AUTHOR=Cianciotti Beatrice Claudia , Magnani Zulma Irene , Ugolini Alessia , Camisa Barbara , Merelli Ivan , Vavassori Valentina , Potenza Alessia , Imparato Antonio , Manfredi Francesco , Abbati Danilo , Perani Laura , Spinelli Antonello , Shifrut Eric , Ciceri Fabio , Vago Luca , Di Micco Raffaella , Naldini Luigi , Genovese Pietro , Ruggiero Eliana , Bonini Chiara 

TITLE=TIM-3, LAG-3, or 2B4 gene disruptions increase the anti-tumor response of engineered T cells

JOURNAL=Frontiers in Immunology

VOLUME=15

YEAR=2024

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2024.1315283

DOI=10.3389/fimmu.2024.1315283

ISSN=1664-3224

ABSTRACT=<sec><title>Background</title><p>In adoptive T cell therapy, the long term therapeutic benefits in patients treated with engineered tumor specific T cells are limited by the lack of long term persistence of the infused cellular products and by the immunosuppressive mechanisms active in the tumor microenvironment. Exhausted T cells infiltrating the tumor are characterized by loss of effector functions triggered by multiple inhibitory receptors (IRs). In patients, IR blockade reverts T cell exhaustion but has low selectivity, potentially unleashing autoreactive clones and resulting in clinical autoimmune side effects. Furthermore, loss of long term protective immunity in cell therapy has been ascribed to the effector memory phenotype of the infused cells.</p></sec><sec><title>Methods</title><p>We simultaneously redirected T cell specificity towards the NY-ESO-1 antigen via TCR gene editing (TCR<sub>ED</sub>) and permanently disrupted <italic>LAG3</italic>, <italic>TIM-3</italic> or <italic>2B4</italic> genes (IR<sub>KO</sub>) via CRISPR/Cas9 in a protocol to expand early differentiated long-living memory stem T cells. The effector functions of the TCR<sub>ED</sub>-IR<sub>KO</sub> and IR competent (TCR<sub>ED</sub>-IR<sub>COMP</sub>) cells were tested in short-term co-culture assays and under a chronic stimulation setting <italic>in vitro</italic>. Finally, the therapeutic efficacy of the developed cellular products were evaluated in multiple myeloma xenograft models.</p></sec><sec><title>Results</title><p>We show that upon chronic stimulation, TCR<sub>ED</sub>-IR<sub>KO</sub> cells are superior to TCR<sub>ED</sub>-IR<sub>COMP</sub> cells in resisting functional exhaustion through different mechanisms and efficiently eliminate cancer cells upon tumor re-challenge <italic>in vivo</italic>. Our data indicate that TIM-3 and 2B4-disruption preserve T-cell degranulation capacity, while LAG-3 disruption prevents the upregulation of additional inhibitory receptors in T cells.</p></sec><sec><title>Conclusion</title><p>These results highlight that TIM-3, LAG-3, and 2B4 disruptions increase the therapeutic benefit of tumor specific cellular products and suggest distinct, non-redundant roles for IRs in anti-tumor responses.</p></sec>