XL888 and pembrolizumab modulate the immune landscape of colorectal tumors in a phase Ib/II clinical trial

Abstract

We conducted a phase Ib/II clinical trial to evaluate the safety, feasibility, and clinical activity of combining pembrolizumab (anti-PD-1) with XL888 (Hsp90 inhibitor) in patients with advanced colorectal cancer (CRC). We hypothesized that this regimen would modulate soluble and cellular immune mediators and enhance clinical outcomes. The trial employed a 3 + 3 open-label design, with an expansion cohort at the recommended phase II dose (RP2D) in treatment-refractory, mismatch repair-proficient CRC patients. Comprehensive analyses of plasma cytokines, peripheral blood mononuclear cells (PBMCs), and spatial immune cell patterns in liver biopsies were performed to identify unique immune signatures resulting from the combined therapy. The combination of pembrolizumab and XL888 proved to be safe and feasible, with a subset of patients achieving stable disease, although no objective responses were observed in this heavily pre-treated population. Correlative studies revealed immunomodulatory effects in tumors and circulation, including a reduction in IL6⁺ cells and macrophages (CD68⁺) within metastatic liver tissue, alterations in blood CD3⁺ cells, and upregulation of numerous inflammatory plasma cytokines. These findings suggest local and systemic immune activation by the combination of pembrolizumab and XL888. While clinical activity was modest in treatment-refractory CRC patients, there were notable effects on the tumor immune environment and systemic immune modulation.

Keywords: Clinical trial; colorectal cancer; cytokines; heat shock protein; immune checkpoint inhibitors; immunotherapy.

Figure 1.

Peripheral blood and metastatic liver biopsy collection timeline for correlative studies. (a) Timeline examines blood and biopsy collection timepoints during cycle 1 for the dose-escalation and colorectal cancer (CRC) dose-expansion phases of the trial. (b) Schematic displays CRC patients receiving the RP2D, detailing analyses performed and the total number of samples processed for each analysis. Images created using BioRender.com.

Figure 2.

Clinical outcomes for patients in the dose-escalation and CRC dose- expansion phases. a-b Kaplan–Meier plots display the PFS (a) and OS (b) outcomes for patients in the dose-escalation and CRC-expansion phase of the trial. (c) Maximum percent change in the size of target metastatic liver lesions from baseline for the CRC dose-expansion phase. Each bar represents an individual patient.

Figure 3.

Multiplex immunohistochemistry (mIHC) analysis of immune and stromal cell populations in paired metastatic liver biopsies at C1D1 and C1D15 with pembrolizumab alone or combined with XL888. (a) Representative mIHC image of paired liver biopsies. Specific biomarkers displayed for panel 1 include, DAPI (royal blue), CD19 (yellow), IL-6 (magenta), ɑ-SMA (light blue), CD68 (red), CK19 (gray), and CD3 (green) as indicated in the top left; white scale bars represent 25µm, 100µm, or 1000µm. b-e. Percentage of IL-6⁺ (b), ɑ-SMA⁺ IL-6⁺ (c), CD68⁺ (d), and CD68⁺ IL-6⁺ (e) as a proportion of DAPI+ cells.

Figure 4.

CyTOF-based identification and quantification of CD19+ cells in liver metastases at C1D1 and C1D15 in patients treated with pembrolizumab alone or in combination with XL888. (a) CyTOF gating strategy demonstrating the sequential identification of CD45⁺ cells followed by CD19⁺ cells within the gated population. (b) Percentage of live CD19⁺ cells from C1D1 to C1D15 in patients treated with pembrolizumab (n = 4) or in combination with XL888 (n = 4).

Figure 5.

Differential plasma cytokine profiles in patients treated with pembrolizumab alone or in combination with XL888. (a) Heat map of plasma cytokine modulation in response to treatment. Relative fold change (log₂ transformed) of multiple cytokines, normalized to treatment baseline (C1D1) in patients receiving pembrolizumab alone (top panel) or in combination with XL888 (bottom panel). Mann–Whitney test was performed to compare cytokine levels between the two treatment groups at each time point, with statistical significance at **p < 0.01. (b) Quantitative analysis of cytokine concentrations in blood plasma for eight cytokines: IL-6 (p = 0.0020), IP-10 (p = 0.0020), GM-CSF (p = 0.0312, 0.0156), MCP-1 (p = 0.0059) (top, left to right), and MDC (p = 0.0488), IL-15 (p = 0.0195), Eotaxin (p = 0.0273), IL-1RA (p = 0.132) (bottom, left to right). Changes in biomarker levels from baseline (C1D1) to post-treatment (C1D15) were assessed using the Wilcoxon matched-pairs signed rank test, statistical significance at *p < 0.05 and **p < 0.01.

Figure 6.

CyTOF-based immune profiling reveals alterations in peripheral blood immune cell subsets. a-d. The percentage of live cells expressing CD45⁺ (a), CD3⁺ (p = 0.0273) (b), CD8⁺ (c), and CD4⁺ (d) Changes in immune cell populations from C1D1 to C1D15 were assessed using the Wilcoxon matched-pairs signed rank test with statistical significance set at p < 0.05.