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Despite reports of striking outcomes, immunotherapy efficacy in melanoma is limited to subsets of patients 1, 2. Combining immunotherapies with other modalities has yielded limited improvements but also adverse events requiring cessation of treatment 1. In addition to ineffective patient stratification, efficacy can be impaired by paucity of tumor-infiltrating lymphocytes (TILs). Thus, effective strategies to safely increase TILs are urgently needed to improve immunotherapies 3. Here, we report that dietary administration of the sugar L-fucose triggers CD4+T cell-mediated increases in TILs, anti-tumor immunity, and enhanced immune checkpoint blockade responses. This is induced by the fucosylation and cell surface enrichment of the MHC-II protein HLA-DRB1 in melanoma. Single-cell immunofluorescent staining analysis of patient melanoma specimens demonstrates that fucosylation and fucosylated HLA-DRB1 is associated with intratumoral T cell abundance and anti-PD1 responder status. Our findings demonstrate that fucosylation is a key mediator of anti-tumor immunity, via regulation of melanoma cell surface HLA-DRB1 and induction of anti-tumor immunity, suggesting use of melanoma fucosylation as a novel strategy to stratify patients for immunotherapies. Importantly, our study suggests that L-fucose represents a powerful, non-toxic agent for safely increasing anti-tumor immunity and immunotherapy efficacy in melanoma.

There is increased talk about using second life batteries in applications. In first life applications, the batteries start from new and a range of life cycle estimation techniques are applied. However, it is not clear how second life batteries should be monitored compared to first life batteries. This paper investigated different algorithms from first life applications, for estimating and forecasting battery cell state of health in conjunction with capacity calculations using second life cells under long term durability testing. The paper looks at how close these models predict capacity fade based on a set of second life batteries that have been undertaking sweat testing over six different applications. The paper concludes that there are two methods that could be suitable candidates for predicting lifespan. One of these needed to be modified from the original.

Adoptive transfer of tumor infiltrating lymphocytes (TIL) is a feasible and effective therapy for melanoma and lung cancer[1,2]. Multiple factors may determine the quality of the TIL product including components of the tumor microenvironment. In this work, we analyzed the role of melanoma infiltrating B cells in the context of TIL expansion based on their documented association with response to other types of immunotherapies[3]. We stimulated melanoma infiltrating B cells using human recombinant CD40L on the first day of ex-vivo TIL expansion. Samples were expanded from cryopreserved melanoma tumor single cell suspensions, in high dose IL-2 alone (standard protocol), or in high dose IL-2 plus CD40L. After 48h, analysis of activation markers on the CD40-expressing cells by flow cytometry was performed. For further investigation of the changes induced by CD40L stimulation, TIL expansion cultures (+/- CD40L) were analyzed using scRNA-seq (10X Genomics Chromium NextGEM Single Cell 5’ v2 and V(D)J Reagent kits; Illumina NovaSeq 6000 instrument with S4 sequencing flow cell) at 48h of culture (n=7 patients). The TIL expansion success rate was 68% with the CD40L treatment condition compared to 36% with the standard protocol. TILs cultured in the presence of CD40L expanded to on average three times more than with the standard protocol (P ≤ 0.01). Treatment with CD40L increased the percentage of CD39- CD69- T cells (P ≤ 0.05). Within the tumor digests, a higher percentage of B cells, including switched memory B cells (CD27+ IgD− ), was associated with successful TIL expansion (P=0.04). scRNA-seq analysis demonstrated different clustering patterns within the B cell compartment based on culture conditions. No clear partition was observed for other cell types, including the myeloid compartment. B cells displayed 126 DEGs associated to CD40L addition, CCL22, CD83, EBI3 and CD58 were among the upregulated genes in the CD40L-treated B cells. Other cell types experienced minimal to no change in transcriptomic profiles. B cell clusters were sub-classified based on CD27 and IgD expression[4], showing a predominance of naïve and switched memory B cells. Our results show that higher presence of B cells within tumors is associated with better TIL expansion, suggesting an interplay between T and B cells, and providing rationale for the design of improved TIL expansion protocols based on B cell stimulation with CD40L. This work has been supported in part by the Flow Cytometry, Genomics and Biostatistics and Bioinformatics Core Facilities at Moffitt Cancer Center, an NCI designated Comprehensive Cancer Center (P30-CA076292). We acknowledge Moffitt’s Melanoma Center of Excellence and the Mark Foundation for the financial support. [1] Sarnaik, A.A. et al. JCO 39, 2656-2666 (2021). [2] Creelan, B. et al. Nat Med 27, 1410-1418 (2021). [3] Cabrita, R. et al. Nature 577, 561-565 (2020). [4] Sanz, I. et al. Front Immunol 10, 2458 (2019). Citation Format: Renata Ariza Marques Rossetti, Leticia Tordesillas, Matthew Beatty, Dongliang Du, Yian Ann Chen, Amod Sarnaik, Shari Pilon-Thomas, Daniel Abate-Daga. CD40L stimulates melanoma infiltrating B cells and enhances ex vivo TIL expansion. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4055.

Adoptive cell transfer (ACT) with tumor-infiltrating lymphocytes (TILs) can generate durable clinical responses in patients with metastatic melanoma and ongoing trials are evaluating efficacy in other advanced solid tumors. The aim of this study was to develop methods for the expansion of tumor-reactive TIL from resected soft tissue sarcoma to a degree required for the ACT. From 2015 to 2018, 70 patients were consented to an institutional review board-approved protocol, and fresh surgical specimens were taken directly from the operating room to the laboratory. Fragments of the tumor (1 mm(3)) or fresh tumor digest were placed in culture for a period of 4 weeks. Successfully propagated TIL from these cultures were collected and analyzed by flow cytometry. TIL were cocultured with autologous tumor and function was assessed by measurement of interferon-γ in the supernatant by enzyme-linked immunosorbent assay. Initial TIL cultures were further expanded using a rapid expansion protocol. Nearly all specimens generated an initial TIL culture (91% fragment method, 100% digest method). The phenotype of the TIL indicated a predominant CD3(+) population after culture (43% fragment, 52% digest) and TIL were responsive to the autologous tumor (56% fragment, 40% digest). The cultured TIL expanded to a degree required for clinical use following rapid expansion protocol (median: 490-fold fragment, 403-fold digest). The data demonstrate the feasibility of TIL culture from fresh soft tissue sarcoma. The derived TIL have tumor-specific reactivity and can be expanded to clinically relevant numbers. An active ACT clinical trial using the methods described in this report is now approved for patients with metastatic soft tissue sarcoma.

The acidic pH of tumors profoundly inhibits effector functions of activated CD8 + T-cells. We hypothesize that this is a physiological process in immune regulation, and that it occurs within lymph nodes (LNs), which are likely acidic because of low convective flow and high glucose metabolism. Here we show by in vivo fluorescence and MR imaging, that LN paracortical zones are profoundly acidic. These acidic niches are absent in athymic Nu/Nu and lymphodepleted mice, implicating T-cells in the acidifying process. T-cell glycolysis is inhibited at the low pH observed in LNs. We show that this is due to acid inhibition of monocarboxylate transporters (MCTs), resulting in a negative feedback on glycolytic rate. Importantly, we demonstrate that this acid pH does not hinder initial activation of naïve T-cells by dendritic cells. Thus, we describe an acidic niche within the immune system, and demonstrate its physiological role in regulating T-cell activation., T-cell activation primarily occurs in the lymph nodes, highly organized and specialized secondary lymphoid organs. Here the authors show that the acidic extracellular pH in lymph node paracortical zones limits cytokine production by effector T-cells, but does not alter their activation by antigen-presenting cells.

BackgroundThe immunogenic nature of melanoma has been exploited for the development of adoptive transfer of ex-vivo expanded tumor infiltrating lymphocytes (TIL). This adoptive cell transfer therapy has overall response rates of around 50%. Multiple factors may determine the quality of the TIL product including components of the tumor microenvironment. B-cells are frequently found in melanoma metastasis, and display signs of antigen experience. Recently, B-cell tumor infiltration has been associated with improved clinical responses to immune checkpoint inhibitors,1 2 but their role in TIL therapy remains unexplored. Considering the potential role of B cells, we aim to develop strategies to enhance the quality of TIL products through B-cell stimulation during ex-vivo TIL expansion.MethodsWe stimulated melanoma infiltrating B-cells using human recombinant CD40L on the first day of ex-vivo TIL expansion. Thirteen samples were expanded from melanoma tumor single cell suspensions, in high dose IL-2 alone (standard protocol), or in high dose IL-2 plus CD40L. After up to four weeks of expansion, the TIL phenotype was analyzed by flow cytometry.ResultsThe expansion success rate from the frozen tumor digests was 69% (95% CI: 38.6–90.9%) in the CD40L treatment condition compared to 23% with the standard protocol. Also, TILs cultured in the presence of CD40L expanded to higher numbers than with the standard protocol (P = 0.02). Interestingly, most of the samples expanded with CD40L had a significant increase in the percentage of CD4+ T cells (P = 0.03), but not to the detriment of the absolute number of CD8+ T cells. Treatment with CD40L increased the percentage of effector memory-like T cells (P = 0.03) and of CD39- CD69- T cells (P < 0.05), which were recently associated with response to TIL therapy.3ConclusionsThis preliminary work demonstrates that the stimulation with CD40L at the initiation of TIL culture leads to enhanced TIL expansion and an increase in CD4+ T cells with an effector memory-like and stem-like phenotype. Our group and others have previously described cases of patients who had tumor regression after receiving TIL therapy that were predominantly CD4+ T cells, suggesting that expansion of the CD4+ TIL repertoire may enhance TIL therapy.4AcknowledgementsThis work has been supported in part by the Flow Cytometry, Genomics and Biostatistics and Bioinformatics Core Facilities at Moffitt Cancer Center, an NCI designated Comprehensive Cancer Center (P30-CA076292). We acknowledge Moffitt’s Melanoma Center of Excellence for the financial support.ReferencesCabrita R, Lauss M, Sanna A. Tertiary lymphoid structures improve immunotherapy and survival in melanoma. Nature 2020;577:561–565.Petitprez F, de Reynies A, Keung EZ. B cells are associated with survival and immunotherapy response in sarcoma. Nature 2020;577:556–560.Krishna S, Lowery FJ, Copeland AR. Stem-like CD8 T cells mediate response of adoptive cell immunotherapy against human cancer. Science 2020;370:1328–1334.Friedman KM, Prieto PA, Devillier LE. Tumor-specific CD4+ melanoma tumor-infiltrating lymphocytes. J Immunother 2012;35:400–408.Ethics ApprovalThe study was approved by Advarra IRB, approval number MCC20559.

Background Chemotherapy regimens that include the utilization of gemcitabine are the standard of care in pancreatic cancer patients. However, most patients with advanced pancreatic cancer die within the first 2 years after diagnosis, even when treated with standard of care chemotherapy. This study aims to explore combination therapies that could boost the efficacy of standard of care regimens in pancreatic cancer patients. Methods In this study, we used PV-10, a 10% solution of rose bengal, to induce the death of human pancreatic tumor cells in vitro. Murine in vivo studies were carried out to examine the effectiveness of the direct injection of PV-10 into syngeneic pancreatic tumors in causing lesion-specific ablation. Intralesional PV-10 treatment was combined with systemic gemcitabine treatment in tumor-bearing mice to investigate the control of growth among treated tumors and distal uninjected tumors. The involvement of the immune-mediated clearance of tumors was examined in immunogenic tumor models that express ovalbumin (OVA). Results In this study, we demonstrate that the injection of PV-10 into mouse pancreatic tumors caused lesion-specific ablation. We show that the combination of intralesional PV-10 with the systemic administration of gemcitabine caused lesion-specific ablation and delayed the growth of distal uninjected tumors. We observed that this treatment strategy was markedly more successful in immunogenic tumors that express the neoantigen OVA, suggesting that the combination therapy enhanced the immune clearance of tumors. Moreover, the regression of tumors in mice that received PV-10 in combination with gemcitabine was associated with the depletion of splenic CD11b+Gr-1+ cells and increases in damage associated molecular patterns HMGB1, S100A8, and IL-1α. Conclusions These results demonstrate that intralesional therapy with PV-10 in combination with gemcitabine can enhance anti-tumor activity against pancreatic tumors and raises the potential for this strategy to be used for the treatment of patients with pancreatic cancer.

We present our next generation clinical dual-modality OCT and near infrared autofluorescence/fluorescence (NIRAF/NIRF) imaging platform. This platform allows combined tissue microstructure visualization (OCT) and obtaining molecular information either by intrinsic tissue near infrared autofluorescence (NIRAF) or by exogenous near infrared fluorescence contrast agents (NIRF). Components of this platform, OCT-NIRAF/NIRF imaging system, rotary junction and catheters, were developed using an industry standard design control processes to enable quality clinical translation. We have identified sources of image degradation in dual-modality catheter-based imaging (e.g. core-cladding crosstalk in OCT, background noise in fluorescence) and present methods to mitigate their effects. We also show catheter fabrication and validation, as well as automated fluorescence sensitivity and distance calibration methods that ensure robust and repeatable system performance.

Background & Aims Tethered capsule endomicroscopy (TCE) involves swallowing a small tethered pill that implements optical coherence tomography (OCT) imaging, procuring high resolution images of the whole esophagus. Here, we demonstrate and evaluate the feasibility and safety of TCE and a portable OCT imaging system in patients with Barrett’s esophagus (BE) in a multi-center (5-site) clinical study. Methods Untreated patients with BE as per endoscopic biopsy diagnosis were eligible to participate in the study. TCE procedures were performed in unsedated patients by either doctors or nurses. After the capsule was swallowed, the device continuously obtained 10-μm-resolution cross-sectional images as it traversed the esophagus. Following imaging, the device was withdrawn through mouth, and disinfected for subsequent reuse. BE lengths were compared to endoscopy findings when available. OCT-TCE images were compared to volumetric laser endomicroscopy (VLE) images from a patient who had undergone VLE on the same day as TCE. Results 147 patients with BE were enrolled across all sites. 116 swallowed the capsule (79%), 95/114 (83.3%) men and 21/33 (63.6%) women (P = .01). High-quality OCT images were obtained in 104/111 swallowers (93.7%) who completed the procedure. The average imaging duration was 5.55 ± 1.92 minutes. The mean length of esophagus imaged per patient was 21.69 ± 5.90 cm. A blinded comparison of maximum extent of BE measured by OCT-TCE and EGD showed a strong correlation (r = 0.77-0.79). OCT-TCE images were of similar quality to those obtained by OCT-VLE. Conclusions The capabilities of TCE to be used across multiple sites, be administered to unsedated patients by either physicians or nurses who are not expert in OCT-TCE, and to rapidly and safely evaluate the microscopic structure of the esophagus make it an emerging tool for screening and surveillance of BE patients. Clinical trial registry website and trial number: NCT02994693 and NCT03459339.

Allogeneic hematopoietic cell transplantation (alloHCT) using haploidentical donors (haploHCT) with post-transplantation cyclophosphamide (PTCy) for augmented graft-versus-host disease (GVHD) prophylaxis has emerged as a robust platform to expand donor options with acceptable levels of GVHD and graft failure. The mechanism by which PTCy mitigates GVHD risk is partly explained by preferential cytotoxicity based on aldehyde dehydrogenase levels and up-regulation of regulatory T cells, but is incompletely understood. Myeloid-derived suppressor cells are important mediators of T-cell function and are up-regulated by cyclophosphamide exposure. We hypothesized that this cell type may play a role in GVHD protection in children undergoing haploHCT/PTCy. We prospectively collected samples in the first month after alloHCT from children undergoing standard of care (SOC) alloHCT with matched donors and tacrolimus-based GVHD prophylaxis (N = 11) and PTCy recipients (N = 11). MDSC recovery was compared using flow cytometry, and MDSC suppressive function was assessed at the peak of MDSC quantitative recovery post-alloHCT. Groups were well matched for conditioning regimen and stem cell source. PTCy recipients exhibited more robust MDSC recovery, particularly polymorphonuclear-MDSCs than SOC recipients, with preservation of T-cell suppressive function. This corresponded to significantly lower incidence of Grade II to IV acute GVHD (9.1% versus 27.3%) and moderate/severe chronic GVHD (0% versus 27.3%) in PTCy recipients. Patients who developed GVHD had decreased MDSC-mediated T-cell suppression, as well as higher levels of IL-10, a cytokine closely linked to GVHD biology. Overall, these findings provide support for the role of MDSCs in mediating GVHD protection after PTCy-based haploHCT. © 2022 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

BACKGROUND Given the established links between an individual’s behaviors and lifestyle factors and potentially adverse health outcomes, univariate or simple multivariate health metrics and scores have been developed to quantify general health at a given point in time and estimate risk of negative future outcomes. However, these health metrics may be challenging for widespread use and are unlikely to be successful at capturing the broader determinants of health in the general population. Hence, there is a need for a multidimensional yet widely employable and accessible way to obtain a comprehensive health metric. OBJECTIVE The objective of the study was to develop and validate a novel, easily interpretable, points-based health score (“C-Score”) derived from metrics measurable using smartphone components and iterations thereof that utilize statistical modeling and machine learning (ML) approaches. METHODS A literature review was conducted to identify relevant predictor variables for inclusion in the first iteration of a points-based model. This was followed by a prospective cohort study in a UK Biobank population for the purposes of validating the C-Score and developing and comparatively validating variations of the score using statistical and ML models to assess the balance between expediency and ease of interpretability and model complexity. Primary and secondary outcome measures were discrimination of a points-based score for all-cause mortality within 10 years (Harrell c-statistic) and discrimination and calibration of Cox proportional hazards models and ML models that incorporate C-Score values (or raw data inputs) and other predictors to predict the risk of all-cause mortality within 10 years. RESULTS The study cohort comprised 420,560 individuals. During a cohort follow-up of 4,526,452 person-years, there were 16,188 deaths from any cause (3.85%). The points-based model had good discrimination (c-statistic=0.66). There was a 31% relative reduction in risk of all-cause mortality per decile of increasing C-Score (hazard ratio of 0.69, 95% CI 0.663-0.675). A Cox model integrating age and C-Score had improved discrimination (8 percentage points; c-statistic=0.74) and good calibration. ML approaches did not offer improved discrimination over statistical modeling. CONCLUSIONS The novel health metric (“C-Score”) has good predictive capabilities for all-cause mortality within 10 years. Embedding the C-Score within a smartphone app may represent a useful tool for democratized, individualized health risk prediction. A simple Cox model using C-Score and age balances parsimony and accuracy of risk predictions and could be used to produce absolute risk estimations for app users.

Background Chemotherapy regimens that include gemcitabine are considered standard of care in patients with advanced pancreatic ductal adenocarcinoma (PDAC). However, most patients with PDAC die within 2 years of diagnosis, even with these standard of care regimens. In this study, we explored the ability of intratumoral injections of PV-10, a 10% solution of rose bengal, to induce lesion-specific ablation and control of metastatic pancreatic tumors in a murine model. Methods PV-10 was cultured with human pancreatic cancer cell lines overnight and cell death was measured via Annexin-V and DAPI staining. Murine pancreatic tumor cells (Panc02) were injected subcutaneously in one flank to establish a single tumor model; to establish a bilateral tumor model, Panc02 tumor cells were implanted in the opposite flanks. On day 7, a single tumor was treated with intralesional PV-10. Gemcitabine (60 mg/kg) was injected intraperitoneally twice per week for 2 weeks. These experiments were repeated using Panc02 cells modified to overexpress the neoantigen ovalbumin (OVA). Control mouse tumor were directly injected with PBS. Tumor growth of PV-10 injected tumors and non-injected bystander tumors on the opposite flank were measured. Damage associated molecular patterns (DAMPs) in serum and immune cell frequencies within the spleens of tumor-bearing mice were measured to identify an associated systemic response with tumor lytic treatment regimen. Results We established that less than 50% of human and murine pancreatic cells were alive after a 24 hour incubation with 200µM PV-10 in vitro. The combination of intralesional PV-10 with the systemic administration of gemcitabine delayed the growth treated tumors and non-injected distal tumors. In contrast, gemcitabine monotherapy failed to delay tumor growth in bilateral Panc02 tumor models. We observed that this treatment strategy was markedly more successful in immunogenic Panc02OVA tumors resulting in lesion-specific ablation in 5/8 mice compared to 0/8 mice that were treated with gemcitabine monotherapy. This suggests that the combination therapy enhanced the immune-mediated clearance of tumors. Moreover, regression of tumors in mice that received PV-10 in combination with gemcitabine was associated with the depletion of splenic CD11b+Gr-1+ cells and increases in damage associated molecular patterns HMGB1, S100A8, and IL-1α. Conclusions Together, these results demonstrate that intralesional therapy with PV-10 can enhance the efficacy gemcitabine against pancreatic tumors. Ethics Approval Studies were performed under approved Institutional Review Board (IRB) laboratory protocols at the H. Lee Moffitt Cancer Center (Tampa, FL).

Immunomodulatory drugs, such as thalidomide and related compounds, potentiate T-cell effector functions. Cereblon (CRBN), a substrate receptor of the DDB1-cullin-RING E3 ubiquitin ligase complex, is the only molecular target for this drug class, where drug-induced, ubiquitin-dependent degradation of known “neosubstrates,” such as IKAROS, AIOLOS, and CK1α, accounts for their biological activity. Far less clear is whether these CRBN E3 ligase-modulating compounds disrupt the endogenous functions of CRBN. We report that CRBN functions in a feedback loop that harnesses antigen-specific CD8+ T-cell effector responses. Specifically, Crbn deficiency in murine CD8+ T cells augments their central metabolism manifested as elevated bioenergetics, with supraphysiological levels of polyamines, secondary to enhanced glucose and amino acid transport, and with increased expression of metabolic enzymes, including the polyamine biosynthetic enzyme ornithine decarboxylase. Treatment with CRBN-modulating compounds similarly augments central metabolism of human CD8+ T cells. Notably, the metabolic control of CD8+ T cells by modulating compounds or Crbn deficiency is linked to increased and sustained expression of the master metabolic regulator MYC. Finally, Crbn-deficient T cells have augmented antigen-specific cytolytic activity vs melanoma tumor cells, ex vivo and in vivo, and drive accelerated and highly aggressive graft-versus-host disease. Therefore, CRBN functions to harness the activation of CD8+ T cells, and this phenotype can be exploited by treatment with drugs.

Adoptive T cell therapy (ACT) in combination with lymphodepleting chemotherapy is an effective strategy to induce the eradication of tumors, providing long-term regression in cancer patients. Despite that lymphodepleting regimens condition the host for optimal engraftment and expansion of adoptively transferred T cells, lymphodepletion concomitantly promotes immunosuppression during the course of endogenous immune recovery. In this study, we have identified that lymphodepleting chemotherapy initiates the mobilization of hematopoietic progenitor cells that differentiate to immunosuppressive myeloid cells, leading to a dramatic increase of peripheral myeloid-derived suppressor cells (MDSCs). In melanoma and lung cancer patients, MDSCs rapidly expanded in the periphery within 1 week after completion of a lymphodepleting regimen and infusion of autologous tumor-infiltrating lymphocytes (TILs). This expansion was associated with disease progression, poor survival, and reduced TIL persistence in melanoma patients. We demonstrated that the interleukin 6 (IL-6)-driven differentiation of mobilized hematopoietic progenitor cells promoted the survival and immunosuppressive capacity of post-lymphodepletion MDSCs. Furthermore, the genetic abrogation or therapeutic inhibition of IL-6 in mouse models enhanced host survival and reduced tumor growth in mice that received ACT. Thus, the expansion of MDSCs in response to lymphodepleting chemotherapy may contribute to ACT failure, and targeting myeloid-mediated immunosuppression may support anti-tumor immune responses.

Upon binding to thalidomide and other immunomodulatory drugs, the E3 ligase substrate receptor cereblon (CRBN) promotes proteosomal destruction by engaging the DDB1–CUL4A–Roc1–RBX1 E3 ubiquitin ligase in human cells but not in mouse cells, suggesting that sequence variations in CRBN may cause its inactivation. Therapeutically, CRBN engagers have the potential for broad applications in cancer and immune therapy by specifically reducing protein expression through targeted ubiquitin-mediated degradation. To examine the effects of defined sequence changes on CRBN's activity, we performed a comprehensive study using complementary theoretical, biophysical, and biological assays aimed at understanding CRBN's nonprimate sequence variations. With a series of recombinant thalidomide-binding domain (TBD) proteins, we show that CRBN sequence variants retain their drug-binding properties to both classical immunomodulatory drugs and dBET1, a chemical compound and targeting ligand designed to degrade bromodomain-containing 4 (BRD4) via a CRBN-dependent mechanism. We further show that dBET1 stimulates CRBN's E3 ubiquitin–conjugating function and degrades BRD4 in both mouse and human cells. This insight paves the way for studies of CRBN-dependent proteasome-targeting molecules in nonprimate models and provides a new understanding of CRBN's substrate-recruiting function.

BackgroundImmunotherapy for cancer has long been focused on the generation of CD8+ cytotoxic T lymphocyte responses, independent of their dynamic CD4+ T cell counterpart. One promising approach, adoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TIL), has yielded response rates ranging from 28–55%.1–2 Investigation into the role of CD4+ TIL in this setting remains critically underexplored as an opportunity to improve upon these successes.MethodsTwo metastatic melanoma patients (PT1 and PT2) were treated with TIL on a completed clinical trial at Moffitt Cancer Center (NCT01005745). Tumor recognition by TIL was assessed via co-culture with tumor. Whole exome (WES) and RNA Sequencing were performed on cryopreserved tumor sections and mutant peptide-MHC binding was predicted. TIL were stimulated with antigen presenting cells (APCs) loaded with neoantigen-derived 25mer peptides and sorted based on 41BB/OX40 upregulation, followed by functional immunologic assays. TCR sequencing was conducted on patient peripheral blood as well as isolated neoantigen-specific TIL clones to determine persistence in vivo and cognate peptide-MHC targets were determined empirically.ResultsPT1, infused with predominantly CD4+ TIL (88%), achieved a complete response (CR) despite lack of IFNγ detection with conventional in vitro tumor co-culture methods. Infusion product TIL were sorted by upregulation of OX40 and 41BB upon stimulation with APCs loaded with the mutant peptide pool. Neoantigen reactivity arose from a single peptide sequence, which conferred recognition by a CD4+ TIL clone, which comprised 17% of the infusion product and enriched to greater than 80% after sorting via FACS. These CD4+ TIL produced IFNγ, TNFα, and granzyme B in response to peptide-loaded APCs in an HLA-DR dependent manner. TCRβ overlap revealed this CD4+ clone peaked at two weeks post-infusion (40%) and persisted after infusion for at least six weeks. PT2 was infused with highly reactive, primarily CD8+ (88%) TIL and also achieved a CR. Isolated CD4+ TIL were also responsive to tumor antigens in the context of MHC Class II in vitro. Tumor-reactive CD4+ TIL were enriched by IFNγ capture and delayed xenograft growth in vivo (pConclusionsInvestigation of these case studies demonstrated evidence of CD4+ TIL involvement in complete clinical responses after ACT. Ongoing studies will define the precise role of tumor-reactive CD4+ T cells in the anti-tumor immune response and provide the framework for future investigation into their function and therapeutic efficacy.Trial RegistrationNCT01005745ReferencesBailey SR, et al. Human CD26high T cells elicit tumor immunity against multiple malignancies via enhanced migration and persistence. Nat Commun 2017;8(1):1961.Tran E, et al. Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer. Science 2014;344(6184):641–5.Ethics ApprovalApproved by USF IRB approval number Ame5_107905. All participants gave informed consent before taking part.

BackgroundAdoptive cell transfer (ACT) using tumor-infiltrating lymphocytes (TIL) has achieved an overall response rate of 39% in metastatic melanoma patients at Moffitt Cancer Center. In these trials, a substantial fraction of patients were non-responders by RECIST, but demonstrated a mixed response to therapy. These results suggest that the infused TIL product contained tumor-reactive T cells with therapeutic potential, which could be further optimized to improve ACT with TIL. We hypothesized that outcomes might be improved by identifying and enriching neoantigen-reactive TIL within bulk products. The purpose of this study is to define approaches to optimize ACT with TIL, by identifying, enriching, and analyzing neoantigen reactive TIL from the ACT infusion product of previously treated metastatic melanoma patients.MethodsPatient-derived cryopreserved tumor tissue, PBMC, and TIL from completed metastatic melanoma TIL trials were used for this study. Whole exome and RNA sequencing were performed on DNA and RNA extracted from tumor tissue and compared to DNA from autologous PBMC. Genetic sequencing and gene expression data were utilized to determine protein-modifying somatic mutations. Peptides were then predicted for their ability to be presented on MHC molecules, prioritized, and up to 192 custom 25-mers were synthesized per patient sample. Neoantigen peptides were loaded onto patient-derived dendritic cells (DC) and co-cultured with autologous TIL. These TIL were then sorted by FACS on their ability to upregulate 41BB and OX40 and expanded through the rapid expansion protocol (REP). Enriched TIL were subsequently screened for neoantigen reactivity by 41BB/OX40 upregulation, cytokine release, and degranulation.ResultsProtein-altering somatic mutations from metastatic melanoma tissues ranged from 49 to 1631 mutations (median = 389). On average, 16.2% of TIL were sorted for upregulation of 41BB/OX40 upon co-culture with DC pulsed with the neoantigen peptide pool (range: 2.7–31.1%). CD4+ TIL displayed a 3.75-fold upregulation of 41BB/OX40, while CD8+ TIL saw a 1.88-fold increase (n=6). This coincided with substantial production of IFNγ, TNFα, and granzyme B (n=6). Neoantigen-reactive (41BB+/OX40+) and non-reactive (41BB-/OX40-) TIL expanded to similar degrees in REP (average of 639-fold vs. 611-fold; n=6). Restimulation of enriched neoantigen-specific TIL resulted in superior pro-inflammatory functionality (granzyme B, IFNγ, and TNFα) when compared to non-reactive TIL.ConclusionsTIL from metastatic melanoma patient samples were successfully enriched for neoantigen-reactive TIL, which maintained increased reactivity against these predicted peptides upon restimulation when compared non-reactive TIL. These data support further investigation into the use of neoantigen-enriched TIL products to enhance efficacy of ACT.Trial RegistrationNCT01005745, NCT01659151, NCT01701674Ethics ApprovalNCT01005745 was approved by USF IRB approval number Ame5_107905.NCT01659151 was approved by Advarra IRB approval number 14.03.0083.NCT01701674 was approved by USF IRB approval number Ame13_Pro00009061.All participants gave informed consent before taking part.

Immunotherapy for cancer has long been focused on the generation of CD8+ cytotoxic T lymphocyte responses, independent of their dynamic CD4+ T cell counterpart. One promising approach, adoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TIL), has yielded response rates ranging from 28-55%. Although lasting and complete responses have been achieved, there is substantial opportunity for improvement. Investigation into the role of CD4+ TIL in this setting remains critically underexplored. CD4+ T cells recognize tumor antigen presented on MHC Class II either directly on tumor cells or indirectly through antigen presenting cells (APCs) and are able to elicit potent anti-tumor responses under the appropriate conditions. Here, we present a case study of a metastatic melanoma patient who received adoptive transfer of a predominantly (88%) CD4+ TIL product. This patient demonstrated a complete response (CR) to therapy despite a lack of detection of IFNg in the infusion product in vitro when these TIL were co-cultured with autologous tumor prior to ACT. Tumor recognition was also absent when CD8+ TIL were isolated and stimulated directly with HLA-matched tumor lines, indicating a lack of recognition of shared melanoma antigens presented on MHC Class I. Longitudinal analysis of the peripheral blood of this patient confirmed that the infused CD4+ TIL persisted after therapy for at least six weeks. Whole exome sequencing (WES) performed on the TIL surgical specimen discovered 88 non-synonymous single nucleotide variants (SNVs) as candidate neoantigens. Predicted binding of the resulting mutant peptides to autologous HLA molecules generated a predominantly MHC Class II restricted profile, with 81.8% of variants capable of MHC Class II presentation and greater than half exclusive to MHC Class II only. CD4+ TIL were screened for tumor antigen recognition by upregulation of OX40 and 41BB after stimulation with autologous APCs loaded with mutant peptides. Nearly half (49.2%) of CD4+ TIL responded to tumor-derived peptides. These CD4+ TIL were then sorted into tumor-reactive and non-reactive subsets for further clonal analysis of phenotype and transcriptional profile (scRNASeq) of these T cells in order to characterize the nature of the CD4+ TIL response to tumor antigen. Overall, thorough interrogation of this patient's case study demonstrated evidence of CD4+ TIL involvement in a complete clinical response after ACT. Ongoing studies will define the precise role of tumor-reactive CD4+ T cells in the anti-tumor immune response and provide the framework for future investigation into their function and therapeutic efficacy. Citation Format: MacLean Hall, Pat Innamarato, Amy Mackay, Holly Branthoover, Alex Alfaro, Carolyn Rich, Allison Richards, Jonathan Hensel, Matthew Beatty, John Mullinax, Amod A. Sarnaik, Shari Pilon-Thomas. A case study investigation into the role of CD4+ tumor-infiltrating lymphocytes in a metastatic melanoma patient with a complete response to adoptive cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1513.

While the most common method used to evaluate and survey patients with Barrett’s Esophagus (BE) is endoscopic biopsy, this procedure is invasive, time-consuming, and suffers from sampling errors. Moreover, it requires patient sedation that increases cost and mandates its operation in specialized settings. Our lab has developed a new imaging tool termed tethered capsule endomicroscopy (TCE) that involves swallowing a tethered capsule which utilizes optical coherence tomography (OCT) to obtain three-dimensional microscopic (10µm) images of the entire esophageal wall as it traverses the luminal organ via peristalsis or is retrieved by pulling up tether. As opposed to endoscopy, TCE procedure is non-invasive, doesn’t require patient sedation and mitigates sampling error by evaluating the microscopic structure of the entire esophagus. The merits of TCE make it a suitable device to investigate the microscopic natural history of BE in a longitudinal manner. Here, we present our initial experience of a multicenter (5-site) clinical trial to study the microscopic natural history of BE. The TCE device used for the study is the new generation capsule with the ball lens optical configuration and a distal scan stepper motor, which provides 30µm (lateral) resolution and 40Hz imaging rate. The portable OCT imaging system is a custom in-house built swept source system and provides 7µm (axial) at a 100 kHz A-line rate with a center wavelength of ~1310 nm. To date, we have successfully enrolled 69 subjects at all sites (MGH: 33, Columbia University: 11, Kansas City VA: 10, Mayo Jacksonville: 8, Mayo Rochester: 7) and 59 have swallowed the capsule (85.5%). There have been no reported adverse events associated with TCE procedure. High-quality OCT images were reliably obtained from patients who swallowed the device, and BE tissues were identified by expert readers. Our initial experience with TCE in a multicenter study demonstrates that this technology is easy to use and efficient in multiple clinical settings. Completion of this longitudinal study is likely to provide new insights on the temporal progression of BE that may impact management strategies.

Protein acetylation is an important contributor to cancer initiation. Histone deacetylase 6 (HDAC6) controls JAK2 translation and protein stability and has been implicated in JAK2-driven diseases best exemplified by myeloproliferative neoplasms (MPNs). By using novel classes of highly selective HDAC inhibitors and genetically deficient mouse models, we discovered that HDAC11 rather than HDAC6 is necessary for the proliferation and survival of oncogenic JAK2-driven MPN cells and patient samples. Notably, HDAC11 is variably expressed in primitive stem cells and is expressed largely upon lineage commitment. Although Hdac11is dispensable for normal homeostatic hematopoietic stem and progenitor cell differentiation based on chimeric bone marrow reconstitution, Hdac11 deficiency significantly reduced the abnormal megakaryocyte population, improved splenic architecture, reduced fibrosis, and increased survival in the MPLW515L-MPN mouse model during primary and secondary transplantation. Therefore, inhibitors of HDAC11 are an attractive therapy for treating patients with MPN. Although JAK2 inhibitor therapy provides substantial clinical benefit in MPN patients, the identification of alternative therapeutic targets is needed to reverse MPN pathogenesis and control malignant hematopoiesis. This study establishes HDAC11 as a unique type of target molecule that has therapeutic potential in MPN.

International audience; Tethered capsule endomicroscopy (TCE) is an emerging screening technology that comprehensively obtains microstructural OCT images of the gastrointestinal (GI) tract in unsedated patients. To advance clinical adoption of this imaging technique, it will be important to validate TCE images with co-localized histology, the current diagnostic gold standard. One method for co-localizing OCT images with histology is image-targeted laser marking, which has previously been implemented using a driveshaft-based, balloon OCT catheter, deployed during endoscopy. In this paper, we present a TCE device that scans and targets the imaging beam using a low-cost stepper motor that is integrated inside the capsule. In combination with a 4-laser-diode, high power 1430/1450 nm marking laser system (800 mW on the sample and 1s pulse duration), this technology generated clearly visible marks, with a spatial targeting accuracy of better than 0.5 mm. A laser safety study was done on swine esophagus ex vivo, showing that these exposure parameters did not alter the submucosa, with a large, 4-5x safety margin. The technology was demonstrated in living human subjects and shown to be effective for co-localizing OCT TCE images to biopsies obtained during subsequent endoscopy.

Penile cancer is a rare but highly lethal cancer, and therapeutic options for patients presenting with lymph nodal disease are very limited. Adoptive cell therapy using tumor infiltrating lymphocytes (TIL) was shown to provide durable objective response in patients with metastatic melanoma and TIL have been expanded from solid tumors at rates between 70 and 90% depending on the specific diagnosis. We evaluated whether TIL could be expanded from surgical specimens of patients with penile cancer. Tumor samples from metastatic lymph nodes obtained at the time of inguinal lymph node dissection were collected, minced into fragments, placed in individual wells of a 24-well plate, and propagated in high dose IL-2 for four weeks. The phenotype of expanded TILs was assessed by flow cytometry and their anti-tumor reactivity was assessedby IFN-γ ELISA. TIL were expanded from 11 out of 12 (91.6%) samples of metastatic lymph nodes. Expanded TIL were predominantly CD3(+) (mean 67.5%, SD 19.4%) with a mean of 46.8% CD8(+) T cells (SD 21.1%). Five out of 11 samples (45.4%) from expanded TIL secreted IFN-γ in response to autologous tumor. TIL expansion and phenotype of expanded T cell lymphocytes were independent of previous HPV infection and treatment with neoadjuvant chemotherapy. This is the first report demonstrating successful expansion of tumor-reactive TIL from penile cancer patients, which support development of adoptive cell therapy strategies using TIL for the treatment of advanced and recurrent penile cancer.

Background Given the established links between an individual’s behaviors and lifestyle factors and potentially adverse health outcomes, univariate or simple multivariate health metrics and scores have been developed to quantify general health at a given point in time and estimate risk of negative future outcomes. However, these health metrics may be challenging for widespread use and are unlikely to be successful at capturing the broader determinants of health in the general population. Hence, there is a need for a multidimensional yet widely employable and accessible way to obtain a comprehensive health metric. Objective The objective of the study was to develop and validate a novel, easily interpretable, points-based health score (“C-Score”) derived from metrics measurable using smartphone components and iterations thereof that utilize statistical modeling and machine learning (ML) approaches. Methods A literature review was conducted to identify relevant predictor variables for inclusion in the first iteration of a points-based model. This was followed by a prospective cohort study in a UK Biobank population for the purposes of validating the C-Score and developing and comparatively validating variations of the score using statistical and ML models to assess the balance between expediency and ease of interpretability and model complexity. Primary and secondary outcome measures were discrimination of a points-based score for all-cause mortality within 10 years (Harrell c-statistic) and discrimination and calibration of Cox proportional hazards models and ML models that incorporate C-Score values (or raw data inputs) and other predictors to predict the risk of all-cause mortality within 10 years. Results The study cohort comprised 420,560 individuals. During a cohort follow-up of 4,526,452 person-years, there were 16,188 deaths from any cause (3.85%). The points-based model had good discrimination (c-statistic=0.66). There was a 31% relative reduction in risk of all-cause mortality per decile of increasing C-Score (hazard ratio of 0.69, 95% CI 0.663-0.675). A Cox model integrating age and C-Score had improved discrimination (8 percentage points; c-statistic=0.74) and good calibration. ML approaches did not offer improved discrimination over statistical modeling. Conclusions The novel health metric (“C-Score”) has good predictive capabilities for all-cause mortality within 10 years. Embedding the C-Score within a smartphone app may represent a useful tool for democratized, individualized health risk prediction. A simple Cox model using C-Score and age balances parsimony and accuracy of risk predictions and could be used to produce absolute risk estimations for app users.

BackgroundThe therapeutic armamentarium of bladder cancer has been recently enriched with the introduction of new therapies including immune checkpoint inhibitors, receptor tyrosine kinase inhibitors and antibody drug conjugates, however treatment responses and duration of responses are still less than expected. Adoptive cellular therapy (ACT) using tumor-infiltrating lymphocytes (TILs) has potential to treat bladder cancer, as previously demonstrated by successful expansion of tumor reactive T cells from human bladder tumors.MethodsA model system using OT-I T cells and an ovalbumin expressing MB49 tumor cell line (MB49OVA) was developed to study ACT in bladder cancer. Systemic ACT-treated mice were given T cells intravenously after lymphodepleting chemotherapy and followed by interleukin (IL)-2 administration. Intravesical ACT treated mice were given T cells directly into the bladder, without chemotherapy or IL-2. TILs were isolated from MB49 orthotopic tumors and expanded ex vivo in IL-2. Immune cell infiltrates were analyzed by flow cytometry. T cell infiltration was studied using a CXCR3 blocking antibody.ResultsSystemic ACT-treated mice had a decrease in tumor growth, increase in T cell infiltration and long-term immune protection compared with control-treated mice. OT-I T cells delivered intravesically were able to control tumor growth without lymphodepleting chemotherapy or IL-2 in MB49OVA orthotopic tumors. Intravesical delivery of TIL expanded from MB49 tumors was also able to decrease tumor growth in mice with MB49 orthotopic tumors. Blocking CXCR3 on OT-I T cells prior to intravesical delivery decreased T cell infiltration into the tumor and prevented the control of tumor growth.ConclusionsThis study demonstrates how TIL therapy can be used in treating different stages of bladder cancer.

Background: Until recently, treatment options for patients with head and neck cancer include a combination of surgery, chemotherapy and radiation. Although the combined use of these treatment options modestly improves survival, combination therapy increases toxicity. Overall survival of these treatment regimens for patients with advanced head and neck cancer is just over 10 months. Even with the newly approved and vastly more tolerable use of anti-PD-1 immunotherapy, survival is only extended by 3-5 months. In comparison, adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) has improved the median overall survival in patients with metastatic melanoma to 52 months. The goal of this study was to test the feasibility of expanding tumor-reactive TIL from head and neck tumors for the development of a clinical trial. Methods: Primary tumors were resected from 20 head and neck cancer patients. Tumors were minced into small fragments (1-3 mm3) and plated in high dose IL-2. After 4 weeks, expanded TIL were phenotyped by flow cytometry and tested for tumor reactivity by IFN-gamma production after co-culture of TIL with autologous tumor digest. IFN-gamma levels were determined by ELISA. Reactive TIL were expanded using a rapid expansion protocol (REP). Results: The mean expansion of TIL per fragment was 1.96 × 106. From 20 primary tumors, seven were contaminated and discarded. TIL were successfully expanded from 11/13 (85%) evaluable tumors. The phenotype of expanded TIL consisted of CD3− CD56+ NK cells and CD3+ T cells. Tumor-specific reactivity was analyzed for seven samples. TIL expanded from three (43%) tumors were predominantly CD8+ and were reactive against autologous tumor as determined by IFN-gamma ELISA. Expansion of TIL during REP resulted in an average 600-fold increase in cell numbers (range of 61 - 1,774 fold) and led to the depletion of NK cells and an increase of CD3+ T cells. Conclusions: In this study, we have demonstrated the feasibility of growing tumor-reactive TIL from head and neck tumors. This study raises the potential for the implementation of ACT with TIL for the treatment of head and neck cancer patients. Citation Format: Luz Nagle, Amy Mackay Weber, MacLean Hall, Matthew Beatty, J. Trad Wadsworth, Caitlin McMullen, Krupal Patel, Kathryn Vorwald, Christine Chung, Shari Pilon-Thomas. Expansion of tumor-specific tumor-infiltrating lymphocytes (TIL) from head and neck tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2178.