Transient TCR-based T cell therapy in a patient with advanced treatment-resistant MSI-high colorectal cancer.

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.

Efficient chimeric antigen receptor targeting of a central epitope of CD22.

Chimeric antigen receptor (CAR) T-cell therapy has had considerable success in the treatment of B-cell malignancies. Targeting the B-lineage marker CD19 has brought great advances to the treatment of acute lymphoblastic leukemia and B-cell lymphomas. However, relapse remains an issue in many cases. Such relapse can result from downregulation or loss of CD19 from the malignant cell population or expression of alternate isoforms. Consequently, there remains a need to target alternative B-cell antigens and diversify the spectrum of epitopes targeted within the same antigen. CD22 has been identified as a substitute target in cases of CD19-negative relapse. One anti-CD22 antibody-clone m971-targets a membrane-proximal epitope of CD22 and has been widely validated and used in the clinic. Here, we have compared m971-CAR with a novel CAR derived from IS7, an antibody that targets a central epitope on CD22. The IS7-CAR has superior avidity and is active and specific against CD22-positive targets, including B-acute lymphoblastic leukemia patient-derived xenograft samples. Side-by-side comparisons indicated that while IS7-CAR killed less rapidly than m971-CAR in vitro, it remains efficient in controlling lymphoma xenograft models in vivo. Thus, IS7-CAR presents a potential alternative candidate for the treatment of refractory B-cell malignancies.

Impact of human telomerase reverse transcriptase peptide vaccine combined with androgen deprivation therapy and radiotherapy in de novo metastatic prostate cancer: Long-term clinical monitoring.

Prostate cancer is considered as poorly immunogenic. In a phase I/II study on de novo metastatic prostate cancer we found that a human telomerase reverse transcriptase (hTERT) vaccine induced an early immune response in most of the patients. Here we present the results from the long-term monitoring of the immune responses and clinical outcomes. Twenty-two men with ISUP 4 to 5 and lymph node and/or bone metastases were treated with androgen deprivation therapy (ADT), radiotherapy and the hTERT vaccine UV1 between January 2013 and July 2014. Immune response was monitored before, during and after vaccination and continued every 6 months until PSA progression. All patients had magnetic resonance imaging (MRI) at baseline, and after 6 months, 1 and 2 years, and at progression. The clinical outcome was time to progression, overall survival and prostate cancer-specific survival. The median follow-up was 62 months (range: 19-101). At the last observation, nine of the 22 patients were still alive. Six have no progression, two have castration-resistant disease treated with second-line ADT and one has castration-refractory disease. Median time to PSA progression was 21 months, median overall survival was 62 months and median prostate cancer-specific survival was 84 months. Lack of immune response was an independent marker of prostate cancer death. The long-term monitoring showed that some patients had unanticipated subsequent high immune responses without developing recurrence. This association indicates that there might be a clinical benefit of hTERT vaccination in a subgroup of men with primary metastatic hormone-sensitive prostate cancer treated with ADT and radiotherapy.

Combinatorial CAR design improves target restriction.

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 costimulatory 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.

Characterization of the T cell receptor repertoire and melanoma tumor microenvironment upon combined treatment with ipilimumab and hTERT vaccination.

BACKGROUND: This clinical trial evaluated a novel telomerase-targeting therapeutic cancer vaccine, UV1, in combination with ipilimumab, in patients with metastatic melanoma. Translational research was conducted on patient-derived blood and tissue samples with the goal of elucidating the effects of treatment on the T cell receptor repertoire and tumor microenvironment. METHODS: The trial was an open-label, single-center phase I/IIa study. Eligible patients had unresectable metastatic melanoma. Patients received up to 9 UV1 vaccinations and four ipilimumab infusions. Clinical responses were assessed according to RECIST 1.1. Patients were followed up for progression-free survival (PFS) and overall survival (OS). Whole-exome and RNA sequencing, and multiplex immunofluorescence were performed on the biopsies. T cell receptor (TCR) sequencing was performed on the peripheral blood and tumor tissues. RESULTS: Twelve patients were enrolled in the study. Vaccine-specific immune responses were detected in 91% of evaluable patients. Clinical responses were observed in four patients. The mPFS was 6.7 months, and the mOS was 66.3 months. There was no association between baseline tumor mutational burden, neoantigen load, IFN-γ gene signature, tumor-infiltrating lymphocytes, and response to therapy. Tumor telomerase expression was confirmed in all available biopsies. Vaccine-enriched TCR clones were detected in blood and biopsy, and an increase in the tumor IFN-γ gene signature was detected in clinically responding patients. CONCLUSION: Clinical responses were observed irrespective of established predictive biomarkers for checkpoint inhibitor efficacy, indicating an added benefit of the vaccine-induced T cells. The clinical and immunological read-out warrants further investigation of UV1 in combination with checkpoint inhibitors. Trial registration Clinicaltrials.gov identifier: NCT02275416. Registered October 27, 2014. https://clinicaltrials.gov/ct2/show/NCT02275416?term=uv1&draw=2&rank=6.

"Built-in" PD-1 blocker to rescue NK-92 activity from PD-L1-mediated tumor escape mechanisms.

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.

Targeting Telomerase with an HLA Class II-Restricted TCR for Cancer Immunotherapy.

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.

Long-term surviving cancer patients as a source of therapeutic TCR.

We have established a platform for the isolation of tumour-specific TCR from T cells of patients who experienced clinical benefit from cancer vaccination. In this review we will present the rationale behind this strategy and discuss the advantages of working with "natural" wild type TCRs. Indeed, the general trend in the field has been to use various modifications to enhance the affinity of such therapeutic TCRs. This was done to obtain stronger T cell responses, often at the cost of safety. We further describe antigen targets and recent in vitro and in vivo results obtained to validate them. We finally discuss the use of MHC class II-restricted TCR in immunotherapy. Typically cellular anti-tumour immune responses have been attributed to CD8 T cells; however, we isolated mainly CD4 T cells. Importantly, these MHC class II-restricted TCRs have the potential to induce broad, long lasting immune responses that enable cancer control. The use of CD4 T cell-derived TCRs for adoptive immunotherapy has so far been limited and we will here discuss their therapeutic potential.

Correction to: Preclinical assessment of transiently TCR redirected T cells for solid tumour immunotherapy.

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.

Colorectal cysts as a validating tool for CAR therapy.

BACKGROUND: Treatment of cancers has largely benefited from the development of immunotherapy. In particular, Chimeric Antigen Receptor (CAR) redirected T cells have demonstrated impressive efficacy against B-cell malignancies and continuous efforts are made to adapt this new therapy to solid tumors, where the immunosuppressive tumor microenvironment is a barrier for delivery. CAR T-cell validation relies on in vitro functional assays using monolayer or suspension cells and in vivo xenograft models in immunodeficient animals. However, the efficacy of CAR therapies remains difficult to predict with these systems, in particular when challenged against 3D organized solid tumors with highly intricate microenvironment. An increasing number of reports have now included an additional step in the development process in which redirected T cells are tested against tumor spheres. RESULTS: Here, we report a method to produce 3D structures, or cysts, out of a colorectal cancer cell line, Caco-2, which has the ability to form polarized spheroids as a validation tool for adoptive cell therapy in general. We used CD19CAR T cells to explore this method and we show that it can be adapted to various platforms including high resolution microscopy, bioluminescence assays and high-throughput live cell imaging systems. CONCLUSION: We developed an affordable, reliable and practical method to produce cysts to validate therapeutic CAR T cells. The integration of this additional layer between in vitro and in vivo studies could be an important tool in the pre-clinical workflow of cell-based immunotherapy.

T cell receptor therapy against melanoma-Immunotherapy for the future?

Malignant melanoma has seen monumental changes in treatment options the last decade from the very poor results of dacarbazine treatment to the modern-day use of targeted therapies and immune checkpoint inhibitors. Melanoma has a high mutational burden making it more capable of evoking immune responses than many other tumours. Even when considering double immune checkpoint blockade with anti-CTLA-4 and anti-PD-1, we still have far to go in melanoma treatment as 50% of patients with metastatic disease do not respond to current treatment. Alternative immunotherapy should therefore be considered. Since melanoma has a high mutational burden, it is considered more immunogenic than many other tumours. T cell receptor (TCR) therapy could be a possible way forward, either alone or in combination, to improve the response rates of this deadly disease. Melanoma is one of the cancers where TCR therapy has been frequently applied. However, the number of antigens targeted remains fairly limited, although advanced personalized therapies aim at also targeting private mutations. In this review, we look at possible aspects of targeting TCR therapy towards melanoma and provide an implication of its use in the future.

SJI 2020 special issue: A catalogue of Ovarian Cancer targets for CAR therapy.

Ovarian Cancer (OC) is currently difficult to cure, mainly due to its late detection and the advanced state of the disease at the time of diagnosis. Therefore, conventional treatments such as debulking surgery and combination chemotherapy are rarely able to control progression of the tumour, and relapses are frequent. Alternative therapies are currently being evaluated, including immunotherapy and advanced T cell-based therapy. In the present review, we will focus on a description of those Chimeric Antigen Receptors (CARs) that have been validated in the laboratory or are being tested in the clinic. Numerous target antigens have been defined due to the identification of OC biomarkers, and many are being used as CAR targets. We provide an exhaustive list of these constructs and their current status. Despite being innovative and efficient, the OC-specific CARs face a barrier to their clinical efficacy: the tumour microenvironment (TME). Indeed, effector cells expressing CARs have been shown to be severely inhibited, rendering the CAR T cells useless once at the tumour site. Herein, we give a thorough description of the highly immunosuppressive OC TME and present recent studies and innovations that have enabled CAR T cells to counteract this negative environment and to destroy tumours.

Immune stimulatory effect of anti-EpCAM immunotoxin - improved overall survival of metastatic colorectal cancer patients.

Introduction: In a recent phase I trial in a heterogeneous group of carcinoma patients with advanced disease, we did not observe objective responses by CT at 8 weeks in patients treated with either the anti-EpCAM immunotoxin MOC31PE alone or administered in combination with the immunosuppressor cyclosporin (CsA). We have now assessed overall survival (OS) data for the two groups to reveal potential differences, and to elucidate putative underlying mechanisms.Material and methods: The OS time of MOC31PE monotherapy (34 patients) and MOC31PE in combination with CsA (23 patients), was assessed. Pre- and post-treatment patient sera were analyzed in a multiplex immunoassay, and the immunogenic effects of MOC31PE were studied in vitro and in a dendritic cell maturation assay.Results: When the data were analyzed for all treated patients regardless of cancer type, the MOC31PE alone group had a median OS of 12.7 months (95% CI = 5.6-19.8 months) compared to 6.2 months (95% CI = 5.6-6.8 months) (p=.066) for the patients treated with MOC31PE + CsA group. For the subgroup of patients with colorectal cancer, the median OS survival was 16.3 months (95% CI = 5.6-27.0) for the MOC31PE only cohort (n = 15), compared to 6.0 months (CI = 5.8-6.2) (p < .001) for the combination group. The cytokine profile in patient sera and the in vitro immunological studies indicate that MOC31PE induced an immunogenic response leading to T-cell activation; a response that was suppressed in patients treated with MOC31PE + CsA.Conclusions: The results reveal a promising clinical benefit of anti-EpCAM immunotoxin treatment in patients with advanced disease, an effect apparently explained by a previously unknown immunogenic effect of MOC31PE.

Preclinical assessment of transiently TCR redirected T cells for solid tumour immunotherapy.

Off-target toxicity due to the expression of target antigens in normal tissue or TCR cross-reactivity represents a major risk when using T cell receptor (TCR)-engineered T cells for treatment of solid tumours. Due to the inherent cross-reactivity of TCRs it is difficult to accurately predict their target recognition pre-clinically. It has become evident that direct testing in a human being represents the best evaluation of the risks. There is, therefore, a clear unmet need for assessing the safety of a therapeutic TCR in a more controllable manner than by the injection of permanently modified cellular products. Using transiently modified T cells combined with dose escalation has already been shown feasible for chimeric antigen receptor (CAR)-engineered T cells, but nothing is yet reported for TCR. We performed a preclinical evaluation of a therapeutic TCR transiently expressed in T cells by mRNA electroporation. We analyzed if the construct was active in vitro, how long it was detectable for and if this expression format was adapted to in vivo efficacy assessment. Our data demonstrate the potential of mRNA engineered T cells, although less powerful than permanent redirection, to induce a significant response. Thus, these findings support the development of mRNA based TCR-therapy strategies as a feasible and efficacious method for evaluating TCR safety and efficacy in first-in-man testing.

Treating osteosarcoma with CAR T cells.

Novel therapies to treat patients with solid cancers that have developed resistance to chemotherapy represent unmet needs of considerable dimensions. In the present review, we will address the attempts to develop chimeric antigen receptor (CAR) targeted immunotherapy against osteosarcoma (OS). This aggressive cancer displays its peak incidence in children and young adults. The main cause of patient death is lung metastases with a 5-year survival as low as 5%-10% in the primary metastatic setting and 30% in the relapse situation, respectively. Effective adjuvant combination chemotherapy introduced more than 40 years ago improved the survival rates from below 20% to around 60% in patients; however, since then, no major breakthroughs have been made. The use of immune checkpoint inhibitors has been disappointing in OS, while other types of immunotherapies such as CAR T cells remain largely unexplored. Indeed, for CAR T-cell therapy to be efficacious, two main criteria need to be fulfilled: (a) CAR T cells should target an epitope selectively expressed on the cell surface of OS in order to prevent toxicities in normal tissues and (b) the target should also be widely expressed on OS metastases. These challenges have already been undertaken in OS and illustrate the difficulties in developing tomorrow's CAR-T treatment in a solid tumour. We will discuss the experiences with CAR-T therapy development and efficacy to combat the clinical challenges in OS.

Human c-SRC kinase (CSK) overexpression makes T cells dummy.

Adoptive cell therapy with T-cell receptor (TCR)-engineered T cells represents a powerful method to redirect the immune system against tumours. However, although TCR recognition is restricted to a specific peptide-MHC (pMHC) complex, increasing numbers of reports have shown cross-reactivity and off-target effects with severe consequences for the patients. This demands further development of strategies to validate TCR safety prior to clinical use. We reasoned that the desired TCR signalling depends on correct pMHC recognition on the outside and a restricted clustering on the inside of the cell. Since the majority of the adverse events are due to TCR recognition of the wrong target, we tested if blocking the signalling would affect the binding. By over-expressing the c-SRC kinase (CSK), a negative regulator of LCK, in redirected T cells, we showed that peripheral blood T cells inhibited anti-CD3/anti-CD28-induced phosphorylation of ERK, whereas TCR proximal signalling was not affected. Similarly, overexpression of CSK together with a therapeutic TCR prevented pMHC-induced ERK phosphorylation. Downstream effector functions were also almost completely blocked, including pMHC-induced IL-2 release, degranulation and, most importantly, target cell killing. The lack of effector functions contrasted with the unaffected TCR expression, pMHC recognition, and membrane exchange activity (trogocytosis). Therefore, co-expression of CSK with a therapeutic TCR did not compromise target recognition and binding, but rendered T cells incapable of executing their effector functions. Consequently, we named these redirected T cells "dummy T cells" and propose to use them for safety validation of new TCRs prior to therapy.

Phase I/IIa clinical trial of a novel hTERT peptide vaccine in men with metastatic hormone-naive prostate cancer.

In newly diagnosed metastatic hormone-naive prostate cancer (mPC), telomerase-based immunotherapy with the novel hTERT peptide vaccine UV1 can induce immune responses with potential clinical benefit. This phase I dose escalation study of UV1 evaluated safety, immune response, effects on prostate-specific antigen (PSA) levels, and preliminary clinical outcome. Twenty-two patients with newly diagnosed metastatic hormone-naïve PC (mPC) were enrolled; all had started androgen deprivation therapy and had no visceral metastases. Bone metastases were present in 17 (77%) patients and 16 (73%) patients had affected lymph nodes. Three dose levels of UV1 were given as intradermal injections combined with GM-CSF (Leukine®). Twenty-one patients in the intention-to-treat population (95%) received conformal radiotherapy. Adverse events reported were predominantly grade 1, most frequently injection site pruritus (86.4%). Serious adverse events considered possibly related to UV1 and/or GM-CSF included anaphylactic reaction in two patients and thrombocytopenia in one patient. Immune responses against UV1 peptides were confirmed in 18/21 evaluable patients (85.7%), PSA declined to <0.5 ng/mL in 14 (64%) patients and in ten patients (45%) no evidence of persisting tumour was seen on MRI in the prostatic gland. At the end of the nine-month reporting period for the study, 17 patients had clinically stable disease. Treatment with UV1 and GM-CSF gave few adverse events and induced specific immune responses in a large proportion of patients unselected for HLA type. The intermediate dose of 0.3 mg UV1 resulted in the highest proportion of, and most rapid UV1-specific immune responses with an acceptable safety profile. These results warrant further clinical studies in mPC.

Enhancing Antitumor Immune Responses by Optimized Combinations of Cell-penetrating Peptide-based Vaccines and Adjuvants.

Cell penetrating peptides (CPPs) from the protein ZEBRA are promising candidates to exploit in therapeutic cancer vaccines, since they can transport antigenic cargos into dendritic cells and induce tumor-specific T cells. Employing CPPs for a given cancer indication will require engineering to include relevant tumor-associated epitopes, administration with an appropriate adjuvant, and testing for antitumor immunity. We assessed the importance of structural characteristics, efficiency of in vitro transduction of target cells, and choice of adjuvant in inducing the two key elements in antitumor immunity, CD4 and CD8 T cells, as well as control of tumor growth in vivo. Structural characteristics associated with CPP function varied according to CPP truncations and cargo epitope composition, and correlated with in vitro transduction efficiency. However, subsequent in vivo capacity to induce CD4 and CD8 T cells was not always predicted by in vitro results. We determined that the critical parameter for in vivo efficacy using aggressive mouse tumor models was the choice of adjuvant. Optimal pairing of a particular ZEBRA-CPP sequence and antigenic cargo together with adjuvant induced potent antitumor immunity. Our results highlight the irreplaceable role of in vivo testing of novel vaccine constructs together with adjuvants to select combinations for further development.

In vitro re-challenge of CAR T cells.

Chimeric antigen receptor (CAR) T cells (CAR T) have emerged as a potential therapy for cancer patients. CAR T cells are capable of recognizing membrane proteins on cancer cells which initiates a downstream signaling in T cells that ends in cancer cell death. Continuous antigen exposure over time, activation of inhibitory signaling pathways and/or chronic inflammation can lead to CAR T cell exhaustion. In this context, the design of CARs can have a great impact on the functionality of CAR T cells, on their potency and exhaustion. Here, using CD19CAR as model, we provide a re-challenge protocol where CAR T cells are cultured weekly with malignant lymphoid cell lines BL-41 and Nalm-6 to simulate them with continuous antigen pressure over a four-week period. This protocol can be value for assessing CAR T cell functionality and for the comparison of different CAR constructs.

CD37 is a safe chimeric antigen receptor target to treat acute myeloid leukemia.

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.