Background: ImmTAC molecules are unique TCR–anti-CD3 bispecifics that redirect T cells against intracellular antigens. IMCgp100, an ImmTAC targeting melanocyte-expressed gp100 antigen, has demonstrated monotherapy activity in advanced melanoma and can cause rash and cytokine-mediated AEs, hypothesized to be on-target (gp100) or effector (CD3) mediated. A preclinical MoA for T cell bispecifics suggests chemokine CXCL10 redirection of CXCR3+ T cells from blood into antigen-positive tissues; this has not been clinically validated. Methods: 84 HLA-A2+ pts with advanced melanoma (n = 61 cutaneous [CM], n = 19 uveal [UM], n = 4 other) received IMCgp100 (NCT01211262). Serum (n = 40) and PBMC (n = 22) samples were taken pre- and post-infusion to analyze changes in cytokines and circulating T cells. Pre- (n = 16) and post-treatment (n = 11) tumor biopsies were analyzed by IHC for CD3, PD-L1 and gp100 expression; tumor RNA (n = 12) was analyzed for gene expression. Results: IMCgp100 induced a transient increase in IFNg-inducible cytokines, most prominently CXCL10. A greater increase in serum CXCL10 was associated with longer OS (p = 0.0002), tumor shrinkage (p = 0.003), and greater transient reduction in peripheral CXCR3+CD8+ T cells (p = 0.001). Reduction in CXCR3+ CD8+ T cells also trended with longer OS (p = 0.02), and tumor shrinkage (p = 0.03). 3/16 pre-treatment biopsies had < 1% gp100 expression (all progressive disease). 8/11 biopsies post-IMCgp100 had increased CD3+ T cells compared with matched pre-treatment samples (associated with baseline gp100 but not PD-L1 expression). Based on tumor biopsy gene expression analysis, IMCgp100 increased T cell markers, IFNg-inducible and cytotoxicity-related genes. Conclusions: The association of clinical benefit with increased serum CXCL10 and decreased peripheral CXCR3+ T cells supports the MoA of IMCgp100-induced T cell redirection and activation. Tumor biopsy results support IMCgp100 redirection of T cells to antigen-positive tumor. A Phase II trial in CM (NCT02535078), a Phase I/II trial in UM (NCT02570308), and a Pivotal RCT in UM (NCT03070392) are ongoing. Clinical trial information: NCT01211262