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Drug resistance remains the major driving factor behind the clinical failure of targeted therapeutics. Current oncology precision medicine approaches rely on targeting known acquired resistance mutations, such as EGFR T790M or ALK/ROS mutations in NSCLC with 2nd and 3rd generation molecules designed to overcome or prevent resistance. These next generation targeted therapeutic approaches have increasingly long and complex drug development timelines and burdensome toxicities from off target effects (e.g. wild-type receptor targeting) or drug-drug interactions (DDI). The toxicities limit tolerability, compliance and combinability of different targeted therapeutics. RNA-based immunotherapy approaches offer an increasingly attractive alternative to next generation small molecule targeted therapeutics approaches: (1) RNA-based approaches only require a known acquired resistance sequence, (2) drug development timelines, cost and complexity can be meaningfully condensed, and (3) multiple acquired resistance mutations can be targeted with the same candidate. RBI-1000 is a candidate using a novel type of self-replicating RNA (srRNA) to generate robust immunity directed against acquired resistance mutations that develop in ER+ breast cancer (ER+ BC) in response to endocrine therapy. RBI-1000 includes on-target mutations within the estrogen receptor ligand binding domain, and bypass mutations either in the form of activating mutations in the PI3K kinase domain or amplifications of HER2/HER3. Here, we demonstrate that this srRNA encapsulated in a lipid nanoparticle primes polyfunctional CD4 and CD8 T cells leading to tumor growth inhibition and improved survival in a mouse model expressing the targeted acquired resistance mutation. Priming of T cells against acquired mutations is also confirmed in human HLA-transgenic mice. The immune cell-mediated elimination of clones expressing the acquired resistance mutations is predicted to prolong endocrine control of ER+BC, in an analogous manner to small molecule or monoclonal antibody targeted therapies, but with a more favorable dosing and adverse event profile due to precise immunologic targeting and no DDI. Citation Format: Zelanna Goldberg, Christian Maine, Gabrielle P. Dailey, Christine Domingo, Gaelle Picarda, Hunter Little, Annie Chou, Jessica Sparks, Darina Spasova, Shigeki Miyake-Stoner, Zachary C. Hartman, Christopher A. Rabiola, Erika J. Crosby, Herbert K. Lyerly, Nathaniel Wang, Parinaz Aliahmad. A self-replicating RNA precision medicine approach to overcoming resistance to endocrine therapy in ER+BC. [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 6403.

Adoptive cellular therapies using engineered chimeric antigen receptor T cells (CAR-T cells) are rapidly emerging as a highly effective treatment option for a variety of life-threatening hematological malignancies. Small molecule-mediated modulation of T cell differentiation during the in vitro CAR-T manufacturing process has great potential as a method to optimize the therapeutic potential of cellular immunotherapies. In animal models, T cells with a central or stem memory (TCM/SCM) phenotype display enhanced in vivoefficacy and persistence relative to other T cell subpopulations. We sought to identify small molecules that promote skewing towards a TCM/SCM phenotype during the CAR-T manufacturing process, with associated enhanced viability, expansion and metabolic profiles of the engineered cells. To this end, we developed a high-throughput functional screening platform with primary human T cells using a combination of high-content immunophenotyping and gene expression-based readouts to analyze cells following a high-throughput T cell culture platform that represents a scaled-down model of clinical CAR-T cell production. Multicolor flow cytometry was used to measure expansion, cell viability and the expression levels of cell surface proteins that define TCM cells (e.g., CCR7, CD62L and CD27) and markers of T cell exhaustion (e.g., PD1, LAG3, and TIM3). In parallel, a portion of each sample was evaluated using high content RNA-Seq based gene expression analysis of ~100 genes representing key biological pathways of interest. A variety of known positive and negative control compounds were incorporated into the high-throughput screens to validate the functional assays and to assess the robustness of the 384-well-based screening. The ability to simultaneously correlate small molecule-induced changes in protein and gene expression levels with impacts on cell proliferation and viability of various T cell subsets, enabled us to identify multiple classes of small molecules that favorably enhance the therapeutic properties of CAR-T cells. Consistent with results previously presented by Perkins et al. (ASH, 2015), we identified multiple PI3K inhibitors that could modify expansion of T cells while retaining a TCM/SCM phenotype. In addition, we identified small molecules, and small molecule combinations, that have not been described previously in the literature that could improve CAR-T biology. Several of the top hits from the screens have been evaluated across multiple in vitro (e.g., expansion, viability, CAR expression, serial restimulation/killing, metabolic profiling, and evaluation of exhaustion markers) and in vivo (e.g., mouse tumor models for persistence and killing) assays. Results from the initial screening hits have enabled us to further refine the optimal target profile of a pharmacologically-enhanced CAR-T cell. In addition, we are extending this screening approach to identify small molecules that enhance the trafficking and persistence of CAR-T cells for treating solid tumors. In conclusion, the approach described here identifies unique small molecule modulators that can modify CAR-T cells during in vitro expansion, such that improved profiles can be tracked and selected from screening through in vitro and in vivo functional assays. Disclosures Rosen: Fate Therapeutics: Employment, Equity Ownership. Rezner:Fate Therapeutics, Inc: Employment, Equity Ownership. Robbins:Fate Therapeutics: Employment, Equity Ownership. Hardy:Fate Therapeutics: Employment, Equity Ownership. Peralta:Fate Therapeutics: Employment, Equity Ownership. Maine:Fate Therapeutics: Employment, Equity Ownership. Sabouri:Fate Therapeutics: Employment, Equity Ownership. Reynal:Fate Therapeutics: Employment. Truong:Fate Therapeutics: Employment, Equity Ownership. Moreno:Fate Therapeutics, Inc.: Employment, Equity Ownership. Foster:Fate Therapeutics: Employment, Equity Ownership. Borchelt:Fate Therapeutics: Employment, Equity Ownership. Meza:Fate Therapeutics: Employment, Equity Ownership. Thompson:Juno Therapeutics: Employment, Equity Ownership. Fontenot:Juno Therapeutics: Employment, Equity Ownership. Larson:Juno Therapeutics: Employment, Equity Ownership. Mujacic:Juno Therapeutics: Employment, Equity Ownership. Shoemaker:Fate Therapeutics: Employment, Equity Ownership.

Mutations in the phosphatase-encoding gene, PTPN22, are associated with a number of autoimmune conditions, including type I diabetes, rheumatoid arthritis and systemic lupus erythematosus. To study how PTPN22 functions in immune cells we have made extensive use of a PTPN22 deficient (KO) mouse. PTPN22 KO mice exhibit increased T cell numbers, activity and function as well as increased germinal center activity and antibody production with no autoimmune phenotype. As such we are currently investigating whether loss of PTPN22 would be of benefit to host immunity against chronic viral infections. To test this hypothesis we infected PTPN22 KO mice with the persistent lymphocytic choriomeningitis virus (LCMV) cl13 virus. LCMV cl13 infection in WT B6 mice leads to chronic infection lasting over 60 days characterized by dysfunctional ‘exhausted’ virus-specific T cells. PTPN22 KO mice were able to clear the cl13 infection whereas WT mice were not. Clearance is associated with increased viral-specific CD4 T cell cytokine production 8 days post infection, as well as increased numbers of Tfh and plasma cells. At later time points after infection, PTPN22 KO mice have increased numbers of functional CD8 T cells that produce IFNγ and TNFα, whereas CD8 cells in WT mice do not. These results suggest that inhibition of PTPN22 may prove beneficial in helping to clear chronic infection.