Potent Cytotoxic Tumor-Infiltrating Lymphocytes Can Be Generated from Immune-Excluded Chondrosarcomas Using Regulatable Membrane-Bound IL15.

Autologous tumor-infiltrating lymphocyte (TIL) cell therapy is showing promising efficacy against immunologically "hot" tumors such as melanoma, cervical cancer, and renal cancer. However, generation of tumoricidal TILs from "cold" tumors with a low tumor mutational burden, such as many sarcoma types, poses a challenge due to limited infiltration of the tumor microenvironment (TME) with lymphocytes, low frequencies of tumor antigen-specific, high-affinity T cells, and incompletely understood mechanisms of immune-resistance prevailing in the TME. Here, we report the successful generation and expansion of TILs engineered with regulatable, membrane-bound IL15 (cytoTIL15 cells) from immune-excluded, paucicellular chondrosarcoma biopsies largely consisting of collagenous matrix and demonstrate that these cells have potent tumor-killing capacity in cell culture and in tumor spheroid models in the absence of exogenous IL2. Comprehensive spatial profiling of the TME revealed ubiquitous collagen and myeloid infiltration as major resistance mechanisms, whereas lymphocytic infiltration was largely restricted to peripheral regions of the tumors, a relevant consideration when sampling these tumors for TIL harvest. Moreover, we demonstrate that IL15 reduced the signaling threshold of T-cell receptors isolated from TIL clonotypes, increasing their infiltration and cytotoxicity in autologous 3D tumor models. These results suggest the possibility of developing an effective IL2-free TIL therapy for patients with immune-excluded tumors.