A compact and simple method of achieving differential transgene expression by exploiting translational readthrough

The development of multicistronic vectors enabling differential transgene expression is a goal of gene therapy and poses a significant engineering challenge. Current approaches rely on the insertion of long regulatory sequences that occupy valuable space in vectors, which have a finite and limited packaging capacity. Here we describe a simple method of achieving differential transgene expression by inserting stop codons and translational readthrough motifs (TRMs) to suppress stop codon termination. TRMs reduced downstream transgene expression ∼sixfold to ∼140-fold, depending on the combination of stop codon and TRM used. We show that a TRM can facilitate the controlled secretion of the highly potent cytokine IL-12 at therapeutically beneficial levels in an aggressive immunocompetent mouse melanoma model to prevent tumor growth. Given their compact size (6 bp) and ease of introduction, we envisage that TRMs will be widely adopted in recombinant DNA engineering to facilitate differential transgene expression

AKT inhibition generates potent polyfunctional clinical grade AUTO1 CAR T-cells, enhancing function and survival

BACKGROUND: AUTO1 is a fast off-rate CD19-targeting chimeric antigen receptor (CAR), which has been successfully tested in adult lymphoblastic leukemia. Tscm/Tcm-enriched CAR-T populations confer the best expansion and persistence, but Tscm/Tcm numbers are poor in heavily pretreated adult patients. To improve this, we evaluate the use of AKT inhibitor (VIII) with the aim of uncoupling T-cell expansion from differentiation, to enrich Tscm/Tcm subsets. METHODS: VIII was incorporated into the AUTO1 manufacturing process based on the semiautomated the CliniMACS Prodigy platform at both small and cGMP scale. RESULTS: AUTO1 manufactured with VIII showed Tscm/Tcm enrichment, improved expansion and cytotoxicity in vitro and superior antitumor activity in vivo. Further, VIII induced AUTO1 Th1/Th17 skewing, increased polyfunctionality, and conferred a unique metabolic profile and a novel signature for autophagy to support enhanced expansion and cytotoxicity. We show that VIII-cultured AUTO1 products from B-ALL patients on the ALLCAR19 study possess superior phenotype, metabolism, and function than parallel control products and that VIII-based manufacture is scalable to cGMP. CONCLUSION: Ultimately, AUTO1 generated with VIII may begin to overcome the product specific factors contributing to CD19+relapse

Development of a Chimeric Antigen Receptor (CAR) - based T cell therapy for glioblastoma

High grade gliomas are aggressive brain tumours for which treatment is highly challenging due to the location within the central nervous system (CNS), which may reduce access of cytotoxic chemotherapy, and their infiltrative growth, which precludes complete surgical resection. Current treatment includes surgical removal – wherever possible - followed by radiotherapy and chemotherapy. However, recurrence is common, resulting in a survival of only 12 to 15 months after diagnosis. This highlights the need for new therapies. Chimeric antigen receptors (CARs) are synthetic molecules which combine the specificity of an antibody to the signalling domains of a T cell receptor (TCR), allowing T cells to directly recognise tumour antigens with no need for co-stimulation. CAR-T cells have shown promising responses in the treatment of haematological malignancies, inducing complete and durable responses in patients with chemo-refractory disease treated with CD19-redirected T cells. This therapeutic approach may be highly suitable for high grade gliomas as T cells have the ability to track to distant tumour sites. However, translation of this technology to solid tumours is proving more difficult, due to several challenges, including: requirement for an effective infiltration of CAR-T cells within the tumour and the immunosuppressive environment provided by solid malignancies. In this work, we developed an immunocompetent animal model of glioma, to study kinetics of migration and infiltration of CAR-T cells and the interplay between CAR-T cells, the tumour and the endogenous immune system to inform the design of T cell immunotherapy for this brain tumours. The tumour specific variant III of the epidermal growth factor receptor (EGFRvIII) – a mutation found in 30% of glioblastomas – was used as model antigen. A murine CAR was constructed based on the single chain fragment variant (ScFv) of EGFRvIII-specific antibody MR1.1 linked with a CD8 stalk to CD28-CD3ζ activation domains. A murine marker gene (truncated CD34) was co-expressed to allow for ex vivo analysis as well as firefly luciferase for in vivo tracking of CAR T-cells. The mouse glioma cell line GL261 was modified to express the mouse version of EGFRvIII and used to establish orthotopic tumours. After validation of function and specificity in vitro, efficacy of CAR-T cells was tested in vivo. Both bioluminescence imaging (BLI) and flow cytometry demonstrated that CAR T cells accumulated within the tumour in an antigen-dependent manner. MRI demonstrated that CAR T cells delayed tumour growth and increased survival. However, tumours were not consistently eradicated. Both immunohistochemistry and BLI indicated lack of long term persistence of T cells within the tumour. Analysis of tumour infiltrating lymphocytes (TILs) phenotype suggested that decreased functionality of CAR-T cells could be a result of their exhaustion in situ. We hypothesised that additional strategies were required to improve efficacy and persistence of CAR-T cells. We postulated that CAR-T cell fitness may be prolonged by: - Incorporation of 41BB as additional co-stimulatory domain in the CAR to provide a pro-survival signal. - Combination therapy with PD1 blockade to overcome T cell exhaustion (both on CAR and endogenous T cells) in situ. While the employment of third-generation CAR did not significantly improve survival and showed increased toxicity, combination therapy of CAR-T cells and PD-1 blockade promoted complete clearance of tumours resulting in long term survival. Immunohistochemistry and flow cytometry analysis suggested that combination therapy may increase persistence of CAR-T cells, leading to a more rapid and consistent tumour eradication compared to CAR-T cell administration alone. However, data presented here did not demonstrate a synergistic effect of CAR-T cell therapy and PD1 blockade, as an effect of PD1 blockade alone was also observed. Therefore, additional experiments are required to examine this further

Computational fluid dynamics with imaging of cleared tissue and of in vivo perfusion predicts drug uptake and treatment responses in tumours

Understanding the uptake of a drug by diseased tissue, and the drug’s subsequent spatiotemporal distribution, are central factors in the development of effective targeted therapies. However, the interaction between the pathophysiology of diseased tissue and individual therapeutic agents can be complex, and can vary across tissue types and across subjects. Here, we show that the combination of mathematical modelling, high-resolution optical imaging of intact and optically cleared tumour tissue from animal models, and in vivo imaging of vascular perfusion predicts the heterogeneous uptake, by large tissue samples, of specific therapeutic agents, as well as their spatiotemporal distribution. In particular, by using murine models of colorectal cancer and glioma, we report and validate predictions of steady-state blood flow and intravascular and interstitial fluid pressure in tumours, of the spatially heterogeneous uptake of chelated gadolinium by tumours, and of the effect of a vascular disrupting agent on tumour vasculature

AKT inhibition generates potent polyfunctional clinical grade AUTO1 CAR T-cells, enhancing function and survival

Background AUTO1 is a fast off-rate CD19-targeting chimeric antigen receptor (CAR), which has been successfully tested in adult lymphoblastic leukemia. Tscm/Tcm-enriched CAR-T populations confer the best expansion and persistence, but Tscm/Tcm numbers are poor in heavily pretreated adult patients. To improve this, we evaluate the use of AKT inhibitor (VIII) with the aim of uncoupling T-cell expansion from differentiation, to enrich Tscm/Tcm subsets.Methods VIII was incorporated into the AUTO1 manufacturing process based on the semiautomated the CliniMACS Prodigy platform at both small and cGMP scale.Results AUTO1 manufactured with VIII showed Tscm/Tcm enrichment, improved expansion and cytotoxicity in vitro and superior antitumor activity in vivo. Further, VIII induced AUTO1 Th1/Th17 skewing, increased polyfunctionality, and conferred a unique metabolic profile and a novel signature for autophagy to support enhanced expansion and cytotoxicity. We show that VIII-cultured AUTO1 products from B-ALL patients on the ALLCAR19 study possess superior phenotype, metabolism, and function than parallel control products and that VIII-based manufacture is scalable to cGMP.Conclusion Ultimately, AUTO1 generated with VIII may begin to overcome the product specific factors contributing to CD19+relapse

Intratumoral IL-12 delivery empowers CAR-T cell immunotherapy in a pre-clinical model of glioblastoma

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain cancer, for which effective therapies are urgently needed. Chimeric antigen receptor (CAR)-based immunotherapy represents a promising therapeutic approach, but it is often impeded by highly immunosuppressive tumor microenvironments (TME). Here, in an immunocompetent, orthotopic GBM mouse model, we show that CAR-T cells targeting tumor-specific epidermal growth factor receptor variant III (EGFRvIII) alone fail to control fully established tumors but, when combined with a single, locally delivered dose of IL-12, achieve durable anti-tumor responses. IL-12 not only boosts cytotoxicity of CAR-T cells, but also reshapes the TME, driving increased infiltration of proinflammatory CD4$^{+}$ T cells, decreased numbers of regulatory T cells (Treg), and activation of the myeloid compartment. Importantly, the immunotherapy-enabling benefits of IL-12 are achieved with minimal systemic effects. Our findings thus show that local delivery of IL-12 may be an effective adjuvant for CAR-T cell therapy for GBM

Potential of magnetic hyperthermia to stimulate localized immune activation

Magnetic hyperthermia (MH) harnesses the heat-releasing properties of superparamagnetic iron oxide nanoparticles (SPIONs) and has potential to stimulate immune activation in the tumor microenvironment whilst sparing surrounding normal tissues. To assess feasibility of localized MH in vivo, SPIONs are injected intratumorally and their fate tracked by Zirconium-89-positron emission tomography, histological analysis, and electron microscopy. Experiments show that an average of 49% (21–87%, n = 9) of SPIONs are retained within the tumor or immediately surrounding tissue. In situ heating is subsequently generated by exposure to an externally applied alternating magnetic field and monitored by thermal imaging. Tissue response to hyperthermia, measured by immunohistochemical image analysis, reveals specific and localized heat-shock protein expression following treatment. Tumor growth inhibition is also observed. To evaluate the potential effects of MH on the immune landscape, flow cytometry is used to characterize immune cells from excised tumors and draining lymph nodes. Results show an influx of activated cytotoxic T cells, alongside an increase in proliferating regulatory T cells, following treatment. Complementary changes are found in draining lymph nodes. In conclusion, results indicate that biologically reactive MH is achievable in vivo and can generate localized changes consistent with an anti-tumor immune response.</p