Humoral response to a viral glycan correlates with survival on PROSTVAC-VF

Abstract

Therapeutic cancer vaccines can be effective for treating patients, but clinical responses vary considerably from patient to patient. Early indicators of a favorable response are crucial for making individualized treatment decisions and advancing vaccine design, but no validated biomarkers are currently available. In this study, we used glycan microarrays to profile antiglycan antibody responses induced by PROSTVAC-VF, a poxvirus-based cancer vaccine currently in phase III clinical trials. Although the vaccine is designed to induce T-cell responses to prostate-specific antigen, we demonstrate that this vaccine also induces humoral responses to a carbohydrate on the poxvirus, the Forssman disaccharide (GalNAcα1-3GalNAcβ). These responses had a statistically significant correlation with overall survival in two independent sample sets (P = 0.015 and 0.008) comprising more than 100 patients. Additionally, anti-Forssman humoral responses correlated with clinical outcome in a separate study of PROSTVAC-VF combined with a radiopharmaceutical (Quadramet). Studies on control subjects demonstrated that the survival correlation was specific to the vaccine. The results provide evidence that antiglycan antibody responses may serve as early biomarkers of a favorable response to PROSTVAC-VF and offer unique insights for improving vaccine design.

Keywords: Forssman antigen; glycan array.

Fig. 1.

Postvaccination changes in levels of serum antiglycan antibodies. Heat maps show postvaccination fold changes in antiglycan antibody levels (IgM + IgG + IgA) for (A) discovery and (B) validation sets measured at a serum dilution of 1:200. Columns correspond to 204 array components (ordered alphabetically within glycan families), and rows are individual patients (arranged by expected survival calculated using the Halabi nomogram). Increases are colored red, and decreases are green. Black indicates no significant change (increase <160% or decrease <61%).

Fig. 2.

Humoral responses to terminal saccharides of blood group A and Forssman glycans. (A) Structures of glycans with largest changes in postvaccination antibody levels. Glycan density on the array was controlled by varying the number of glycans conjugated to the albumin carrier of the neoglycoprotein. Presented data were obtained with median conjugation ratios (glycans/albumin molecule) of 4, 4, and 19 for Fs_di, BG-A_di, and BG-A_tri, respectively. (B) Levels of total Ig for BG-A_di, BG-A_tri, and Fs_di were stable in healthy subjects (orange) and in nonvaccinated prostate cancer patients treated with radiation (purple). However, humoral responses to these glycans occurred in patients inoculated with PROSTVAC-VF (blue and red) or its viral vectors (green). Subjects with no significant change in antibody levels (<2.6-fold change) are labeled as n.c. (C) Venn diagram showing that fourfold or greater humoral responses to these three glycans typically co-occurred in the same patients.

Fig. 3.

Kaplan–Meier survival curves for patients stratified by postvaccination response to Fs_di. Kaplan–Meier curves were generated using actual overall survival data (A–C) or HPS data (D–F) for patients stratified according to anti-Fs_di responses (IgG+IgM+IgA). (A and B) In both discovery (A) and validation (B) sets, humoral responses to Fs_di were associated with improved overall survival. (C) Increased anti-Fs_di antibody levels also were seen after inoculation with wild-type viral vectors; however, humoral responses did not correlate with improved survival for control patients. (D–F)To verify that there were no systematic differences between the strata at baseline, Kaplan–Meier survival curves were generated using the HPS based on prevaccination prognostic data.

Fig. 4.

Correlation between humoral response to Fs_di and other prognostic factors. There was no association between anti-Fs_di humoral responses and (A) age, (B) HPS, (C) on-study PSA levels, or (D) Gleason score of the primary prostate tumor. The HPS calculation was based on seven prognostic factors: PSA, Gleason score, serum lactate dehydrogenase, AP, hematocrit, presence of visceral disease, and Eastern Cooperative Oncology Group functional status. Correlations were assessed with Kendall correlation coefficients (τ) and their associated P values.

Fig. 5.

Correlation between humoral response to Fs_di and other measures of immune function. Sera collected before and after vaccination with PROSTVAC-VF were analyzed to determine changes in total Ig (IgG+IgM+IgA) reactive with Fs_di. Fold-changes in anti-Fs_di Ig were plotted against other immunological data. Correlations with (A) peripheral levels of T-regulatory cells, overall postvaccination IgG titers for (B) vaccinia and (C) fowlpox, and (D) fold increase in PSA-specific T cells by ELISPOT assay. τ, Kendall correlation coefficients.

Fig. 6.

Kaplan–Meier survival curves for combination therapy stratified by postvaccination response to Fs_di. Patients (n = 13) treated with PROSTVAC-VF and Quadramet were stratified according to their postvaccination humoral anti-Fs_di responses. Probability of survival is plotted for patients with significant (solid blue) or little to no (dashed red) fold change in total Ig in response to Fs_di. The Kaplan–Meier curves were generated using the measurement technique and threshold used for stratifying Forssman responses in Fig. 3.

Fig. 7.

Glycan profiling of PROSTVAC-VF’s viral vectors. (A) IgG antibody binding to glycans after preincubating serum with PROSTVAC-VF’s viral vectors was normalized to IgG binding under standard conditions in the absence of the viral vectors. The peptide sequence was Ac-S-S-S-G, and the clustered Tn peptide was Ac-Tn(Thr)-Tn(Thr)-Tn(Thr)-G (20/BSA). Error bars ± SD (n = 4); P = 2e-7 by two-way ANOVA.] (B) Additionally, the vectors differentially bound monoclonal antibodies specific for Forssman antigen (Fs) or control (Tn antigen). (C) Comparison of vaccinia and fowlpox binding to lectins reactive for Forssman antigen (DBA) or blood group H (UEA). Error bars ± SD (n = 3). **P < 0.001 for Forssman-specific binding relative to control.