λ, we designed a second-generation CAR targeting Igκ, IGK CAR. This construct demonstrated high target specificity, but displayed reduced efficacy in the presence of serum IgG. Since CD19 CAR is insensitive to serum IgG, we designed various combinatorial CAR constructs in order to maintain the CD19 CAR T cell efficacy, but with IGK CAR target selectivity. The Kz-19BB design, combining CD19 CAR containing a 4-1BB co-stimulatory domain with an IGK CAR containing a CD3zeta stimulatory domain, maintained the target specificity of IgK CAR and was resistant to the presence of soluble IgG. Our results demonstrate that a combinatorial CAR approach can improve target selectivity and efficacy.

T cell receptor (TCR)-engineered T cell therapy is a promising cancer treatment approach. Human telomerase reverse transcriptase (hTERT) is overexpressed in the majority of tumors and a potential target for adoptive cell therapy. We isolated a novel hTERT-specific TCR sequence, named Radium-4, from a clinically responding pancreatic cancer patient vaccinated with a long hTERT peptide. Radium-4 TCR-redirected primary CD4(+) and CD8(+) T cells demonstrated in vitro efficacy, producing inflammatory cytokines and killing hTERT(+) melanoma cells in both 2D and 3D settings, as well as malignant, patient-derived ascites cells. Importantly, T cells expressing Radium-4 TCR displayed no toxicity against bone marrow stem cells or mature hematopoietic cells. Notably, Radium-4 TCR+ T cells also significantly reduced tumor growth and improved survival in a xenograft mouse model. Since hTERT is a universal cancer antigen, and the very frequently expressed HLA class II molecules presenting the hTERT peptide to this TCR provide a very high (>75%) population coverage, this TCR represents an attractive candidate for immunotherapy of solid tumors.

324 Background: Patients with very high-risk prostate cancer (VHR-PC) features experience worse outcome after radical prostatectomy. This study was designed to assess biochemical failure and toxicity of adjuvant dendritic cells vaccine (DCV) in prostate cancer patients who are at greatest risk for cancer progression. Methods: Twenty patients with pathological stage pT2 - pT3b and Gleason score 7B-10, pN0, pN+ or pNx were enrolled into the approved study DC-005. The primary end point was clinical failure. Ten patients were tested for disseminated tumor cells (DTCs) to the bone marrow before inclusion to the study. Three patients out of 10 patients had positive DTCs detection in bone marrow. The mean age of the cohort was 63 years (SD 6.9 years), and three patients had postsurgical pN1 status. Eighteen patients had two or more high-risk factors (ISUP grade 5, T3- stage and or PSA > 20 ng/mL). Autologous dendritic cells were transfected with mRNA for hTERT, survivin and tumor mRNA. The DCV product was applied intradermally after curative intended surgery once per week the first months, then once per months the first year, thereafter every 3 months for two years or until biochemical progression (PSA relapse cut-off ≥ 0.3). Results: After 5 years follow-up (FU) 62% (12/20 patients) had not biochemically progressed and with a median FU of 69 months all patients included in the study are alive. Five patients were treated with salvage and one patient with adjuvant radiation treatment, three patients received limited ADT, and three patients are on first line ADT, none of those eight patients have experienced castration resistant prostate cancer. The toxicity was mild with no serious adverse event related to DCV. Conclusions: Adjuvant DCV mitigates the time to biochemical progression. These results appear favorably compared to historical controls in VHR-PC. The clinical outcomes of this study warrants a future enlarged clinical trial. Clinical trial information: NCT01197625.