Intratumoral G100, a TLR4 Agonist, Induces Antitumor Immune Responses and Tumor Regression in Patients with Merkel Cell Carcinoma

Abstract

Purpose: G100 is a toll-like receptor 4 (TLR4) agonist that triggers innate and adaptive antitumor immune responses in preclinical models. This pilot study assessed the safety, efficacy, and immunologic activity of intratumoral (IT) administration of G100 in patients with Merkel cell carcinoma (MCC).

Patients and methods: Patients with locoregional MCC (n = 3; cohort A) received neoadjuvant IT G100 (2 weekly doses at 5 μg/dose) followed by surgery and radiotherapy; patients with metastatic MCC (n = 7; cohort B) received 3 doses in a 6-week cycle and could receive additional cycles with/without radiotherapy.

Results: IT G100 was safe and feasible in both neoadjuvant and metastatic settings. Treatment-related adverse events were mostly grade 1 or 2 injection-site reactions. IT G100 led to increased inflammation in the injected tumors with infiltration of CD8⁺ and CD4⁺ T cells and activation of immune-related genes. These proinflammatory changes were associated with local tumor regression and appeared to promote systemic immunity. All 3 cohort A patients successfully completed therapy; 2 patients remain recurrence free at 44+ and 41+ months, including 1 with a pathologic complete response after G100 alone. In cohort B, 2 patients achieved sustained partial responses, both lasting 33+ months after 2 cycles of therapy.

Conclusions: In this first-in-human study, IT G100 induced antitumor immune responses, demonstrated acceptable safety, and showed encouraging clinical activity.See related commentary by Marquez-Rodas et al., p. 1127.

Fig. 1.. Patient characteristics, treatment details and clinical outcomes in 10 patients with MCC treated with intratumoral G100.

Each horizontal bar represents a patient. All patients were alive at last follow-up. All 3 patients with locoregional disease had no evidence of disease (NED) after definitive treatment with surgery/radiation therapy. Two patients with metastatic disease achieved partial response (PR) and 5 had progressive disease (PD).

Fig. 2.. Pathologic complete response (CR) with single-agent G100 (Patient G2).

Representative H&E staining and IHC staining for CK20 is shown for pre-treatment biopsy samples (baseline) and for the residual mass that was surgically removed post-treatment with 2 doses of G100. Absence of staining for CK20 post-treatment demonstrates pathologic complete response after G100 alone. Scale bars denote a 100 μm region at 20x magnification.

Fig. 3.. Immunohistochemical (IHC) pre- and post-treatment with intratumoral G100 in a patient with MCC (Patient G6).

(A) Fluorescent IHC staining for CD4⁺ and CD8⁺ T cells demonstrates restriction of T cells to tumor vasculature and tumor edges prior to treatment, contrasted with diffuse tumor infiltration after treatment with G100. (B) Multispectral IHC staining for CD4⁺ T cells, CD8⁺ T cells, CD68 (monocytes/macrophages), and PD-1/L1 expressing cells within tumors before and after treatment demonstrates increased inflammatory response within the tumor environment post-treatment. Scale bars denote a 50 μm region at 20x magnification.

Fig. 4.. Treatment-inducted changes in gene expression and T-cell receptor (TCR) clonotypes.

The heatmap in Panel A depicts gene expression levels for 770 genes related to innate and adaptive immune responses in biopsy samples from a responding patient (Patient G2). Expression levels in the tumor mass (biopsy-proven, tumor-involved right superficial inguinal lymph node; SN) at baseline and post-G100 treatment are shown as well as post-treatment levels in a deeper, untreated draining lymph node (DN). Panel B displays the frequency of clonotypes in pre-treatment (A; x-axis) and post-treatment (B; y-axis) tumor biopsies from patient G6, who achieved an overall partial response to therapy. Both newly identified clones and expansion of previously detected TCR clones were observed in post-treatment tumor biopsies compared with pre-treatment biopsies. New clones are shown along the y-axis (undetected in the pre-treatment sample but present post-treatment). Clones that were present in the pre-treatment sample but were expanded post-treatment are shown above the 45° line. TCR clones with similar or decreased frequency post-treatment are shown on or below the 45° line. Clones shown on the x-axis indicate T cells that were below the limit of detection post-treatment. There was an overall shift toward greater clone frequencies post-treatment, with 21 clonotypes significantly enriched in the post-treatment sample compared with 8 in the pre-treatment sample.