Signaling via a CD28/CD40 chimeric costimulatory antigen receptor (CoStAR™), targeting folate receptor alpha, enhances T cell activity and augments tumor reactivity of tumor infiltrating lymphocytes.

Transfer of autologous tumor infiltrating lymphocytes (TIL) to patients with refractory melanoma has shown clinical efficacy in a number of trials. However, extending the clinical benefit to patients with other cancers poses a challenge. Inefficient costimulation in the tumor microenvironment can lead to T cell anergy and exhaustion resulting in poor anti-tumor activity. Here, we describe a chimeric costimulatory antigen receptor (CoStAR) comprised of FRα-specific scFv linked to CD28 and CD40 intracellular signaling domains. CoStAR signaling alone does not activate T cells, while the combination of TCR and CoStAR signaling enhances T cell activity resulting in less differentiated T cells, and augmentation of T cell effector functions, including cytokine secretion and cytotoxicity. CoStAR activity resulted in superior T cell proliferation, even in the absence of exogenous IL-2. Using an in vivo transplantable tumor model, CoStAR was shown to improve T cell survival after transfer, enhanced control of tumor growth, and improved host survival. CoStAR could be reliably engineered into TIL from multiple tumor indications and augmented TIL activity against autologous tumor targets both in vitro and in vivo. CoStAR thus represents a general approach to improving TIL therapy with synthetic costimulation.

Engineering strategies to optimise adoptive cell therapy in ovarian cancer.

Ovarian cancer is amongst the ten most common cancer types in women, and it is one of the leading causes of death. Despite the promising results of targeted therapies, including anti-angiogenic agents and poly (ADP-ribose) polymerase inhibitors (PARPi), the majority of patients will relapse and develop treatment resistance, implying that novel therapeutic strategies are required. Adoptive cell therapy (ACT) refers to the process by which autologous immune cells are used to eliminate cancer. Examples include tumour infiltrating lymphocytes (TILs), T cells genetically engineered with T cell receptors (TCR), or chimeric antigen receptor (CAR)-T cells. Recently, ACT has revealed promising results in the treatment of haematological malignancies, however, its application to solid tumours is still limited due to lack of functionality and persistence of T cells, prevalence of an exhausted phenotype and impaired trafficking towards the tumour microenvironment (TME). In this review we explore the potential of ACT for the treatment of ovarian cancer and strategies to overcome its principal limitations.

Clinical feasibility and treatment outcomes with nonselected autologous tumor-infiltrating lymphocyte therapy in patients with advanced cutaneous melanoma.

Nonselected autologous tumor-infiltrating lymphocytes (TILs) may provide advantages over other treatments for solid tumors, including checkpoint inhibitor-refractory melanoma. This retrospective analysis reports a single-center experience of nonselected autologous TILs derived from digested tumors for compassionate use treatment of advanced cutaneous melanoma, including after programmed cell death protein 1 (PD-1) inhibition. Patients with histologically confirmed metastatic cutaneous melanoma and no standard-of-care treatment options underwent tumor resection for TIL product manufacturing. Patients received lymphodepleting chemotherapy with cyclophosphamide for 2 days and fludarabine for 5 days, followed by a single TIL infusion and post-TIL high-dose interleukin (IL)-2. Safety assessments included clinically significant adverse events (AEs). Efficacy assessments included overall response rate (ORR), complete response (CR) rate, disease control rate (DCR), and overall survival. Between October 2011 and August 2019, 21 patients underwent treatment (median follow-up time, 52.2 months from TIL infusion). Among all treated patients, median age was 45 years, median number of disease sites was 4, 100% had M1c or M1d disease, and 90% received prior checkpoint inhibitor. Twelve patients received TILs after prior PD-1 inhibition. The safety profile among all treated patients and the prior PD-1 inhibitor subgroup was generally consistent with lymphodepletion and high-dose IL-2. No treatment-related deaths occurred. Among all patients, the ORR was 67%, CR rate was 19%, and the DCR was 86%, which was consistent with that observed in the prior PD-1 inhibitor subgroup (58%, 8%, and 75%, respectively). Median overall survival in all treated patients and the prior PD-1 inhibitor subgroup was 21.3 months. In total, 5 patients (24%) had durable ongoing responses (>30 months post-TIL infusion) at data cutoff, and all patients who achieved CR remained alive and disease free. To further illustrate how TIL therapy may integrate into established treatment paradigms, several case studies of patients treated in this series were included. Overall, these data demonstrate that manufacturing of nonselected autologous TILs from tumor digests is feasible and resulted in high rates of durable response in poor-risk patient populations, which may address significant unmet medical need.

Transgenic T-cell receptor immunotherapy for cancer: building on clinical success.

With the advent of immunotherapy as a realistic and promising option for cancer treatment, adoptive cellular therapies are gaining significant interest in the clinic. Whilst the recent successes of chimeric antigen receptor T-cell therapies for haematological malignancies are widely known, they have yet to show great success in solid cancers. However, immune cells transduced with T-cell receptors have been shown to traffic to and exert anti-cancer effects on solid tumour cells with some great successes. In this review, we explore the field of transgenic T-cell receptor immunotherapy, highlighting some of the key clinical trials which have paved the way for this type of cellular immunotherapy. Some trials have shown amazing clinical results, including long-term remissions and minimal toxicity, and can be looked at as an exemplar for this adoptive cell therapy. There have also been key trials where unexpected, fatal, off-tumour toxicity has occurred, and these trials have also been instrumental in shaping safer clinical trials, particularly regarding preclinical testing. In addition to previous trials, we analysed the current clinical trial space for T-cell receptor T-cell therapy, showing which trials are dominating in the clinic and which targets are being prioritised by researchers around the world. By looking at both past and current trials, we have been able to identify key drivers in developing transgenic T-cell receptor immunotherapy for the future.

Impact of Polymer-TLR-7/8 Agonist (Adjuvant) Morphology on the Potency and Mechanism of CD8 T Cell Induction.

Small molecule Toll-like receptor-7 and -8 agonists (TLR-7/8a) can be used as vaccine adjuvants to induce CD8 T cell immunity but require formulations that prevent systemic toxicity and focus adjuvant activity in lymphoid tissues. Here, we covalently attached TLR-7/8a to polymers of varying composition, chain architecture and hydrodynamic behavior (∼300 nm submicrometer particles, ∼10 nm micelles and ∼4 nm flexible random coils) and evaluated how these parameters of polymer-TLR-7/8a conjugates impact adjuvant activity in vivo. Attachment of TLR-7/8a to any of the polymer compositions resulted in a nearly 10-fold reduction in systemic cytokines (toxicity). Moreover, both lymph node cytokine production and the magnitude of CD8 T cells induced against protein antigen increased with increasing polymer-TLR-7/8a hydrodynamic radius, with the submicrometer particle inducing the highest magnitude responses. Notably, CD8 T cell responses induced by polymer-TLR-7/8a were dependent on CCR2+ monocytes and IL-12, whereas responses by a small molecule TLR-7/8a that unexpectedly persisted in vaccine-site draining lymph nodes (T1/2 = 15 h) had less dependence on monocytes and IL-12 but required Type I IFNs. This study shows how modular properties of synthetic adjuvants can be chemically programmed to alter immunity in vivo through distinct immunological mechanisms.

Optogenetic control of iPS cell-derived neurons in 2D and 3D culture systems using channelrhodopsin-2 expression driven by the synapsin-1 and calcium-calmodulin kinase II promoters.

Development of an optogenetically controllable human neural network model in three-dimensional (3D) cultures can provide an investigative system that is more physiologically relevant and better able to mimic aspects of human brain function. Light-sensitive neurons were generated by transducing channelrhodopsin-2 (ChR2) into human induced pluripotent stem cell (hiPSC) derived neural progenitor cells (Axol) using lentiviruses and cell-type specific promoters. A mixed population of human iPSC-derived cortical neurons, astrocytes and progenitor cells were obtained (Axol-ChR2) upon neural differentiation. Pan-neuronal promoter synapsin-1 (SYN1) and excitatory neuron-specific promoter calcium-calmodulin kinase II (CaMKII) were used to drive reporter gene expression in order to assess the differentiation status of the targeted cells. Expression of ChR2 and characterisation of subpopulations in differentiated Axol-ChR2 cells were evaluated using flow cytometry and immunofluorescent staining. These cells were transferred from 2D culture to 3D alginate hydrogel functionalised with arginine-glycine-aspartate (RGD) and small molecules (Y-27632). Improved RGD-alginate hydrogel was physically characterised and assessed for cell viability to serve as a generic 3D culture system for human pluripotent stem cells (hPSCs) and neuronal cells. Prior to cell encapsulation, neural network activities of Axol-ChR2 cells and primary neurons were investigated using calcium imaging. Results demonstrate that functional activities were successfully achieved through expression of ChR2- by both the CaMKII and SYN1 promoters. The RGD-alginate hydrogel system supports the growth of differentiated Axol-ChR2 cells whilst allowing detection of ChR2 expression upon light stimulation. This allows precise and non-invasive control of human neural networks in 3D.

A Syngeneic Mouse B-Cell Lymphoma Model for Pre-Clinical Evaluation of CD19 CAR T Cells.

The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen receptors (CARs) for acute lymphoblastic leukemia (ALL) andnon-Hodgkin lymphoma (NHL). The focus of the field is now on emulating these successes in other hematological malignancies where less impressive complete response rates are observed. Further engineering of CAR T cells or co-administration of other treatment modalities may successfully overcome obstacles to successful therapy in other cancer settings. We therefore present a model in which others can conduct pre-clinical testing of CD19 CAR T cells. Results in this well tested B-cell lymphoma model are likely to be informative CAR T-cell therapy in general. This protocol allows the reproducible production of mouse CAR T cells through calcium phosphate transfection of Plat-E producer cells with MP71 retroviral constructs and pCL-Eco packaging plasmid followed by collection of secreted retroviral particles and transduction using recombinant human fibronectin fragment and centrifugation. Validation of retroviral transduction, and confirmation of the ability of CAR T cells to kill target lymphoma cells ex vivo, through the use of flow cytometry, luminometry and enzyme-linked immunosorbent assay (ELISA), is also described. Protocols for testing CAR T cells in vivo in lymphoreplete and lymphodepleted syngeneic mice, bearing established, systemic lymphoma are described. Anti-cancer activity is monitored by in vivo bioluminescence and disease progression. We show typical results of eradication of established B-cell lymphoma when utilizing 1st or 2nd generation CARs in combination with lymphodepleting pre-conditioning and a minority of mice achieving long term remissions when utilizing CAR T cells expressing IL-12 in lymphoreplete mice. These protocols can be used to evaluate CD19 CAR T cells with different additional modification, combinations of CAR T cells and other therapeutic agents or adapted for the use of CAR T cells against different target antigens.

CD19 CAR T Cells Expressing IL-12 Eradicate Lymphoma in Fully Lymphoreplete Mice through Induction of Host Immunity.

Chimeric antigen receptor (CAR) T cell therapy represents a significant advancement in cancer therapy. Larger studies have shown ∼90% complete remission rates against chemoresistant and/or refractory CD19+ leukemia or lymphoma. Effective CAR T cell therapy is highly dependent on lymphodepleting preconditioning, which is achieved through chemotherapy or radiotherapy that carries with it significant toxicities. These can exclude patients of low performance status. In order to overcome the need for preconditioning, we constructed fully mouse first and second generation anti-murine CD19 CARs with or without interleukin-12 (IL-12) secretion. To test these CARs, we established a mouse model to reflect the human situation without preconditioning. Murine second generation CAR T cells expressing IL-12 were capable of eradicating established B cell lymphoma with a long-term survival rate of ∼25%. We believe this to be the first study in a truly lymphoreplete model. We provide evidence that IL-12-expressing CAR T cells not only directly kill target CD19+ cells, but also recruit host immune cells to an anti-cancer immune response. This finding is critical because lymphodepletion regimens required for the success of current CAR T cell technology eliminate host immune cells whose anti-cancer activity could otherwise be harnessed by strategies such as IL-12-secreting CAR T cells.

Oncolytic Group B Adenovirus Enadenotucirev Mediates Non-apoptotic Cell Death with Membrane Disruption and Release of Inflammatory Mediators.

Enadenotucirev (EnAd) is a chimeric group B adenovirus isolated by bioselection from a library of adenovirus serotypes. It replicates selectively in and kills a diverse range of carcinoma cells, shows effective anticancer activity in preclinical systems, and is currently undergoing phase I/II clinical trials. EnAd kills cells more quickly than type 5 adenovirus, and speed of cytotoxicity is dose dependent. The EnAd death pathway does not involve p53, is predominantly caspase independent, and appears to involve a rapid fall in cellular ATP. Infected cells show early loss of membrane integrity; increased exposure of calreticulin; extracellular release of ATP, HSP70, and HMGB1; and influx of calcium. The virus also causes an obvious single membrane blister reminiscent of ischemic cell death by oncosis. In human tumor biopsies maintained in ex vivo culture, EnAd mediated release of pro-inflammatory mediators such as TNF-α, IL-6, and HMGB1. In accordance with this, EnAd-infected tumor cells showed potent stimulation of dendritic cells and CD4+ T cells in a mixed tumor-leukocyte reaction in vitro. Whereas many viruses have evolved for efficient propagation with minimal inflammation, bioselection of EnAd for rapid killing has yielded a virus with a short life cycle that combines potent cytotoxicity with a proinflammatory mechanism of cell death.

CCR7+ selected gene-modified T cells maintain a central memory phenotype and display enhanced persistence in peripheral blood in vivo.

BACKGROUND: Adoptive T cell immunotherapy (ATCT) for cancer entails infusing patients with T cells that recognise and destroy tumour cells. Efficient engraftment of T cells and persistence in the circulation correlate with favourable clinical outcomes. T cells of early differentiation possess an increased capacity for proliferation and therefore persistence, using these cells for ATCT could therefore lead to improved clinical outcomes. METHOD: We describe a method to enrich T cells of early differentiation status using paramagnetic beads and antibodies targeting cells expressing C-C motif chemokine receptor 7 (CCR7). RESULTS: Selection of cells expressing CCR7 enriches T cells of bearing markers of early differentiation status. This was validated through analysis of an array of surface markers and an observed reduction in effector cell functions ex vivo. CCR7 selection resulted in dramatic 83.6 and 137 fold increases in circulating levels of CD4 and CD8 T cells respectively compared to non-sorted T cells 3 weeks after adoptive transfer to NSG mice. We observed no significant difference in the engraftment levels of CCR7 or CD62L selected cells in the NSG mouse model. Comparison of cells ex vivo, however, suggests CCR7 selection is superior to CD62L selection in enriching T cells of early differentiation status. CONCLUSIONS: CCR7 selection offers a means to enrich T cells of early differentiation status for ACTC. Together our data suggests that these T cells are likely to display enhanced engraftment and persistence in patients in vivo and could therefore improve therapeutic efficacy of ACTC.

CAR T-cell therapy: toxicity and the relevance of preclinical models.

Chimeric antigen receptor (CAR) T cells form part of a broad wave of immunotherapies that are showing promise in early phase cancer clinical trials. This clinical delivery has been based upon preclinical efficacy testing that confirmed the proof of principle of the therapy. However, CAR T-cell therapy does not exist alone as T cells are generally given in combination with patient preconditioning, most commonly in the form of chemotherapy, and may also include systemic cytokine support, both of which are associated with toxicity. Consequently, complete CAR T-cell therapy includes elements where the toxicity profile is well known, but also includes the CAR T cell itself, for which toxicity profiles are largely unknown. With recent reports of adverse events associated with CAR T-cell therapy, there is now concern that current preclinical models may not be fit for purpose with respect to CAR T-cell toxicity profiling. Here, we explore the preclinical models used to validate CAR T-cell function and examine their potential to predict CAR T-cell driven toxicities for the future.

Tissue-specific attenuation of oncolytic sindbis virus without compromised genetic stability.

Wild-type Sindbis virus (SV) shows promise as an oncolytic agent, although potential off-target replication is a safety concern. To remove possible pathology reflecting virus replication in liver, muscle, and/or hematopoietic cells, microRNA (miR)-response elements (MREs) to liver-specific miR122, muscle-specific miR133a and miR206, or hematopoietic-specific miR142-3p were inserted into the Sindbis viral genome. We compared the effectiveness of MREs in two distinct genomic locations and found better tissue-specific attenuation when they were inserted into the structural polyprotein coding region (up to 6000-fold selectivity with miR142-3p) rather than into the 3' untranslated region (up to 850-fold with miR142-3p). While this degree of tissue-specific attenuation may be effective for relieving pathology in vivo, genetic instability of RNA viruses raises concerns over the mutation or loss of MREs conferring safety. Genetically modified SVs containing a reporter transgene, used as a surrogate for virus replication, mutated quickly in vitro, losing 50% transgene sequence within 6.2 passages. Using a shorter insert containing MREs but no transgene, complete genetic stability was observed over at least 10 passages. We conclude that SV may be genetically modified to improve clinical properties, but attention must be paid to ensure that genetic stability is sufficient for intended applications.

Blood compatibility of enveloped viruses.

A significant limitation to the use of viruses as systemic vectors is the susceptibility of the vector to inactivation and clearance by various blood components. Despite much focus on antibodies as the primary neutralizing molecules in blood, other mechanisms inactivate and clear virus particles from the bloodstream in both naïve and pre-immune hosts. This review provides an overview of the major blood components that interact with enveloped viruses. The mechanisms of action of these blood components by which virus particles are inactivated are also discussed. In addition, important blood components that act as barriers to the systemic delivery of therapeutic viruses are identified, and recent advances in overcoming these barriers are highlighted. Particular attention is given to the field of oncolytic virotherapy in which adequate intravenous virus delivery is critical for therapeutic success.