Tebentafusp, A TCR/Anti-CD3 Bispecific Fusion Protein Targeting gp100, Potently Activated Antitumor Immune Responses in Patients with Metastatic Melanoma

Abstract

Purpose: Tebentafusp is a first-in-class bispecific fusion protein designed to target gp100 (a melanoma-associated antigen) through a high affinity T-cell receptor (TCR) binding domain and an anti-CD3 T-cell engaging domain, which redirects T cells to kill gp100-expressing tumor cells. Here, we report a multicenter phase I/II trial of tebentafusp in metastatic melanoma (NCT01211262) focusing on the mechanism of action of tebentafusp.

Patients and methods: Eighty-four patients with advanced melanoma received tebentafusp. Treatment efficacy, treatment-related adverse events, and biomarker assessments were performed for blood-derived and tumor biopsy samples obtained at baseline and on-treatment.

Results: Tebentafusp was generally well-tolerated and active in both patients with metastatic uveal melanoma and patients with metastatic cutaneous melanoma. A 1-year overall survival rate of 65% was achieved for both patient cohorts. On-treatment cytokine measurements were consistent with the induction of IFNγ pathway-related markers in the periphery and tumor. Notably, tebentafusp induced an increase in serum CXCL10 (a T-cell attractant) and a reduction in circulating CXCR3⁺ CD8⁺ T cells together with an increase in cytotoxic T cells in the tumor microenvironment. Furthermore, increased serum CXCL10 or the appearance of rash (likely due to cytotoxic T cells targeting gp100-expressing skin melanocytes) showed a positive association with patient survival.

Conclusions: These data suggest that redirecting T cells using a gp100-targeting TCR/anti-CD3 bispecific fusion protein may provide benefit to patients with metastatic melanoma. Furthermore, the activity observed in these two molecularly disparate melanoma classes hints at the broad therapeutic potential of tebentafusp.

Fig. 1:

1-year Overall survival (OS) for Tebentafusp treated mUV and mCM patients

Fig. 2:. Tebentafusp induced a pharmacodynamic response in multiple peripheral immune markers

a. Maximal post-dose (log2) fold-change, relative to baseline concentration, in response to first dose in serum markers in a subset of 40 patients: IL-8, MMP-1, CXCL10, MCP-1, VEGF, BLC, IL-1RA, Galectin 3, MIP-1α, MIP-1β, IL-4, IL-17, GM-CSF, IL-2 Rα, RANK L, CXCL9, IL-5, G-CSF, IL-12 p70, ICAM-1, CXCL11, HGF, VEGF-D, CTACK, Eotaxin (n=40); IFNγ, TNFα, IL-1β, IL-2, IL-6, IL-10 and IL-15 (n=31/40). (ECM, extracellular matrix). b. Temporal profile of post 1st, 4th and 8th dose fold-change response in IFNγ (), IL-10 (), IL-6 (), CXCL10 () and CXCL11 () in a subset of 15 patients treated weekly with 600 ng/kg/ 50 mcg tebentafusp. Plots represents mean ± standard error of the mean [SEM] c. Percentage differences in CXCR3+ CD4+ and CD8+ parent populations (A), CD4+ subsets (B) and CD8+ subsets (C) at ~24 h post 1st dose tebentafusp compared with baseline. Heatmaps constructed using the ComplexHeatmap R library(13). N, naïve; EM, effector memory; CM, central memory; Tscm, stem-cell memory T cell; and LD, late differentiated effector memory. d. Correlation of fold increase in serum CXCL10 with fold decrease in peripheral CXCR3+ CD8+ cell population 24 h following first dose of tebentafusp (Spearman R= −0.66; p=0.00104; n=21).

Fig. 2:. Tebentafusp induced a pharmacodynamic response in multiple peripheral immune markers

a. Maximal post-dose (log2) fold-change, relative to baseline concentration, in response to first dose in serum markers in a subset of 40 patients: IL-8, MMP-1, CXCL10, MCP-1, VEGF, BLC, IL-1RA, Galectin 3, MIP-1α, MIP-1β, IL-4, IL-17, GM-CSF, IL-2 Rα, RANK L, CXCL9, IL-5, G-CSF, IL-12 p70, ICAM-1, CXCL11, HGF, VEGF-D, CTACK, Eotaxin (n=40); IFNγ, TNFα, IL-1β, IL-2, IL-6, IL-10 and IL-15 (n=31/40). (ECM, extracellular matrix). b. Temporal profile of post 1st, 4th and 8th dose fold-change response in IFNγ (), IL-10 (), IL-6 (), CXCL10 () and CXCL11 () in a subset of 15 patients treated weekly with 600 ng/kg/ 50 mcg tebentafusp. Plots represents mean ± standard error of the mean [SEM] c. Percentage differences in CXCR3+ CD4+ and CD8+ parent populations (A), CD4+ subsets (B) and CD8+ subsets (C) at ~24 h post 1st dose tebentafusp compared with baseline. Heatmaps constructed using the ComplexHeatmap R library(13). N, naïve; EM, effector memory; CM, central memory; Tscm, stem-cell memory T cell; and LD, late differentiated effector memory. d. Correlation of fold increase in serum CXCL10 with fold decrease in peripheral CXCR3+ CD8+ cell population 24 h following first dose of tebentafusp (Spearman R= −0.66; p=0.00104; n=21).

Fig. 2:. Tebentafusp induced a pharmacodynamic response in multiple peripheral immune markers

a. Maximal post-dose (log2) fold-change, relative to baseline concentration, in response to first dose in serum markers in a subset of 40 patients: IL-8, MMP-1, CXCL10, MCP-1, VEGF, BLC, IL-1RA, Galectin 3, MIP-1α, MIP-1β, IL-4, IL-17, GM-CSF, IL-2 Rα, RANK L, CXCL9, IL-5, G-CSF, IL-12 p70, ICAM-1, CXCL11, HGF, VEGF-D, CTACK, Eotaxin (n=40); IFNγ, TNFα, IL-1β, IL-2, IL-6, IL-10 and IL-15 (n=31/40). (ECM, extracellular matrix). b. Temporal profile of post 1st, 4th and 8th dose fold-change response in IFNγ (), IL-10 (), IL-6 (), CXCL10 () and CXCL11 () in a subset of 15 patients treated weekly with 600 ng/kg/ 50 mcg tebentafusp. Plots represents mean ± standard error of the mean [SEM] c. Percentage differences in CXCR3+ CD4+ and CD8+ parent populations (A), CD4+ subsets (B) and CD8+ subsets (C) at ~24 h post 1st dose tebentafusp compared with baseline. Heatmaps constructed using the ComplexHeatmap R library(13). N, naïve; EM, effector memory; CM, central memory; Tscm, stem-cell memory T cell; and LD, late differentiated effector memory. d. Correlation of fold increase in serum CXCL10 with fold decrease in peripheral CXCR3+ CD8+ cell population 24 h following first dose of tebentafusp (Spearman R= −0.66; p=0.00104; n=21).

Fig. 3:. Increased presence of T cells observed in on-treatment tumors.

Image analysis quantified the expression of CD3+, CD4+, or CD8+ T cells together with PD-L1 expression. (a) Number of CD3+, CD8+, CD4+ and PD-L1+ cells/ mm² tumor in paired baseline and early on-treatment biopsies (taken Cycle 1 Day 3–17) from up to 11 patients; line per patient. (b) Example immunohistochemistry (IHC) images of CD3+ staining in baseline and on-treatment (C1D3) biopsies from three patients: non-uveal Patient A (Rectus abdominal muscle) and B (L abdomen); uveal Patient C (abdominal wall). (c) Heatmap representation of genes identified from enrichment analysis with significantly different expression in on-treatment tumor biopsy (taken Cycle 1 Day 3–17) relative to baseline sample from partial response compared with progressive disease patients. These genes belonged to NanoString categories ‘Antigen processing’, ‘Cytotoxicity’ or ‘T cell function’ (*HLA-C, -A, -B also in Antigen Processing and Cytotoxicity category). Data scale represents log2 fold-change relative to associated baseline.

Fig. 3:. Increased presence of T cells observed in on-treatment tumors.

Image analysis quantified the expression of CD3+, CD4+, or CD8+ T cells together with PD-L1 expression. (a) Number of CD3+, CD8+, CD4+ and PD-L1+ cells/ mm² tumor in paired baseline and early on-treatment biopsies (taken Cycle 1 Day 3–17) from up to 11 patients; line per patient. (b) Example immunohistochemistry (IHC) images of CD3+ staining in baseline and on-treatment (C1D3) biopsies from three patients: non-uveal Patient A (Rectus abdominal muscle) and B (L abdomen); uveal Patient C (abdominal wall). (c) Heatmap representation of genes identified from enrichment analysis with significantly different expression in on-treatment tumor biopsy (taken Cycle 1 Day 3–17) relative to baseline sample from partial response compared with progressive disease patients. These genes belonged to NanoString categories ‘Antigen processing’, ‘Cytotoxicity’ or ‘T cell function’ (*HLA-C, -A, -B also in Antigen Processing and Cytotoxicity category). Data scale represents log2 fold-change relative to associated baseline.

Fig. 4:. On-treatment biomarkers associated with clinical response.

A greater maximal fold increase in serum CXCL10 level, and maximal fold decrease in circulating CXCR3+ CD8+ T cell population in response to first dose of tebentafusp, was associated with longer OS and tumour shrinkage. Kaplan–Meier survival of patients by: (a) serum CXCL10 (n=40, p=0.00019) and (c) CXCR3+ CD8+ T cell population (n=22, p=0.0086); both ≥median vs <median. Waterfall plots depicting the maximum % reduction in the sum of longest diameters (SLD) of target tumor measurements from baseline for change in (b) serum CXCL10 (Fisher’s Exact test, p=0.0029) and (d) CXCR3+ CD8+ T cells (Fisher’s Exact test, p=0.03), both ≥median vs <median.

Fig. 5:. Rash associated with patient survival and elevated serum CXCR10

a. Kaplan–Meier survival of patients with any ‘rash’ (refer to methods) within 21 days of treatment start (n=69) versus no rash reported (n=15), (p=0.028). Patients with ‘rash’ have tendency for greater on-treatment maximal fold-change in (b) serum CXCL10 (‘rash’ n=34 vs. no rash n=6, p= 0.001) and (c) circulating CXCR3+CD8+ T cells (‘rash’ n=17 vs. no rash n=4, p= 0.04). Box plot representation of fold-change relative to baseline, showing median and quartiles for each group.