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The incidence of lymphoma is rising, now representing 3% of cancers in the UK alone. Current treatments include chemotherapy and radiotherapy, but whilst significant remission is often observed, responses frequently prove not to be durable. Thus, alternative treatments are sought. One such alternative is the use of bispecific antibodies (BsAb), which show dual specificity for both a target antigen on the tumour cell, and a cytotoxic trigger molecule on an effector cell. To further investigate these issues, we have designed a panel of F(ab')₂ BsAb against a range of murine B cell lymphoma antigens (idiotype, MHC class II, CD19, CD22 and D40). We have assessed the relative merits in targeting both T cells and neutrophils against these antigens. In the first case, BsAb were directed towards the CD3 and CD2 molecules of the T cell receptor. In the second, a transgenic mouse was utilised, to investigate the therapeutic potential of targeting G-CSF primed neutrophils via human FcγRI. Extensive analysis of these derivatives both in vitro and in vivo has revealed that it is the nature of the tumor antigen itself that has most bearing on therapy. This work indicates that effector mechanisms such as antibody-dependent cellular cytotoxicity, whilst certainly contributing to tumor removal, are not sufficient to eradicate the malignant clone. Instead, it appears likely that a combination of growth inhibitory signals, and generation of T cell immunity are more important to successful therapy. In light of this, Ab dimers were produced in an effort to increase the signalling potential of mAb. Following dimerisation, mAb were able to induce growth arrest and cell death in human lymphoma lines, as well as showing increased complement activation. Dimers were also able to protect against in vivo tumor challenge.

Natural killer (NK) cells contribute to immune surveillance against cancer, but the impact of common treatments, such as radiotherapy, on their interaction with cancer cells is poorly understood. Here the response of human peripheral blood NK cells against cancer cell lines treated with X-ray irradiation was investigated. Irradiation of cancer cells was found to significantly reduce NK cell cytotoxicity induced either constitutively or via antibody-dependent cellular cytotoxicity against cell lines from multiple cancers. Resistance appeared 72 hours post-irradiation and persisted for two weeks. Resistance was found to be linked to cell cycle arrest, since pharmacological inhibitors of the cell cycle induced similar resistance as radiotherapy. Although radiotherapy induced resistance to NK cell cytotoxicity, NK cell degranulation, conjugation, and detachment were found to be unaffected. These results were confirmed by live imaging of NK cells and cancer cells, which revealed that the dynamics of the NK cell-cancer cell interactions were not affected, but irradiated cancer cells were less likely to be killed. Instead, radiotherapy was found to induce an intrinsic resistance against perforin. Irradiated cancer cells were highly resistant to perforin-induced calcium flux and lytic death, but their susceptibility to death receptor mediated cytotoxicity was enhanced. Surprisingly, radiotherapy did not affect the binding of perforin or membrane repair in response to pore formation. Instead, functional pore formation, which was observed through the uptake of small dyes directly through the pore, was impaired. Radiation-induced time-dependent up-regulation of phosphatidylserine on the surface of cancer cells correlated with resistance to perforin. This link between phosphatidylserine and perforin inhibition was further demonstrated by the finding that increasing concentrations of phosphatidylserine on synthetic liposomes inhibited lysis by perforin. Finally, irradiation of cancer cells was also shown to inhibit cytotoxicity by CD19-specific CAR T cells. Together, these data establish a novel treatment-induced resistance to lymphocyte cytotoxicity, which may be important for the design of radiotherapy-immunotherapy protocols.

There is an urgent clinical need to improve outcomes for patients with muscle-invasive bladder cancer (MIBC). Radiotherapy (RT) plays an important role in the management of many of these patients. Data from pre-clinical studies show that RT may induce anti-tumour immune stimulatory changes in the tumour microenvironment and systemically. However, these effects seldom manifest clinically due to inherent and adaptive immune suppression within tumours. Combination strategies employing standard-of-care anti-PD-1/PD-L1 agents to enhance RT-induced cancer-specific immunity have demonstrated robust anti-tumour effects in pre-clinical models, even leading to systemic 'abscopal effects' out of the irradiated field. Despite success in murine models, results from early clinical trials testing this premise in patients with metastatic MIBC have been less impressive. The addition of alternative immune modulatory agents to RT may drive local and systemic anti-tumour immune effects, leading to improved survival outcomes in these patients. BO-112 is a novel, intratumourally-administered dsRNA analogue which has been shown to promote anti-tumour immune effects in murine models, and is being tested in an early phase clinical trial in combination with anti-PD-1 treatment in patients with advanced malignancies. It was hypothesised that the efficacy of RT could be improved by combination with BO-112 in murine models of bladder cancer, and enhanced further by the addition of anti-PD-1 treatment. Here, murine bladder cancer cells exhibited hallmarks of immunogenic cell death and may have induced a weak vaccination effect after RT, as well as upregulating several putative proteins which enhance T cell cytotoxicity. Preliminary data revealed that RT may have increased maturation of dendritic cells (DC) and activation of T cells but did not increase the proportion of CD8+ cells in the immune microenvironment of tumours. The majority of immune cells in non-treated and irradiated tumours were myeloid-derived populations. BO-112 reduced the rate of tumour growth in heterotopic MBT2 and MB49 models. A novel system was developed to administer BO-112 to orthotopic MBT2 tumours, which also slowed tumour growth. BO-112 induced tumour cell death and release of type I interferon in vitro, and a signal emerged that it led to activation of T cells and maturation of DC in vivo. Preliminary data suggest it may have increased the proportion of CD8+ cells in the immune microenvironment, but did not consistently modulate markers associated with macrophage polarisation. A signal emerged from further pilot studies that the addition of BO-112 to RT improved local tumour control in MB49 models after treatment. Dual therapy led to increased maturation of DC and activation of CD8+ cells in tumour-draining lymph nodes. However, the majority of mice developed distant metastases, suggesting a lack of robust systemic anti-tumour immune responses. There was no preliminary evidence that the efficacy of combination treatment was not enhanced by employing alternative sequential scheduling. Although combination treatment led to upregulation of PD-L1 on tumour cells in vitro, and myeloid-derived populations in vivo, there was no preliminary evidence that the addition of anti-PD-1 treatment to dual therapy did not further enhance local tumour control or induce systemic anti-tumour immunity. These results provide compelling preliminary data that justify further pre-clinical work into this novel treatment combination. If these findings are confirmed, this should provide proof-of-principle for future early phase trials investigating the efficacy of BO-112 in combination with RT in patients with MIBC. A clinical study determining the feasibility of cystoscopy-guided intratumoural delivery of BO-112 in patients with MIBC is currently in the final stages of development.

Immunotherapy has proven capable of delivering unheralded clinical benefit in many cancers, resulting in a transformation in research and treatment approaches. However, many challenges remain. Currently, immunotherapy only provides significant benefit in immunogenic tumours, and is only efficacious in a minority of patients. Research efforts are therefore focused on improving tumour immunogenicity in less immunogenic tumours such as breast cancer. Triple negative breast cancer (TNBC) has a poor prognosis, but may also have potential for generation of a robust anti-tumour immune response. Epigenetic alterations in anti-tumour immunity are thought to contribute to reduced immunogenicity; treatment with epigenetic agents including histone deacetylase inhibitors (HDACi) could therefore restore robust anti-tumour immunity through an immune priming effect. It was therefore hypothesised that HDACi could induce immune changes in TNBC leading to an effective anti-tumour immune response. To investigate this, a 4T1 orthotopic murine model was developed and characterised. A progressive systemic myeloid infiltrate was noted in the tumour microenvironment and systemic organs, alongside development of lung metastases. 4T1 cells released pro-myeloid cytokines in vitro, suggesting that increasing myeloid infiltration was likely driven by cytokines released by tumour cells in vivo. Treatment of the 4T1 model with the HDACi entinostat led to a non-statistically significant decrease in tumour volume and a trend towards reduction in lung metastatic burden. Since no entinostat-induced overall changes to immune cell population frequencies were detected, it was hypothesised that functional changes could be more important to treatment outcomes. Entinostat was able to reduce immunosuppressive function of CD11b+ cells within the spleen. However, there was also a suggestion that entinostat could increase the immunosuppressive function of CD11b+ cells within the lung. Combination therapy with entinostat and radiation revealed that single agent radiation could delay tumour growth but did not result in long-term tumour control or control of metastatic disease. Importantly, addition of entinostat to single dose, but not fractionated radiation, enhanced outcomes in this radio-resistant tumour model. Fractionated radiation led to increases in myeloid derived suppressor cells (MDSCs), particularly in the spleen. Addition of entinostat to fractionated radiation led to further increases in some MDSC populations. These data suggest that entinostat delivered either as monotherapy, or in combination with radiation, can alter the immune landscape. This has important implications for combination with other immunomodulatory therapies in future studies, to achieve maximum translational relevance and ensure that the potential of HDACi therapy can be realised.