Combined Anti-VEGF and Anti-CTLA-4 Therapy Elicits Humoral Immunity to Galectin-1 Which Is Associated with Favorable Clinical Outcomes

Abstract

The combination of anti-VEGF blockade (bevacizumab) with immune checkpoint anti-CTLA-4 blockade (ipilimumab) in a phase I study showed tumor endothelial activation and immune cell infiltration that were associated with favorable clinical outcomes in patients with metastatic melanoma. To identify potential immune targets responsible for these observations, posttreatment plasma from long-term responding patients were used to screen human protein arrays. We reported that ipilimumab plus bevacizumab therapy elicited humoral immune responses to galectin-1 (Gal-1), which exhibits protumor, proangiogenesis, and immunosuppressive activities in 37.2% of treated patients. Gal-1 antibodies purified from posttreatment plasma suppressed the binding of Gal-1 to CD45, a T-cell surface receptor that transduces apoptotic signals upon binding to extracellular Gal-1. Antibody responses to Gal-1 were found more frequently in the group of patients with therapeutic responses and correlated with improved overall survival. In contrast, another subgroup of treated patients had increased circulating Gal-1 protein instead, and they had reduced overall survival. Our findings suggest that humoral immunity to Gal-1 may contribute to the efficacy of anti-VEGF and anti-CTLA-4 combination therapy. Gal-1 may offer an additional therapeutic target linking anti-angiogenesis and immune checkpoint blockade. Cancer Immunol Res; 5(6); 446-54. ©2017 AACR.

Figure 1

Ipilimumab plus bevacizumab (Ipi-Bev) elicited antibody responses to Gal-1 in patients with metastatic melanoma. A and B, Detection of Gal-1 antibodies in pretreatment and posttreatment plasma samples of representative patients by immunoblot analyses (A) and ELISA (B). Clinical responses of the patients are also indicated (CR, complete response; PR, partial response; SD, stable disease; and PD, progressive disease). C, Gal-1 antibody titers in the pretreatment and posttreatment plasma samples of 43 Ipi-Bev-treated patients measured by ELISA. Patients were grouped by Gal-1 antibody fold change ≥ 1.5 or < 1.5. The numbers of patients in each group are indicated. D, Proportions of patients treated with Ipi-Bev (n = 43), ipilimumab (n = 35), and PD-1 blockade (n = 31) with Gal-1 antibody fold change ≥ 1.5. Statistically significant differences are noted between treatment groups with Ipi-Bev patients having the greatest incidence of Gal-1 antibody fold change ≥ 1.5 (P = 0.044 to ipilimumab alone; P = 0.0005 to PD-1 blockade).

Figure 2

Antibody responses to Gal-1 correlate with clinical outcomes in metastatic melanoma patients treated with Ipi-Bev. A, Patients were plotted based on their Gal-1 antibody fold changes. Each bar represents a patient and the color of the bar indicates clinical response of the patient (CR, complete response; PR, partial response; SD, stable disease; and PD, progressive disease). There were 8 CR/PR (1 CR and 7 PR), 22 SD, and 13 PD patients. Gal-1 antibody titer was considered as increased when fold change ≥ 1.5. B, Composition of the 16 patients with a Gal-1 antibody increase. Numbers (and %) of CR/PR, SD and PD patients with Gal-1 antibody fold changes ≥ 1.5 are shown. C, Frequencies of Gal-1 antibody increase by clinical responses. D, Kaplan-Meier survival curves of patients based on Gal-1 antibody fold change ≥ 1.5 or < 1.5 (P = 0.0031). The median survival of the patients with Gal-1 antibody fold change < 1.5 was 70 weeks (95% CI, 47–81), whereas that of patients with fold change ≥ 1.5 was unreached.

Figure 3

Anti-Gal-1 antibodies isolated from an Ipi-Bev-treated patient abrogate Gal-1 binding to CD45. Anti-Gal-1 antibodies were affinity purified from plasma. Biotinylated HAS-Gal-1 (250 ng/ml) was incubated with a commercial anti-Gal-1 polyclonal antibody (Gal-1 Ab) or control antibody (10 μg/ml), enriched endogenous Gal-1 antibodies (Gal-1 Ig) or normal human IgG (1.98 μg/ml) prior to incubation with coated CD45. The binding of HAS-Gal-1 to CD45 was detected with streptavidin-HRP. Sucrose and lactose were added to the reaction at 5 mM. Data are presented as Mean ± SD of 3 experiments.

Figure 4

Ipi-Bev therapy altered Gal-1 expression in tumors and in the circulation. A and B, Patient P27. Gal-1 expression was hardly detected in the pretreatment tumors (A) but was intensive in tumor cells and tumor associated endothelial cells (TEC) posttreatment (B). Representative TEC and tumor cells are indicated with arrows and arrow heads respectively. C and D, Patient P20. Gal-1 expression was highly expressed in tumor cells but not TEC pretreatment (C). Strong Gal-1 expression was observed in tumor cells and TEC posttreatment (D). E and F, Patient P28. Gal-1 was highly expressed in tumor cells and TEC pretreatment (E). Reduced and no Gal-1 expression was detected in tumor cells and TEC posttreatment (F). G, Pretreatment and posttreatment circulating Gal-1 concentrations of the patients (n = 43). H, Different subsets of patients had increased circulating Gal-1 and antibody responses to Gal-1 in response to Ipi-Bev. Patients are grouped by clinical responses. I, Kaplan-Meier survival curves of patients based on circulating Gal-1 fold changes as a result of Ipi-Bev treatment. Increases in circulating Gal-1 were significantly associated with shortened overall survival (P = 0.012). The median survival of the patients with circulating Gal-1fold change ≥ 1.2 was 13.9 months (95% CI, 5–19), while that of patients with fold change < 1.2 was 21.6 months (95% CI, 16 to ∞).