Oncolytic Virotherapy Promotes Intratumoral T Cell Infiltration and Improves Anti-PD-1 Immunotherapy

Abstract

Here we report a phase 1b clinical trial testing the impact of oncolytic virotherapy with talimogene laherparepvec on cytotoxic T cell infiltration and therapeutic efficacy of the anti-PD-1 antibody pembrolizumab. Twenty-one patients with advanced melanoma were treated with talimogene laherparepvec followed by combination therapy with pembrolizumab. Therapy was generally well tolerated, with fatigue, fevers, and chills as the most common adverse events. No dose-limiting toxicities occurred. Confirmed objective response rate was 62%, with a complete response rate of 33% per immune-related response criteria. Patients who responded to combination therapy had increased CD8+ T cells, elevated PD-L1 protein expression, as well as IFN-γ gene expression on several cell subsets in tumors after talimogene laherparepvec treatment. Response to combination therapy did not appear to be associated with baseline CD8+ T cell infiltration or baseline IFN-γ signature. These findings suggest that oncolytic virotherapy may improve the efficacy of anti-PD-1 therapy by changing the tumor microenvironment. VIDEO ABSTRACT.

Keywords: T lymphocytes; anti-PD-1; biomarkers; cytotixic; interferon gamma; melanoma; oncolytic immunotherapy; oncolytic viruses; pembrolizumab; talimogene laherparepvec; tumor; tumor microenvironment.

Copyright © 2017 Elsevier Inc. All rights reserved.

Figures

Figure 1.. Melanoma Study Design and Clinical Response to Combination of Talimogene Laherparepvec and Pembrolizumab

(A) Phase 1b study design schema. Stars indicate the time of scheduled tumor biopsies. (B) Computed tomography scans of two patients with response to the combination therapy. Melanoma metastases are marked with a blue arrow at baseline. (C) Waterfall plot of best response change in tumor burden from baseline. Patients were required to have baseline and ≥ 1 postbaseline tumor assessments to be included. (D) Change in tumor burden over time. (E) Kaplan-Meier analysis of progression-free survival. (F) Kaplan-Meier analysis of overall survival. See also Figures S1 and S2.

Figure 2.. Combination of Talimogene Laherparepvec and Pembrolizumab Is Effective in Patients with Low Tumor CD8+ Density

(A) Baseline CD8+ density in tumor biopsies according to response rate. Magnitude of bars indicates baseline tumor CD8+ density in each patient’s baseline biopsy, and best overall response is indicated on x axis and by bar color. Gold, CR; pink, PR; blue, PD. (B) Baseline PD-L1 by IHC status (1% cutoff) and IFN-γ signature score by NanoString analysis is shown under each patient’s CD8 result. Best overall response per investigator is shown as of cutoff date of August 2016. Abbreviations: CR, complete response; IFN-γ, interferon γ; IHC, immunohistochemistry; NA, result not available; PD, progressive disease; PR, partial response.

Figure 3.. Talimogene Laherparepvec Increases Tumor CD8+ Density in Patients Responding to Combination of Talimogene Laherparepvec and Pembrolizumab

(A) Examples of pre (week 1) and post (week 6) talimogene laherparepvec and talimogene laherparepvec plus pembrolizumab (week 30) CD8+ density in tumor biopsies: visualization of cells stained with CD8 antibody with red chromogen. Staining was quantified for tissue regions of interest including CD8+ density in the tumor as shown for talimogene laherparepvec-injected tumors. (B and C) CD8+ density (B) and granzyme B H-score (C) is shown for baseline and postbaseline biopsies. The left side in each panel shows postbaseline results from injected lesions, and the right side in each panel shows results from noninjected lesions. Open circles indicate results from tumor biopsies that were depleted of melanoma cells but had pathologic features of having previously been infiltrated by melanoma cells such as melanin deposits. Response is color coded for best overall response per investigator (complete or partial response in red and nonresponse in blue). (D and E) CD8α (D) and IFN-γ normalized (E) mRNA transcript count were measured in the NanoString Pan Cancer Immune Profiling Panel. IFN-γ = interferon γ. See also Figure S3.

Figure 4.. Talimogene Laherparepvec Increases Tumor-Infiltrating Lymphocyte Density and PD-L1 Expression in Tumors

Twelve-color immunofluorescence staining was performed on a single slide from paired pre- and post-talimogene laherparepvec tumor biopsies from each of 13 patients. Markers evaluated included S100 (as melanoma segmentation marker), CD3, CD4, CD8, PD-1, PD-L1, CTLA-4, CD45RO, Foxp3, CD56, CD68, and CD20. (A) A subset of changes at week 6 from baseline in marker cell positive cell density for results with statistical significance (PD-L1, PD-1, CD8, CD4, CD56, CD20, CD45RO, and Foxp3) are graphed for noninjected (left) and injected (right) samples. Median change for each subset is shown with a horizontal line. Response is color coded for best overall response per investigator: complete or partial response in red and nonresponse in blue. (B) Example of the combination of S100 (blue), CD8 (green), and PD-L1 (red) staining is shown at low (top) and high (bottom) magnification for a baseline biopsy from a patient who went on to have a partial response (week 1), week 6 after injection of talimogene laherparepvec, and at week 30 after long-term treatment with the combination of talimogene laherparepvec and pembrolizumab. Abbreviations: CTLA-4, cytotoxic T cell-associated antigen 4; I, biopsy of an injected metastasis; PD-1, programmed death protein 1; PD-L1, programmed death ligand 1; NI, biopsy of a noninjected metastasis. See also Figures S4–S6.

Figure 5.. Circulating T Cell Subsets and Expression of Activation Markers

Peripheral blood cells obtained from baseline, week 1, week 6, week 8, and week 30 were analyzed by flow cytometry. (A) Fold change in absolute CD3+CD8+ cells. (B) Fold change in absolute CD3+CD4+ cells. (C) Percentage change in Ki67+ (CD3+CD8+) cells. (D) Percentage change in PD-1+ (CD3+CD8+) cells at week 1 and week 6 only; after starting on pembrolizumab, the staining antibody competed for the same epitope. (E) Percentage change in TIM3+ (CD3+CD8+) cells. (F) Percentage change in BTLA+ (CD3+CD8+) cells. p values for comparison with baseline are shown below data for each postbaseline visit, based on contrasts from linear mixed-effects modeling. Response is color-coded for best overall response per investigator (complete or partial response in red and nonresponse in blue). Abbreviations: BTLA, B- and T-lymphocyte attenuator; PD-1, programmed death protein 1.