Payload-delivering engineered γδ T cells display enhanced cytotoxicity, persistence, and efficacy in preclinical models of osteosarcoma.

T cell-based cancer immunotherapy has typically relied on membrane-bound cytotoxicity enhancers such as chimeric antigen receptors expressed in autologous αß T cells. These approaches are limited by tonic signaling of synthetic constructs and costs associated with manufacturing. γδ T cells are an emerging alternative for cellular therapy, having innate antitumor activity, potent antibody-dependent cellular cytotoxicity, and minimal alloreactivity. We present an immunotherapeutic platform technology built around the innate properties of the Vγ9Vδ2 T cell, harnessing specific characteristics of this cell type and offering an allocompatible cellular therapy that recruits bystander immunity. We engineered γδ T cells to secrete synthetic tumor-targeting opsonins in the form of an scFv-Fc fusion protein and a mitogenic IL-15Rα-IL-15 fusion protein (stIL15). Using GD2 as a model antigen, we show that GD2-specific opsonin-secreting Vγ9Vδ2 T cells (stIL15-OPS-γδ T cells) have enhanced cytotoxicity and promote bystander activity of other lymphoid and myeloid cells. Secretion of stIL-15 abrogated the need for exogenous cytokine supplementation and further mediated activation of bystander natural killer cells. Compared with unmodified γδ T cells, stIL15-OPS-γδ T cells exhibited superior in vivo control of subcutaneous tumors and persistence in the blood. Moreover, stIL15-OPS-γδ T cells were efficacious against patient-derived osteosarcomas in animal models and in vitro, where efficacy could be boosted with the addition of zoledronic acid. Together, the data identify stIL15-OPS-γδ T cells as a candidate allogeneic cell therapy platform combining direct cytolysis with bystander activation to promote tumor control.

Trellis tree-based analysis reveals stromal regulation of patient-derived organoid drug responses.

Patient-derived organoids (PDOs) can model personalized therapy responses; however, current screening technologies cannot reveal drug response mechanisms or how tumor microenvironment cells alter therapeutic performance. To address this, we developed a highly multiplexed mass cytometry platform to measure post-translational modification (PTM) signaling, DNA damage, cell-cycle activity, and apoptosis in >2,500 colorectal cancer (CRC) PDOs and cancer-associated fibroblasts (CAFs) in response to clinical therapies at single-cell resolution. To compare patient- and microenvironment-specific drug responses in thousands of single-cell datasets, we developed "Trellis"-a highly scalable, tree-based treatment effect analysis method. Trellis single-cell screening revealed that on-target cell-cycle blockage and DNA-damage drug effects are common, even in chemorefractory PDOs. However, drug-induced apoptosis is rarer, patient-specific, and aligns with cancer cell PTM signaling. We find that CAFs can regulate PDO plasticity-shifting proliferative colonic stem cells (proCSCs) to slow-cycling revival colonic stem cells (revCSCs) to protect cancer cells from chemotherapy.

A role for macrophages under cytokine control in mediating resistance to ADI-PEG20 (pegargiminase) in ASS1-deficient mesothelioma.

BACKGROUND: Pegylated arginine deiminase (ADI-PEG20; pegargiminase) depletes arginine and improves survival outcomes for patients with argininosuccinate synthetase 1 (ASS1)-deficient malignant pleural mesothelioma (MPM). Optimisation of ADI-PEG20-based therapy will require a deeper understanding of resistance mechanisms, including those mediated by the tumor microenvironment. Here, we sought to reverse translate increased tumoral macrophage infiltration in patients with ASS1-deficient MPM relapsing on pegargiminase therapy. METHODS: Macrophage-MPM tumor cell line (2591, MSTO, JU77) co-cultures treated with ADI-PEG20 were analyzed by flow cytometry. Microarray experiments of gene expression profiling were performed in ADI-PEG20-treated MPM tumor cells, and macrophage-relevant genetic "hits" were validated by qPCR, ELISA, and LC/MS. Cytokine and argininosuccinate analyses were performed using plasma from pegargiminase-treated patients with MPM. RESULTS: We identified that ASS1-expressing macrophages promoted viability of ADI-PEG20-treated ASS1-negative MPM cell lines. Microarray gene expression data revealed a dominant CXCR2-dependent chemotactic signature and co-expression of VEGF-A and IL-1α in ADI-PEG20-treated MPM cell lines. We confirmed that ASS1 in macrophages was IL-1α-inducible and that the argininosuccinate concentration doubled in the cell supernatant sufficient to restore MPM cell viability under co-culture conditions with ADI-PEG20. For further validation, we detected elevated plasma VEGF-A and CXCR2-dependent cytokines, and increased argininosuccinate in patients with MPM progressing on ADI-PEG20. Finally, liposomal clodronate depleted ADI-PEG20-driven macrophage infiltration and suppressed growth significantly in the MSTO xenograft murine model. CONCLUSIONS: Collectively, our data indicate that ADI-PEG20-inducible cytokines orchestrate argininosuccinate fuelling of ASS1-deficient mesothelioma by macrophages. This novel stromal-mediated resistance pathway may be leveraged to optimize arginine deprivation therapy for mesothelioma and related arginine-dependent cancers.

Targeting Triple Negative Breast Cancer With Oncolytic Adenoviruses.

Breast cancer (BC) is the most common cancer globally, accounting for 685,000 deaths in 2020. Triple-negative breast cancers (TNBC) lack oestrogen (ER) and progesterone (PR) hormone receptor expression and HER2 overexpression. TNBC represent 10-15% of all BC with high incidence in women under 50-years old that have BRCA mutations, and have a dismal prognosis. African American and Hispanic women are at higher risk partly due to the common occurrence of BRCA mutations. The standard treatment for TNBC includes surgery, radiotherapy, and chemotherapy although, resistance to all standard-of-care therapies eventually develops. It is crucial to identify and develop more efficacious therapeutics with different mechanisms of action to improve on survival in these women. Recent findings with oncolytic adenoviruses (OAds) may generate a new strategy to improve on the outcomes for women afflicted by TNBC and other types of BC. OAds are genetically engineered to selectively lyse, eliminate and recruit the host antitumour immune responses, leaving normal cells unharmed. The most common modifications are deletions in the early gene products including the E1B55 KDa protein, specific regions of the E1A protein, or insertion of tumour-specific promoters. Clinical trials using OAds for various adenocarcinomas have not yet been sufficiently evaluated in BC patients. Preclinical studies demonstrated efficacy in BC cell lines, including TNBC cells, with promising novel adenoviral mutants. Here we review the results reported for the most promising OAds in preclinical studies and clinical trials administered alone and in combination with current standard of care or with novel therapeutics. Combinations of OAds with small molecule drugs targeting the epidermal growth factor receptor (EGFR), androgen receptor (AR), and DNA damage repair by the novel PARP inhibitors are currently under investigation with reported enhanced efficacy. The combination of the PARP-inhibitor Olaparib with OAds showed an impressive anti-tumour effect. The most promising findings to date are with OAds in combination with antibodies towards the immune checkpoints or expression of cytokines from the viral backbone. Although safety and efficacy have been demonstrated in numerous clinical trials and preclinical studies with cancer-selective OAds, further developments are needed to eliminate metastatic lesions, increase immune activation and intratumoural viral spread. We discuss shortcomings of the OAds and potential solutions for improving on patient outcomes.

HDAC Inhibitors Enhance Efficacy of the Oncolytic Adenoviruses Ad∆∆ and Ad-3∆-A20T in Pancreatic and Triple-Negative Breast Cancer Models.

The prognosis for triple-negative breast cancer (TNBC) and pancreatic ductal adenocarcinoma (PDAC) is dismal. TNBC and PDAC are highly aggressive cancers with few treatment options and a potential for rapid resistance to standard-of-care chemotherapeutics. Oncolytic adenoviruses (OAds) represent a promising tumour-selective strategy that can overcome treatment resistance and eliminate cancer cells by lysis and host immune activation. We demonstrate that histone deacetylase inhibitors (HDACi) potently enhanced the cancer-cell killing of our OAds, Ad∆∆ and Ad-3∆-A20T in TNBC and PDAC preclinical models. In the TNBC cell lines MDA-MB-436, SUM159 and CAL51, cell killing, viral uptake and replication were increased when treated with sublethal doses of the Class-I-selective HDACis Scriptaid, Romidepsin and MS-275. The pan-HDACi, TSA efficiently improved OAd efficacy, both in vitro and in SUM159 xenograft models in vivo. Cell killing and Ad∆∆ replication was also significantly increased in five PDAC cell lines when pre-treated with TSA. Efficacy was dependent on treatment time and dose, and on the specific genetic alterations in each cell line. Expression of the cancer specific αvß6-integrin supported higher viral uptake of the integrin-retargeted Ad-3∆-A20T in combination with Scriptaid. In conclusion, we demonstrate that inhibition of specific HDACs is a potential means to enhance OAd activity, supporting clinical translation.

Payload Delivery: Engineering Immune Cells to Disrupt the Tumour Microenvironment.

Although chimeric antigen receptor (CAR) T cells have shown impressive clinical success against haematological malignancies such as B cell lymphoma and acute lymphoblastic leukaemia, their efficacy against non-haematological solid malignancies has been largely disappointing. Solid tumours pose many additional challenges for CAR T cells that have severely blunted their potency, including homing to the sites of disease, survival and persistence within the adverse conditions of the tumour microenvironment, and above all, the highly immunosuppressive nature of the tumour milieu. Gene engineering approaches for generating immune cells capable of overcoming these hurdles remain an unmet therapeutic need and ongoing area of research. Recent advances have involved gene constructs for membrane-bound and/or secretable proteins that provide added effector cell function over and above the benefits of classical CAR-mediated cytotoxicity, rendering immune cells not only as direct cytotoxic effectors against tumours, but also as vessels for payload delivery capable of both modulating the tumour microenvironment and orchestrating innate and adaptive anti-tumour immunity. We discuss here the novel concept of engineered immune cells as vessels for payload delivery into the tumour microenvironment, how these cells are better adapted to overcome the challenges faced in a solid tumour, and importantly, the novel gene engineering approaches required to deliver these more complex polycistronic gene constructs.

Advances in oncolytic adenovirus therapy for pancreatic cancer.

Survival rates for pancreatic cancer patients have remained unchanged for the last four decades. The most aggressive, and most common, type of pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC), which has the lowest 5-year survival rate of all cancers globally. The poor prognosis is typically due to late presentation of often non-specific symptoms and rapid development of resistance to all current therapeutics, including the standard-of-care cytotoxic drug gemcitabine. While early surgical intervention can significantly prolong patient survival, there are few treatment options for late-stage non-resectable metastatic disease, resulting in mostly palliative care. In addition, a defining feature of pancreatic cancer is the immunosuppressive and impenetrable desmoplastic stroma that blocks access to tumour cells by therapeutic drugs. The limited effectiveness of conventional chemotherapeutics reveals an urgent need to develop novel therapies with different mechanisms of action for this malignancy. An emerging alternative to current therapeutics is oncolytic adenoviruses; these engineered biological agents have proven efficacy and tumour-selectivity in preclinical pancreatic cancer models, including models of drug-resistant cancer. Safety of oncolytic adenoviral mutants has been extensively assessed in clinical trials with only limited toxicity to normal healthy tissue being reported. Promising efficacy in combination with gemcitabine was demonstrated in preclinical and clinical studies. A recent surge in novel adenoviral mutants entering clinical trials for pancreatic cancer indicates improved efficacy through activation of the host anti-tumour responses. The potential for adenoviruses to synergise with chemotherapeutics, activate anti-tumour immune responses, and contribute to stromal dissemination render these mutants highly attractive candidates for improved patient outcomes. Currently, momentum is gathering towards the development of systemically-deliverable mutants that are able to overcome anti-viral host immune responses, erythrocyte binding and hepatic uptake, to promote elimination of primary and metastatic lesions. This review will cover the key components of pancreatic cancer oncogenesis; novel oncolytic adenoviruses; clinical trials; and the current progress in overcoming the challenges of systemic delivery.