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BACKGROUNDHead and neck squamous cell carcinoma not associated with HPV (HPV-unrelated HNSCC) is associated with a high rate of recurrence and poor survival.METHODSWe 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 TGF-β.RESULTSBintrafusp alfa was well tolerated, and no treatment-associated surgical delays or complications occurred. Objective pathologic responses (PRs) were observed, and 12 of the 14 (86%) patients were alive and disease free at 1 year. Alterations in Treg infiltration and spatial distribution relative to proliferating CD8+ T cells indicated a reversal of Treg immunosuppression in the primary tumor. Detection of neoepitope-specific tumor T cell responses, but not virus-specific responses, correlated with the development of a PR. Detection of neoepitope-specific responses and PRs 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-β blockade can safely enhance tumor antigen-specific immunity and highlight the feasibility of multimechanism neoadjuvant immunotherapy for patients with HPV-unrelated HNSCC.CONCLUSIONOur studies provide 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 antitumor immunity following such treatment.TRIAL REGISTRATIONClinicalTrials.gov NCT04247282.FUNDINGThis work was funded by the Center for Cancer Research, the NCI, and the Intramural Research Program of the NIDCD, NIH. Bintrafusp alfa was provided by the health care business of Merck KGaA (Darmstadt, Germany), through a Cooperative Research and Development Agreement with the NCI. Additional funding was provided by ImmunityBio through a Cooperative Research and Development Agreement with the NIDCD.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer type in the world, with Human Papilloma virus (HPV)-negative patients having a ~50% recurrence rate and poor median overall survival of ~13 months in the recurrent/metastatic setting. Pembrolizumab, a programmed-death-1 (PD-1) checkpoint inhibitor is active but with a response rate as low as 19%. Our preliminary work has identified SET and MYND Domain containing 3 (SMYD3) as a chromatin modifier that is significantly overexpressed in HPV-negative HNSCC tumor tissues compared to normal epithelium and is associated with low CD8+ T-cell infiltration in HPV-negative HNSCC. Furthermore, we have shown that knockdown of human SMYD3 upregulates the expression of multiple type I IFN response and antigen presentation machinery (APM) genes. This study aims to investigate whether Smyd3 depletion potentiates immune-mediated antitumor efficacy in anti-PD-1 resistant syngeneic mouse models of HPV-negative HNSCC (MOC1), as well as relevant mechanisms. SiRNA-mediated knockdown of Smyd3 in mouse HPV-negative MOC1 cells followed by RNA-seq revealed upregulation of multiple type I IFN response and APM genes in vitro. Multicolor flow cytometry of MOC1 tumors treated with control anti-sense oligonucleotides (ASOs) or Smyd3 ASOs showed that Smyd3 depletion in a syngeneic, heterotopic mouse model (C57BL/6) of flank MOC1 tumors induced intratumoral infiltration of CD8+ T-cells, as well as upregulation of H2-Kb (MHC class I) and mouse Pd-l1 in MOC1 cells. Combined treatment of Smyd3 ASOs with anti-PD-1 induced complete regressions of flank MOC1 tumors in 4/8 treated mice and significant tumor growth restriction in 2/8 mice, while 2/8 tumors “escaped” the treatment effect. Single-cell RNA seq of MOC1 tumors treated with control or Smyd3 ASOs is ongoing to evaluate the effects of Smyd3 depletion on MOC1 cancer cells and immune cell subsets of the tumor microenvironment and to identify potential mechanisms of resistance of Smyd3 ASO + anti-PD-1 combination treatment. Genome-wide mapping of Smyd3 and the repressive mark H4K20me3, which is known to be written by Smyd3, in MOC1 cells using CUT&RUN assays is also ongoing and aims to decipher whether Smyd3 directly binds to and regulates the expression of immune-related genes through H4K20me3. Ex-vivo cytotoxicity assays aim to evaluate whether CRISPR Smyd3 KO in MOC1 cells sensitizes cancer cells to TIL-mediated cytotoxicity. Smyd3 depletion induces an inflamed tumor microenvironment and may potentiate the immune-mediated antitumor efficacy of anti-PD-1 in syngeneic mouse models of HPV-negative HNSCC. This work may lay the biological rationale to combine SMYD3 inhibition with T-cell based immunotherapeutic approaches in HPV-negative HNSCC. Citation Format: Daniel E. Tsai, Yvette Robbins, Angel Huynh, Andrew Sinkoe, Cem Sievers, Madhavi Murali, Xiaolin Luo, Clint T. Allen, Vassiliki Saloura. Investigating the immunomodulatory mechanisms of Smyd3 depletion in HPV-negative head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6250.

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 alfa 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 alfa in adults with RRP. Papilloma samples before and after treatment with bintrafusp alfa 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.

Metabolic engineering and synthetic biology have enabled the use of microbial production platforms for the renewable production of many high-value natural products. Titers and yields, however, are often too low to result in commercially viable processes. Microbial co-cultures have the ability to distribute metabolic burden and allow for modular specific optimization in a way that is not possible through traditional monoculture fermentation methods. Here, we present an Escherichia coli co-culture for the efficient production of flavonoids in vivo, resulting in a 970-fold improvement in titer of flavan-3-ols over previously published monoculture production. To accomplish this improvement in titer, factors such as strain compatibility, carbon source, temperature, induction point, and inoculation ratio were initially optimized. The development of an empirical scaled-Gaussian model based on the initial optimization data was then implemented to predict the optimum point for the system. Experimental verification of the model predictions resulted in a 65% improvement in titer, to 40.7±0.1mg/L flavan-3-ols, over the previous optimum. Overall, this study demonstrates the first application of the co-culture production of flavonoids, the most in-depth co-culture optimization to date, and the first application of empirical systems modeling for improvement of titers from a co-culture system.

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.

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.

In this study, a data-driven computational procedure was utilized in order to identify candidate transcription factors involved in the gene regulation network associated with mitogenactivated protein kinase (MAPK) signal transduction. Because the network reconstruction algorithm used for this study is relatively new, one purpose of the study was to apply it toward a real problem to gain insight as to its performance. The study resulted in identification of numerous candidate gene network interactions, and simultaneously provided clues regarding the quality of network reconstruction that can be expected from the algorithm at its current stage of development.

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.