Preoperative CTLA-4 blockade: tolerability and immune monitoring in the setting of a presurgical clinical trial

Abstract

Purpose: Cytotoxic T lymphocyte associated antigen (CTLA-4) blockade is being explored in numerous clinical trials as an immune-based therapy for different malignancies. Our group conducted the first preoperative clinical trial with the anti-CTLA-4 antibody ipilimumab in 12 patients with localized urothelial carcinoma of the bladder.

Experimental design: Six patients were treated with 3 mg/kg/dose of anti-CTLA-4 and six patients were treated with 10 mg/kg/dose of antibody. Primary end points of the study were safety and immune monitoring.

Results: Most drug-related adverse events consisted of grade 1/2 toxicities. All patients had measurable immunologic pharmacodynamic effects, consisting of an increased frequency of CD4+ICOShi T cells in tumor tissues and the systemic circulation. To determine if CD4+ICOShi T cells could be a correlative marker for clinical outcome after treatment with anti-CTLA-4, a cohort of metastatic melanoma patients was studied retrospectively for frequency of CD4+ICOShi T cells and survival. Data from this small cohort of patients indicated that an increased frequency of CD4+ICOShi T cells, sustained over a period of 12 weeks of therapy, correlates with increased likelihood of clinical benefit consisting of overall survival.

Conclusions: Our trial shows that anti-CTLA-4 therapy has a tolerable safety profile in the presurgical setting and that a preoperative model can be used to obtain biological data on human immune responses, which can efficiently guide the monitoring of patients treated in the metastatic disease setting.

Figure 1. Increased frequency of CD4 ⁺ICOS^hi and CD8⁺ ICOS^hi T cells in tumor tissues after treatment with 10mg/kg/dose of anti-CTLA-4

Representative patient samples demonstrating that CD4⁺ICOS^hi (Upper Panel) and CD8⁺ICOS^hi (Lower Panel) T cells were increased in frequency in tumor tissues after treatment with 10 mg/kg/dose of anti-CTLA-4 as compared to untreated tumor tissues (A). Compilation of data showing statistically significant increased frequencies of CD4⁺ICOS^hi and CD8⁺ICOS^hi T cells in tumor tissues from anti-CTLA-4 treated patients (N=5) as compared to tumor tissues obtained from untreated patients (N=10) (B).

Figure 2. Perivascular infiltration of cells into tumor tissues after treatment with 10 mg/kg/dose of anti-CTLA-4

Representative pictures demonstrating an absence of perivascular infiltration of cells in untreated tumor tissues (0/11) and tumor tissues obtained from patients treated with 3 mg/kg/dose of anti-CTLA-4 (0/6) as compared to the presence of perivascular infiltration noted in tumor tissues obtained from patients treated with anti-CTLA-4 at 10 mg/kg/dose (2/5) (Upper Panel). Immunohistochemistry revealed that the infiltrating cells were positive for CD3, CD8, CD4 and granzyme but predominantly negative for CD20 and CD56 (Lower Panel).

Figure 3. Increased frequency of CD4⁺ICOS^hi and CD8⁺ICOS^hi T cells in the peripheral blood after treatment with 10 mg/kg/dose of anti-CTLA-4

Representative peripheral blood samples taken after the first dose of anti-CTLA-4 at week 3 and after the second dose of anti-CTLA-4 at week 7 showed an increased frequency of CD4⁺ICOS^hi (Upper Panel) and CD8⁺ICOS^hi T cells (Lower Panel) as compared to baseline (pre-therapy) samples (A). Compilation of data showing pre-therapy values for frequency of CD4⁺ICOS^hi T cells (B) and CD8⁺ICOS^hi T cells (C) in bladder cancer patients (N=6) and average frequency in healthy donors (HD, N=10) with statistically significant increases in frequencies of ICOS^hi T cells at weeks 3 and 7 after treatment with anti-CTLA-4.

Figure 4. Increased frequency of CD4⁺ ICOS^hi T cells is detected after patients with metastatic melanoma are treated with anti-CTLA-4

A representative dot plot of ICOS staining in a melanoma patient at baseline and after anti-CTLA-4 therapy, as well as the mouse IgG₁ isotype control (A). All samples analyzed for % CD4⁺ICOS^hi expression in the different donor and patient groups at different time points (B) (HD, healthy donor; Mel-Pt, control melanoma patients; Pre-tx, pre-treatment; SAV, streptavidin).

Figure 5. Correlation of clinical outcome with frequency of CD4⁺ICOS^hi T cells after patients were treated with anti-CTLA-4

Multiparameter flow cytometric analyses were performed on fresh patient PBMCs at baseline, week 7 and 12. Seven of seven patients with clinical benefit at week 24 had persistent elevation in % of CD4⁺ICOS^hi cells, defined as a ≥2-fold increase in % CD4⁺ICOS^hi expression at week 7 or 12 over baseline that was sustained at week 12. In comparison, one of seven patients without clinical benefit, i.e. with progressive disease or death at week 24, had persistent elevation in % of CD4⁺ICOS^hi cells. (* denotes patients with persistent CD4⁺ICOS^hi increase) (A). Kaplan-Meier curve showing the difference in OS for patients with and without persistent % CD4⁺ICOS^hi increase is statistically significant (median OS not reached vs. 27 weeks, p=0.03) (B).