BackgroundT-cell-engaging CD3-bispecific antibodies (CD3-bsAbs) are promising modalities for cancer immunotherapy. Although this therapy has reached clinical practice for hematological malignancies, the absence of sufficient infiltrating T cells is a major barrier for efficacy in solid tumors. In this study, we exploited oncolytic reovirus as a strategy to enhance the efficacy of CD3-bsAbs in immune-silent solid tumors.MethodsThe mutant p53 and K-ras induced murine pancreatic cancer model KPC3 resembles human pancreatic ductal adenocarcinomas with a desmoplastic tumor microenvironment, low T-cell density and resistance to immunotherapy. Immune-competent KPC3 tumor-bearing mice were intratumorally injected with reovirus type 3 Dearing strain and the reovirus-induced changes in the tumor microenvironment and spleen were analyzed over time by NanoString analysis, quantitative RT-PCR and multicolor flow cytometry. The efficacy of reovirus in combination with systemically injected CD3-bsAbs was evaluated in immune-competent mice with established KPC3 or B16.F10 tumors, and in the close-to-patient human epidermal growth factor receptor 2 (HER2)+ breast cancer model BT474 engrafted in immunocompromised mice with human T cells as effector cells.ResultsReplication-competent reovirus induced an early interferon signature, followed by a strong influx of natural killer cells and CD8+ T cells, at the cost of FoxP3+ Tregs. Viral replication declined after 7 days and was associated with a systemic activation of lymphocytes and the emergence of intratumoral reovirus-specific CD8+ T cells. Although tumor-infiltrating T cells were mostly reovirus-specific and not tumor-specific, they served as non-exhausted effector cells for the subsequently systemically administered CD3-bsAbs. Combination treatment of reovirus and CD3-bsAbs led to the regression of large, established KPC3, B16.F10 and BT474 tumors. Reovirus as a preconditioning regimen performed significantly better than simultaneous or early administration of CD3-bsAbs. This combination treatment induced regressions of distant lesions that were not injected with reovirus, and systemic administration of both reovirus and CD3-bsAbs also led to tumor control. This suggests that this therapy might also be effective for metastatic disease.ConclusionsOncolytic reovirus administration represents an effective strategy to induce a local interferon response and strong T-cell influx, thereby sensitizing the tumor microenvironment for subsequent CD3-bsAb therapy. This combination therapy warrants further investigation in patients with non-inflamed solid tumors.

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BackgroundPancreatic ductal adenocarcinoma (PDAC) is notoriously resistant to treatment including checkpoint-blockade immunotherapy. We hypothesized that a bimodal treatment approach consisting of dendritic cell (DC) vaccination to prime tumor-specific T cells, and a strategy to reprogram the desmoplastic tumor microenvironment (TME) would be needed to break tolerance to these pancreatic cancers. As a proof-of-concept, we investigated the efficacy of combined DC vaccination with CD40-agonistic antibodies in a poorly immunogenic murine model of PDAC. Based on the rationale that mesothelioma and pancreatic cancer share a number of tumor associated antigens, the DCs were loaded with either pancreatic or mesothelioma tumor lysates.MethodsImmune-competent mice with subcutaneously or orthotopically growing KrasG12D/+;Trp53R172H/+;Pdx-1-Cre (KPC) PDAC tumors were vaccinated with syngeneic bone marrow-derived DCs loaded with either pancreatic cancer (KPC) or mesothelioma (AE17) lysate and consequently treated with FGK45 (CD40 agonist). Tumor progression was monitored and immune responses in TME and lymphoid organs were analyzed using multicolor flow cytometry and NanoString analyzes.ResultsMesothelioma-lysate loaded DCs generated cross-reactive tumor-antigen-specific T-cell responses to pancreatic cancer and induced delayed tumor outgrowth when provided as prophylactic vaccine. In established disease, combination with stimulating CD40 antibody was necessary to improve survival, while anti-CD40 alone was ineffective. Extensive analysis of the TME showed that anti-CD40 monotherapy did improve CD8 +T cell infiltration, but these essential effector cells displayed hallmarks of exhaustion, including PD-1, TIM-3 and NKG2A. Combination therapy induced a strong change in tumor transcriptome and mitigated the expression of inhibitory markers on CD8 +T cells.ConclusionThese results demonstrate the potency of DC therapy in combination with CD40-stimulation for the treatment of pancreatic cancer and provide directions for near future clinical trials.

Due to the high coverage of international vaccination programs, most people worldwide have been vaccinated against common pathogens, leading to acquired pathogen-specific immunity with a robust memory T cell repertoire. While CD8+ anti-tumor cytotoxic T lymphocytes (CTL) are the preferred effectors of cancer immunotherapy, CD4+ T cell help is also required for an optimal antitumor immune response to occur. Hence, we investigated whether the pathogen-related CD4+ T cell memory populations could be re-engaged to support the CTLs, converting a weak primary antitumor immune response into a stronger secondary one. To this end, we used our PeptiCRAd technology that consists of an oncolytic adenovirus coated with MHC-I-restricted tumor-specific peptides, and developed it further by introducing pathogen-specific MHC-II-restricted peptides. Mice pre-immunized with tetanus vaccine were challenged with B16.OVA tumors and treated with the newly developed hybrid TT-OVA-PeptiCRAd containing both tetanus toxoid-and tumorspecific peptides. Treatment with the hybrid PeptiCRAd significantly enhanced anti-tumor efficacy and induced TT-specific, CD40 ligand-expressing CD4+ T helper cells and maturation of antigen presenting cells (APCs). Importantly, this approach could be extended to naturally occurring tumor peptides (both tumor-associated antigens and neoantigens), as well as to other pathogens beyond tetanus, highlighting the usefulness of this technique to take full advantage of CD4+ memory T cell repertoires when designing immunotherapeutic treatment regimens. Finally, the anti-tumor effect was even more prominent when combined with the immune checkpoint inhibitor anti-PD1, strengthening the rationale behind combination therapy with oncolytic viruses. Research.

Recent approaches in the treatment of cancer focus on involving the immune system to control the tumor growth. The administration of immunotherapies, like checkpoint inhibitors, has shown impressive results in the long term survival of patients. Cancer vaccines are being investigated as further tools to prime tumor-specific immunity. Biomaterials show potential as adjuvants in the formulation of vaccines, and biomimetic elements derived from the membrane of tumor cells may widen the range of antigens contained in the vaccine. Here, we show how mice presenting an aggressive melanoma tumor model treated twice with the complete nanovaccine formulation showed control on the tumor progression, while in a less aggressive model, the animals showed remission and control on the tumor progression, with a modification in the immunological profile of the tumor microenvironment. We also prove that co-administration of the nanovaccine together with a checkpoint inhibitor increases the efficacy of the treatment (87.5% of the animals responding, with 2 remissions) compared to the checkpoint inhibitor alone in the B16.OVA model. Our platform thereby shows potential applications as a cancer nanovaccine in combination with the standard clinical care treatment for melanoma cancers.

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Background and PurposeAgonists for the liver X receptor (LXR) are considered promising therapeutic moieties in cholesterol‐driven diseases by promoting cellular cholesterol efflux pathways. However, current clinical application of these agents is hampered by concomitant LXR‐induced activation of a lipogenic transcriptional network, leading to hepatic steatosis. Recent studies have suggested that protein arginine methyltransferase 3 (PRMT3) may act as a selective co‐activator of LXR activity. Here, we verified the hypothesis that PRMT3 inhibition selectively disrupts the ability of LXR to stimulate lipogenesis while maintaining its capacity to modulate macrophage cholesterol homeostasis.Experimental ApproachA combination of the LXR agonist T0901317 and palm oil was administered to C57BL/6 mice to maximally stimulate LXR and PRMT3 activity. PRMT3 activity was inhibited using the allosteric inhibitor SGC707.Key ResultsTreatment with SGC707 did not negatively influence the T0901317/palm oil‐induced up‐regulation of the cholesterol efflux ATP‐binding cassette transporter genes, ABCA1 and ABCG1, in peritoneal cells. In contrast, SGC707 treatment was associated with a significant decrease in the hepatic expression of the lipogenic gene fatty acid synthase (−64%). A similar trend was observed for stearoyl‐coenzyme A desaturase and acetyl CoA carboxylase expression (−43%; −56%). This obstruction of lipogenic gene transcription coincided with a significant 2.3‐fold decrease in liver triglyceride content as compared with the T0901317 and palm oil‐treated control group.Conclusion and ImplicationsWe showed that inhibition of PRMT3 activity by SGC707 treatment selectively impairs LXR‐driven transcription of hepatic lipogenic genes, while the positive effect of LXR stimulation on macrophage cholesterol efflux pathways is maintained.

The surface inhibitory receptor NKG2A forms heterodimers with the invariant CD94 chain and is expressed on a subset of activated CD8 T cells. As antibodies to block NKG2A are currently tested in several efficacy trials for different tumor indications, it is important to characterize the NKG2A+ CD8 T cell population in the context of other inhibitory receptors. Here we used a well‐controlled culture system to study the kinetics of inhibitory receptor expression. Naïve mouse CD8 T cells were synchronously and repeatedly activated by artificial antigen presenting cells in the presence of the homeostatic cytokine IL‐7. The results revealed NKG2A as a late inhibitory receptor, expressed after repeated cognate antigen stimulations. In contrast, the expression of PD‐1, TIGIT and LAG‐3 was rapidly induced, hours after first contact and subsequently down regulated during each resting phase. This late, but stable expression kinetics of NKG2A was most similar to that of TIM‐3 and CD39. Importantly, single‐cell transcriptomics of human tumor‐infiltrating lymphocytes (TILs) showed indeed that these receptors were often coexpressed by the same CD8 T cell cluster. Furthermore, NKG2A expression was associated with cell division and was promoted by TGF‐β in vitro, although TGF‐β signaling was not necessary in a mouse tumor model in vivo. In summary, our data show that PD‐1 reflects recent TCR triggering, but that NKG2A is induced after repeated antigen stimulations and represents a late inhibitory receptor. Together with TIM‐3 and CD39, NKG2A might thus mark actively dividing tumor‐specific TILs.