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Recurrent respiratory papillomatosis (RRP) is a debilitating neoplastic disorder of the upper aerodigestive tract caused by chronic infection with low-risk human papillomavirus types 6 or 11. Patients with severe RRP can require hundreds of lifetime surgeries to control their disease and pulmonary papillomatosis can be fatal. Here we report the comprehensive genomic and transcriptomic characterization of respiratory papillomas. We discovered and characterized distinct subtypes with transcriptional resemblance to either a basal or differentiated cell state that associate with disease aggressiveness and differ in key molecular, immune and APOBEC mutagenesis profiles. Through integrated comparison with high-risk HPV-associated head and neck squamous cell carcinoma, our analysis revealed divergent molecular and immune papilloma subtypes that form independent of underlying genomic alterations. Cumulatively our results support the development of dysregulated cellular proliferation and suppressed anti-viral immunity through distinct programs of squamous cell differentiation and associated expression of low-risk HPV genes. These analyses provide insight into the pathogenesis of respiratory papillomas and provide a foundation for the development of therapeutic strategies.

Background: Head and neck squamous cell carcinoma not associated with human papillomavirus (HPV-unrelated HNSCC) is associated with high rates of recurrence and poor survival.Methods: We conducted a clinical trial in 14 patients with newly diagnosed, HPV-unrelated HNSCC to evaluate the safety and efficacy of neoadjuvant bintrafusp alfa, a bifunctional fusion protein that blocks programmed death-ligand 1 (PD-L1) and neutralizes transforming growth factor-beta (TGF-).Results: Bintrafusp alfa was well tolerated, and no treatment-associated surgical delays or complications occurred. Objective pathologic responses were observed and 12 of 14 patients (86%) were alive and disease free at one year. Alterations in regulatory T cell infiltration and spatial distribution relative to proliferating CD8 T cells indicated reversal of Treg immunosuppression in the primary tumor. Detection of neoepitope-specific tumor T cell responses, but not viral-specific responses, correlated with development of a pathologic response. Detection of neoepitope-specific responses and pathologic responses in tumors was not correlated with genomic features or tumor antigenicity but was associated with reduced pretreatment myeloid cell tumor infiltration. These results indicate that dual PD-L1 and TGF- blockade can safely enhance tumor antigen-specific immunity and highlight the feasibility of multi-mechanism neoadjuvant immunotherapy in patients with HPV-unrelated HNSCC. Conclusion: Our studies provide new insight into the ability of neoadjuvant immunotherapy to induce polyclonal neoadjuvant-specific T cell responses in tumors and suggest that features of the tumor microenvironment, such as myeloid cell infiltration, may be a major determinant of enhanced anti-tumor immunity following such treatment.

IL-6 signaling plays an important role in inflammatory processes in the body. While a number of models for IL-6 signaling are available, the parameters associated with these models vary from case to case as they are non-trivial to determine. In this study, optimal experimental design is utilized to reduce the parameter uncertainty of an IL-6 signaling model consisting of ordinary differential equations, thereby increasing the accuracy of the estimated parameter values and, potentially, the model itself. The D-optimality criterion, operating on the Fisher information matrix and, separately, on a sensitivity matrix computed from the Morris method, was used as the objective function for the optimal experimental design problem. Optimal input functions for model parameter estimation were identified by solving the optimal experimental design problem, and the resulting input functions were shown to significantly decrease parameter uncertainty in simulated experiments. Interestingly, the determined optimal input functions took on the shape of PRBS signals even though there were no restrictions on their nature. Future work should corroborate these findings by applying the determined optimal experimental design on a real experiment.

101 Background: Genetically engineered T-cell therapy has shown remarkable clinical activity in hematologic malignancies. It is not known if this type of treatment can be applied effectively to epithelial cancers, which account for 80% to 90% of human malignancies. Methods: We conducted a phase I clinical trial with a 3 + 3 dose escalation in which patients with metastatic HPV-16+ epithelial cancers were treated with a one-time infusion of genetically engineered T cells expressing a T-cell receptor targeting an HLA-A*02:01-restricted epitope of HPV-16 E7 (E7 TCR-T cells). A lymphocyte-depleting conditioning regimen was administered before cell infusion, and high-dose systemic aldesleukin was administered after cell infusion. Results: Twelve patients, previously treated with a median of 4 (range, 3 to 7) anticancer agents, were treated. The cell dose was not limited by toxicity. Six patients demonstrated objective clinical responses, which included regression of bulky tumors and complete elimination of some tumors. Responses occurred in patients with vulvar, anal, head and neck, and cervical cancer. Four patients who previously received PD-1-based therapy responded. Response duration ranged from 3 to 9 months. Sustained, high-level engraftment of E7 TCR-T cells in peripheral blood was observed (median after approximately 6 weeks, 66% of total T cells, range 1% to 88%) and correlated with cell dose but not with clinical response. Infused T cell characteristics did not correlate strongly with response. Of the 4 resistant tumors that were studied, 3 demonstrated genetic defects in HLA-A*02:01 or B2M (necessary components of the target complex) and 1 demonstrated copy loss with decreased expression of antigen presentation and interferon response molecules (i.e. TAP1, TAP2, IFNGR1, IFNGR2). Of the 3 sensitive tumors studied, 0 showed genetic defects in these molecules. Conclusions: E7 TCR-T cells demonstrated safety and clinical activity in the treatment of highly refractory metastatic HPV-16+ cancers. Treatment resistance was linked to definitive genetic defects in the targeted peptide-HLA complex and to manifold defects in antigen processing and interferon response. Clinical trial information: NCT02858310.

BackgroundRecurrent respiratory papillomatosis (RRP) is a human papillomavirus (HPV) driven neoplastic disorder of the upper aerodigestive tract that causes significant morbidity and can lead to fatal airway obstruction. Prior clinical study demonstrated clinical benefit with the programmed death-ligand 1 (PD-L1) monoclonal antibody avelumab. Bintrafusp alpha is a bifunctional inhibitor of PD-L1 and transforming growth factor-beta (TGF-b) that has shown clinical activity in several cancer types.MethodsWe conducted a phase II clinical trial evaluating bintrafusp alpha in adults with RRP. Papilloma samples before and after treatment with bintrafusp alpha were assessed for correlates of response with multiplex immunofluorescence as well as immunological and genomic analyses. Post hoc analyses of papilloma samples before and after treatment with avelumab were assessed for comparison.ResultsDual PD-L1/TGF-b inhibition failed to abrogate papilloma growth in most subjects and increased the frequency of clinically indicated interventions after treatment in four of eight subjects based on each subject’s own historical control. TGF-b neutralization consistently decreased pSMAD3 and p21 and increased Ki67 expression within the basal layers of papillomas, indicating that TGF-b restrained proliferation. These alterations were not observed in papillomas treated with PD-L1 blockade alone. Dual PD-L1/TGF-b inhibition did not enhance anti-HPV immunity within papillomas beyond that observed with PD-L1 blockade. Genomic alterations in TGF-b superfamily genes were infrequent in papillomas and normal mucosa but present in a significant fraction of head and neck carcinomas.ConclusionsIntact TGF-b signaling restrains proliferation within papillomas, and the use of clinical agents that abrogate this pathway should be avoided in patients with RRP.Trial registration numbersNCT03707587 and NCT02859454.

The isolation of T cells, followed by differentiation into Regulatory T cells (Tregs), and re-transplantation into the body has been proposed as a therapeutic option for inflammatory bowel disease. A key requirement for making this a viable therapeutic option is the generation of a large population of Tregs. However, cytokines in the local microenvironment can impact the yield of Tregs during differentiation. As such, experimental design is an essential part of evaluating the importance of different cytokine concentrations for Treg differentiation. However, currently only single, constant concentrations of the cytokines have been investigated. This work addresses this point by performing experimental design in silico which seeks to maximize the predicted induction of Tregs relative to Th17 cells, by selecting an optimal input function for the concentrations of TGF-β, IL-2, IL-6, and IL-23. While this approach sounds promising, the results show that only marginal improvements in the concentration of Tregs can be achieved for dynamic cytokine profiles as compared to optimal constant concentrations. Since constant concentrations are easier to implement in experiments, it is recommended for this particular system to keep the concentrations constant where IL-6 should be kept low and high concentrations of TGF-β, IL-2, and IL-23 should be used.