Garcia, J. M. | bioRxiv Science

bioRxiv2021

Enhanced Solid Tumor Recognition and T cell Stemness with SynNotch CAR Circuits

The lack of highly tumor-specific antigens limits the development of engineered T cell therapeutics because of life-threatening "on-target/off-tumor" toxicities. Here we identify ALPPL2 as a tumor-specific antigen expressed in a spectrum of solid tumors, including mesothelioma. ALPPL2 can act as a sole target for chimeric antigen receptor (CAR) therapy or be combined with tumor-associated antigens such as MCAM or mesothelin in synthetic Notch (synNotch) CAR combinatorial antigen circuits. SynNotch CAR T cells display superior tumor control when compared to CAR T cells to the same antigens by prevention of CAR-mediated tonic signaling allowing T cells to maintain a long-lived memory and non-exhausted phenotype. Collectively, we establish ALPPL2 as a clinically viable target for multiple solid tumors and demonstrate the multi-faceted therapeutic benefits of synNotch CAR T cells. ONE SENTENCE SUMMARYSynNotch CAR circuits targeting novel solid tumor antigens enhance specificity and improve therapeutic efficacy by regulating T cell exhaustion.

immunology↗

bioRxiv2020

A functional genomic screen in Saccharomyces cerevisiae reveals divergent mechanisms of resistance to different alkylphosphocholine chemotherapeutic agents.

The alkylphosphocholine (APC) class of antineoplastic and antiprotozoal drugs, such as edelfosine and miltefosine, are structural mimics of lyso-phosphatidylcholine (lyso-PC), and are inhibitory to the yeast Saccharomyces cerevisiae at low micromolar concentrations. Cytotoxic effects related to inhibition of phospholipid synthesis, induction of an unfolded protein response, inhibition of oxidative phosphorylation, and disruption of lipid rafts have been attributed to members of this drug class, however the molecular mechanisms of action of these drugs remain incompletely understood. Cytostatic and cytotoxic effects of the alkylphosphocholines exhibit variability with regard to chemical structure, leading to differences in effectiveness against different organisms or cell types. We now report the comprehensive identification of Saccharomyces cerevisiae titratable-essential gene and haploid non-essential gene deletion mutants that are resistant to the APC drug miltefosine (hexadecyl-O-phosphocholine). 58 strains out of ~5600 tested displayed robust and reproducible resistance to miltefosine. This gene set was heavily enriched in functions associated with vesicular transport steps, especially those involving endocytosis and retrograde transport of endosome derived vesicles to the Golgi or vacuole, suggesting a role for these trafficking pathways in transport of miltefosine to potential sites of action in the endoplasmic reticulum (ER) and mitochondrion. In addition, we identified mutants with defects in phosphatidylinositol-4-phosphate synthesis (TetO::STT4) and hydrolysis (sac1{Delta}), an oxysterol binding protein homolog (osh2{Delta}), a number of ER resident proteins, and multiple components of the eisosome. These findings suggest that ER-plasma membrane contact sites and retrograde vesicle transport are involved in the interorganelle transport of lyso-PtdCho and related lyso-phospholipid-like analogs to their intracellular sites of cytotoxic activity.

genetics↗