Intratumoral regulatory T cell (Treg) abundance associates with diminished anti-tumor immunity and poor prognosis in human cancers. Recent work demonstrates that CD25, the high affinity receptor subunit for IL-2, is a selective target for Treg depletion in mouse and human malignancies; however, anti-human CD25 antibodies have failed to deliver clinical responses against solid tumors due to bystander IL-2 receptor signaling blockade on effector T cells, which limits their anti-tumor activity. Here we demonstrate potent single-agent activity of anti-CD25 antibodies optimized to deplete Tregs whilst preserving IL-2-STAT5 signaling on effector T cells, and demonstrate synergy with immune checkpoint blockade in vivo. Pre-clinical evaluation of an anti-human CD25 (RG6292) antibody with equivalent features demonstrates, in both non-human primates and humanized mouse models, efficient Treg depletion with no overt immune-related toxicities. Our data supports the clinical development of RG6292 and evaluation of novel combination therapies incorporating non-IL-2 blocking anti-CD25 antibodies in clinical studies.

HVCN1 (Hydrogen voltage-gated channel 1) is the only mammalian voltage-gated proton channel. In human B lymphocytes, HVCN1 associates with the B-cell receptor (BCR) and is required for optimal BCR signaling and redox control. HVCN1 is expressed in malignant B cells that rely on BCR signaling, such as chronic lymphocytic leukemia (CLL) cells. However, little is known about its regulation in these cells. We found that HVCN1 was expressed in B cells as two protein isoforms. The shorter isoform (HVCN1 S ) was enriched in B cells from a cohort of 76 CLL patients. When overexpressed in a B-cell lymphoma line, HVCN1 S responded more profoundly to protein kinase C-dependent phosphorylation. This more potent enhanced gating response was mediated by increased phosphorylation of the same residue responsible for enhanced gating in HVCN1 L , Thr 29 . Furthermore, the association of HVCN1 S with the BCR was weaker, which resulted in its diminished internalization upon BCR stimulation. Finally, HVCN1 S conferred a proliferative and migratory advantage as well as enhanced BCR-dependent signaling. Overall, our data show for the first time, to our knowledge, the existence of a shorter isoform of HVCN1 with enhanced gating that is specifically enriched in malignant B cells. The properties of HVCN1 S suggest that it may contribute to the pathogenesis of BCR-dependent B-cell malignancies. is a small protein that conducts protons across membranes selectively (1, 2) and in a regulated manner. Previously, we described its function in B lymphocytes, where proton channels sustain B-cell receptor (BCR) signaling via regulation of reactive oxygen species production by the NADPH oxidase enzyme complex (3). In addition, we found HVCN1 to be directly associated with the BCR. Upon receptor stimulation, the BCR and HVCN1 were cointernalized to late endosomal/lysosomal organelles called "MIICs," or MHC class II-containing compartments, where antigens bound to the BCR are digested into small peptides and loaded onto MHC class II molecules for presentation to T cells (3).HVCN1 is expressed not only by normal but also by malignant B cells, such as those in chronic lymphocytic leukemia (CLL) (3). CLL cells are characterized by their reliance on BCR signaling for survival and growth (4), so it is possible that they maintain or upregulate HVCN1 expression to sustain their growth. Other tumor cells, such as those in breast (5) and colorectal cancer (6), have been found to rely on HVCN1 for survival. In these tumor cells, proton channels prevent excessive acidification of the cytoplasm and allow increased cell migration. In malignant B cells, HVCN1 may regulate intracellular pH and at the same time sustain BCR signaling. However, its precise roles remain to be elucidated.We show here that CLL cells and other B-cell lines specifically express higher levels of a shorter isoform of HVCN1, HVCN1 S . We identified the existence of two distinct isoforms of relatively similar size when immunoblotting B-cell lysates with an HVCN1-specific antibody (3). HVCN1 S ...

epithelium prone to infection and in intestine leads to obstruction. Patients homozygous for F508del, have a tremendous variation in the severity of disease. Recent Genome-wide association studies indicate that this variation is due to presence of modifier genes, with SLC6A14 as the top modifier (Sun et.al.,2012). SLC6A14 is a Naþ/Cl-dependent cationic/neutral amino-acid transporter on the surface of lung and colonic epithelium. As both transporters are expressed apically, we hypothesized that SLC6A14 would modify the fluid secretory capacity of the epithelium. So in collaboration with TCP, we generated a SLC6A14 knock-out mouse. We can measure in-vivo fluid secretion in mice, using an intestinal closed-loop assay. SLC6A14 knock-out mice exhibited a decrease in cAMP stimulated fluid secretion mediated via CFTR relative to Wt control. To explore the mechanism by which this modification occurs, we utilized a BHK heterologous expression system, overexpressing CFTR and SLC6A14. Interestingly, the functional interaction can be recapitulated in this system, suggesting that it not tissue-type dependent. Preliminary biochemical and anion-flux studies support the hypothesis that SLC6A14 does not affect the processing or stability of Wt or F508del-CFTR proteins rather it augments the activity of these channel proteins once localized to the cell surface. Future studies will focus on understanding if this augmentation is related to modification of CFTR's phosphorylation dependent gating. These results show a positive impact of SLC6A14 on CFTR channel function and fluid secretion, providing an alternative drug target for CF patients. 96-PlatEnhanced Activation of an Amino-Terminally Truncated Isoform of Voltage-Gated Proton Channel HVCN1 Enriched in Malignant B cells

Potential–density pair basis sets can be used for highly efficient N‐body simulation codes, but they suffer from a lack of versatility, i.e. a basis set has to be constructed for each different class of stellar system. We present numerical techniques for generating a biorthonormal potential–density pair basis set that has a general specified pair as its lowest‐order member. We go on to demonstrate how the set can be used to construct N‐body equilibria, which we then evolve using an N‐body code that calculates forces using the basis set.

Adoptive cell therapy (ACT) using ex vivo expanded tumor infiltrating lymphocytes (TIL) has shown great promise as a treatment for metastatic melanoma and has the potential to deliver durable responses in other solid tumors. Clonal neoantigens, which are derived from mutations occurring very early in the tumor development, are present in all cancer cells within a patient and therefore could be the optimal targets for TIL-based therapies. Recently it was shown that the number of clonal neoantigens within a tumor is associated with improved clinical outcomes following checkpoint inhibition in patients with non-small cell lung cancer (NSCLC) and melanoma. An approach that targets multiple clonal neoantigens with specific T cells has the potential to demonstrate high specificity and efficacy whilst mitigating the risk of immune escape. Achilles Therapeutics is developing a personalized ACT product, ATL001, to target clonal neoantigens, which are identified using tumor exome sequencing and the PELEUS™ bioinformatics platform. Clonal neoantigen reactive T cells (cNeTs) are then manufactured from TIL using the VELOS™ manufacturing process. Two Phase I/IIa clinical trials of ATL001 are ongoing in patients with advanced NSCLC and metastatic or recurrent melanoma. In common with the development of other ACT products, the key to characterizing and improving cNeT products relies on evaluating a diverse set of exploratory endpoints in early clinical trials, including understanding the procedural, clinical and biological factors that influence cNeT manufacturing rate and product reactivity; monitoring the expansion, persistence and phenotype of the infused cells in vivo and identifying potential biomarkers of clinical activity or safety of cNeTs in treated patients. These insights may suggest further improvements to cNeT product development in ensuing iterations. The evaluation of these endpoints requires the collection of a rich longitudinal dataset that traces each patient's journey from tissue procurement and cNeT manufacture, to final product infusion and follow up. The data collected will include clinical and disease characteristics, tumor microenvironment insights from exome sequencing and immunohistochemistry of procured tumor, and metrics from the VELOS™ manufacturing process, along with a comprehensive immune-monitoring programme comprising immuno-sequencing, immunophenotyping, bespoke ctDNA panels and reactivity assays at specified timepoints, all to be evaluated against clinical outcomes data. The amalgamation of diverse streams of data requires the development of robust processes and systems for data collection, processing and storage. Furthermore, the evaluation of multiple exploratory endpoints will require integration and modelling of baseline covariates, time-series immune-monitoring and efficacy data, all of which will be described Citation Format: Michael Epstein, Rebecca Pike, Emma Leire, Jen Middleton, Megan Wileman, Lylia Ouboussad, Leah Manning, Theres Oakes, Eva Pekle, Amy Baker, Mark Brown, Daisy Melandri, Pablo Becker, Anabel Ramirez, Natasa Hadjistephanou, Samra Turaljic, Mariam Jamal-Hanjani, Martin Forster, Iraj Ali, Jane Robertson, Karl Peggs, Sergio Quezada. Characterization of a novel clonal neoantigen reactive T cell (cNeT) product through a comprehensive translational research program [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 1508.