314 Engineering CAR T cells to eliminate solid tumors through improved tumor-specific homing

<h3>Background</h3> Adoptive transfer of genetically engineered chimeric antigen receptor (CAR) T cells is one of the most effective immunotherapies against hematological malignancies but has limited clinical success in solid tumors. A recent meta-analysis revealed a ~9% pooled response rate among solid tumor patients treated with CAR-T cell therapy.¹ This represents an enormous unmet clinical need, as solid tumors comprise 90% of adult human cancer diagnoses.² Indeed, although CAR-T cells targeting solid tumors display significant anti-cancer functions in pre-clinical studies, CAR-T cell immunotherapies have mostly failed to show promising clinical trial outcomes.³Patient response rates to T cell immunotherapy strongly correlate with the number of adoptively transferred T cells that successfully reach target tumors. Therefore, the efficacy of CAR-T cell therapy in solid tumors is mainly limited by physical access of T cells to the tumors, with very few able to reach their target sites, resulting in treatment failure. Currently, no clear strategies exist for improving tumor-specific CAR-T cell homing. <h3>Methods</h3> We developed an <i>in vitro</i> model of 3D-cell migration to image CAR-T cell recruitment to target tumoroids by fluorescence and light-sheet microscopy. To address CAR-T cell-specific migration signals, a ‘Multifunctional Approach to Pharmacological Screening (MAPS)’⁴ platform was used to evaluate 3D-migration inhibition in the presence of &gt;1200 chemical suppressors of GPCRs. Receptor hits were corroborated using primary T cell CRISPR knockouts (KO). ELISA and qPCR were used to characterize relevant lipid expression in cancer and immune cells. Wildtype and genetically modified mouse models were evaluated with <i>in vivo</i> tumor models by flow cytometry and intravital two-photon microscopy. <h3>Results</h3> Our migration assay revealed massive CAR-T cell swarming to target tumoroids. Strikingly, this pattern was observed in several human tumor models, suggesting the presence of a highly conserved CAR-T cell recruitment signal applicable to many solid tumors. High-throughput GPCR chemical suppressor library screening identified lipid receptors, including the Leukotriene B4 (LTB₄) receptor BLT1, as key mediators of the CAR-T cell homing to solid tumor targets. Compared to wildtype, BLT1 KO CAR-T cells exhibited significantly diminished homing to tumors <i>in vivo</i> and poorer tumor control, whereas BLT1 overexpression CAR-T cells home significantly better, with improved tumor control. LTB₄ expression characterized by qPCR and ELISA shows a significant increase in the presence of an inflammatory stimulus and higher expression at the tumor compared to healthy tissue. <h3>Conclusions</h3> BLT1 expression in CAR-T cells can improve CAR-T cell homing to solid tumors, thus promoting broad therapeutic efficacy and patient survival. <h3>References</h3> Hou B, Tang Y, Li W, Zeng Q, Chang D. Efficiency of CAR-T therapy for treatment of solid tumor in clinical trials: a meta-analysis. <i>Dis Markers</i>. 2019 Feb 11;<b>2019</b>:3425291. doi: 10.1155/2019/3425291. PMID: 30886654; PMCID: PMC6388318. Siegel RL, Miller KD, Wagle NS, Jemal A. Cancer statistics, 2023. <i>CA Cancer J Clin</i>. 2023 Jan;<b>73</b>(1):17–48. doi: 10.3322/caac.21763. PMID: 36633525. Patel U, Abernathy J, Savani BN, Oluwole O, Sengsayadeth S, Dholaria B. CAR T cell therapy in solid tumors: a review of current clinical trials. <i>EJHaem</i>. 2021 Dec 7;<b>3</b>(Suppl 1):24–31. doi: 10.1002/jha2.356. PMID: 35844304; PMCID: PMC9175685. Harris IS, Endress JE, Coloff JL, Selfors LM, McBrayer SK, Rosenbluth JM, Takahashi N, Dhakal S, Koduri V, Oser MG, Schauer NJ, Doherty LM, Hong AL, Kang YP, Younger ST, Doench JG, Hahn WC, Buhrlage SJ, DeNicola GM, Kaelin WG Jr, Brugge JS. Deubiquitinases maintain protein homeostasis and survival of cancer cells upon glutathione depletion. <i>Cell Metab</i>. 2019 May 7;<b>29</b>(5):1166–1181.e6. doi: 10.1016/j.cmet.2019.01.020. Epub 2019 Feb 21. PMID: 30799286; PMCID: PMC6506399. <h3>Ethics Approval</h3> The animal protocols related to this study were approved by the University Committee on Animal Resources (study ID: UCAR-2008-096E) at the University of Rochester, Rochester NY.