CD4+ tumor-infiltrating lymphocytes secreting T cell-engagers induce regression of autologous patient-derived non-small cell lung cancer xenografts

Adoptive transfer of tumor-infiltrating lymphocytes (TIL) has shown remarkable results in melanoma, but only modest clinical benefit in other cancers, even after TIL have been genetically modified to improve their tumor homing, cytotoxic potential or overcoming cell exhaustion. The required ex vivo TIL expansion process may induce changes in the T cell clonal composition, which could likely compromise the tumor reactivity of TIL preparations and ultimately the success of TIL therapy. A promising approach based on the production of bispecific T cell engagers (TCE) by engineered T cells (STAb-T therapy) improves the efficacy of current T cell redirection strategies against tumor-associated antigens in hematological tumors. We studied the TCRβ repertoire in non-small cell lung cancer (NSCLC) tumors and in ex vivo expanded TIL from two unrelated patients. We generated TIL secreting anti-epidermal growth factor receptor (EGFR) x anti-CD3 TCE (TILSTAb) and tested their antitumor efficacy in vitro and in vivo using a NSCLC patient-derived xenograft (PDX) model in which tumor fragments and TIL from the same patient were transplanted into hIL-2 NOG mice. We confirmed that the standard TIL expansion protocol promotes the loss of tumor-dominant T cell clones and the overgrowth of virus-reactive TCR clonotypes that were marginally detectable in primary tumors. We demonstrated the antitumor activity of TILSTAb both in vitro and in vivo when administered intratumorally and systemically in an autologous immune-humanized PDX EGFR+ NSCLC mouse model, where tumor regression was mediated by TCE-redirected CD4+ TIL bearing non-tumor dominant clonotypes. SIGNIFICANCE Epithelial tumor-derived TIL can be engineered to secrete TCE capable of redirecting T cells bearing non-tumor-dominant clonotypes regardless of their phenotype, which could have broad applications in immunotherapy for solid tumors. ### Competing Interest Statement B.B., L.P-A and L.A-V are co-founders and shareholders of STAb Therapeutics, a spin-off company from the imas12. B.B. and L.A-V are inventors on the patent EP21708942 pending. L.D-A. and L.A-V are inventors on the patent EP23383410.0 pending. L.A-V is cofounder and shareholder of Leadartis, a company focused on unrelated interest. L.A-V reports speaker honoraria from MSD, Merck KGaA, BMS, Janssen, GSK, and Miltenyi, and receives grant support from Merck KGaA, all outside the submitted work. J.Z. has served as a consultant for Sanofi, Pfizer, Astra Zeneca, BMS, Novartis, NanoString, and Guardant Health; reports speaker honoraria from Pfizer, BMS, Roche, Astra Zeneca, NanoString and Guardant Health; and receives grant support from Astra Zeneca, Roche, and BMS, all outside the submitted work. L.P-A. is shareholder of Altum Squencing and STAb Therapeutics, has served as a consultant for Lilly, MSD, Roche, Pharmamar, Merck KGaA, Astra Zeneca, Novartis, Amgen, Pfizer, Sanofi, Bayer, BMS, Mirati, GSK, Janssen, Takeda, Regeneron, and Sanofi. L.P-A. received grant support from MSD, Astra Zeneca, BMS, Pfizer and Pharmamar, all outside the submitted work. R.T. and E.P. are consultants for the company NISOLAB.