Differential antibody response to EBV proteome following EBVST immunotherapy in EBV-associated lymphomas

Abstract

Epstein-Barr virus (EBV) is associated with a diverse range of lymphomas. EBV-specific T-cell (EBVST) infusions have shown promise in safety and clinical effectiveness in treating EBV-associated lymphomas; however, not all patients respond to T-cell immunotherapies. To identify EBV antigen-specific antibody responses associated with clinical outcomes, we comprehensively characterized antibody responses to the complete EBV proteome using a custom protein microarray in 56 patients with EBV-associated lymphoma who received EBVST infusions in phase 1 clinical trials. Responders (nonprogressors) and nonresponders (progressors) had distinct antibody profiles against EBV. Twenty-five immunoglobulin G (IgG) antibodies were significantly elevated in higher levels in nonresponders than in responders at 3 months after EBVST infusion. Ten of these remained significant after adjustment for sex, age, and cancer type, including LMP2A (4 variants), BGRF1/BDRF1 (2 variants), LMP1, BKRF2, BKRF4, and BALF5. Random forest analysis identified these 10 IgG antibodies as key predictors of clinical response. Paired analyses using blood samples collected at both before infusion and 3 months after EBVST infusion indicated an increase in the mean antibody level for 6 other anti-EBV antibodies (IgG [BGLF2, LF1, and BGLF3]; IgA [BGLF3, BALF2, and BBLF2/3) in nonresponders. Overall, our findings suggest that these EBV-directed antibodies as potential serological markers for predicting clinical responses to EBVST infusions and as therapeutic targets for immunotherapy in EBV-positive lymphomas. These trials were registered at www.clinicaltrials.gov as #NCT01555892 (Cytotoxic T-Lymphocytes for EBV-positive Lymphoma [GRALE]), #NCT02973113 (Nivolumab With Epstein Barr Virus Specific T Cells [EBVSTS], Relapsed/Refractory EBV Positive Lymphoma [PREVALE]), and #NCT02287311 (Most Closely Matched 3rd Party Rapidly Generated LMP, BARF1, and EBNA1 Specific CTL, EBV-Positive Lymphoma [MABEL]).

Graphical abstract

Figure 1.

Differential Ab responses between responders and nonresponders to EBVST immunotherapy. The volcano plots show the difference in IgA and IgG levels (SSI) between responders (n = 36) and non-responders (n = 20) to EBVST immunotherapy at preinfusion and 2-week (2 wk), 4-week (4 wk), and 3-month (3 mo) postinfusion time points. The x-axis represents the fold change (ratio of SSI for responders vs nonresponders), whereas the y-axis shows the corresponding t test log 10 (P values.) IgA Abs are shown in red, and IgG Abs are shown in blue. Abs with a coefficient of variation (CV) <30% are included in the analysis (IgA = 202; IgG = 74). The dashed lines represent the nominally significant P value threshold.

Figure 2.

Multiway variable importance plot of IgA and IgG responses at 3 months after infusion (mean decrease accuracy and mean decrease Gini). This figure illustrates the importance of individual Abs in differentiating between responders and nonresponders, as assessed using the random forest model. The x-axis represents the mean decrease in accuracy, and the y-axis represents the mean decrease in Gini. Higher values on both axes indicate greater importance in the model. Abs in the upper right of the plot are identified as important by both metrics, indicating more substantial contributions to model performance. Notably, Abs that were nominally significant at the 3-month time point (P < .05) in the logistic regression models are also among the most important variables identified by the random forest as the most important variables, demonstrating robust agreement across methods.

Figure 3.

Change in Ab response between preinfusion and 3 months postinfusion. Ribbon plots showing the mean change (with 25th to 75th percentile range) in Ab response (SSI) for EBV-specific antibody markers in responders (n = 36; yellow) and nonresponders (n = 20; purple). Only antibodies with nominal P value of < .05 in adjusted logistic regression models are shown.