Two 7-mer cyclic peptides—Rp15-C and Rp13-C—which bear the antigenic motif recognized by the anti-CD20 mAb rituximab, but have different motif-surrounding amino acids, show a comparable avidity for rituximab and inhibit the binding of rituximab to raft-associated CD20 and rituximab-induced membrane ceramide on human lymphoid Daudi cells. Their immunogenic profiles differed: Abs recognizing CD20 were induced in two and five of five BALB/c mice immunized with Rp15-C and Rp13-C, respectively. Analysis of immunogenic motif, performed by panning a 7-mer phage-display peptide library with purified anti-peptide IgGs, showed that the motif defined by anti-Rp15-C mostly included amino acids surrounding the rituximab-specific antigenic motif <aNPS>, whereas that defined by anti-Rp13-C was <NPS>. These data indicate that their motif-surrounding amino acids can markedly influence the specificity of Abs, even when elicited with a short 7-mer peptide. Because these anti-peptide Abs are of IgG isotype, their specificity is likely to reflect how peptides are processed at the T cell level and suggest that, within a short peptide, the motifs defined by T cells during the initial phase and upon their stimulation may be different. Our findings may account for the failure of most forms of peptide-based immunotherapy in cancer and autoimmune diseases in which anti-mimotope Abs are expected to play a relevant therapeutic effect. They also suggest strategies to implement the specificity of peptide-induced Abs against the target Ag.

Peptide mimotopes of the CD20 epitope recognized by rituximab are useful tools for studying this therapeutic mAb’s functional properties. We previously identified two structurally different peptides that are both effective mimotopes: a 7-mer cyclic peptide (Rp15-C) bearing the antigenic motif a/sNPS that matches 170ANPS173 of the extracellular loop of CD20, and a 12-mer linear peptide (Rp5-L) containing the antigenic motif WPxWLE lacking sequence homology to CD20. In this study, we investigated whether the different structures of Rp15-C and Rp5-L reflect the mimicry of the same or different CD20 epitopes recognized by rituximab. Using immunochemical methods, we found that, like Rp15-C, Rp5-L mimics the raft-associated form of CD20 (by inhibiting rituximab binding to CD20 in vitro). Rp5-L and Rp15-C elicit, in immunized mice, anti-CD20 Abs that stain CD20+ cells with a punctate pattern similar to that of rituximab. However, only anti-Rp5-L Abs recognize denatured CD20. When phage-display peptide libraries were panned with anti-Rp5-L, phage clones were enriched that expressed the consensus qWPxwL, similar to the antigenic motif WPxWLE, but not matching a/sNPS. Finally, WPxWLE and ANPS share some, but not all, contact sites within the rituximab Ag-combining site, indicating that WPxWLE is not an exact replica of Rp15-C (or CD20) ANPS. Altogether, these results indicate that the two structurally different peptides are also conformationally different, and suggest that rituximab recognizes two different CD20-associated epitopes.

Tumor-infiltrating lymphocytes (TILs) are a valuable indicator of the immune microenvironment that plays the central role in new anticancer drugs. TILs have a strong prognostic role in triple negative breast cancer (TNBC). Little is known about the interaction with the androgen receptor (AR) and forkhead box A1 (FOXA1). We analyzed the relationships between TIL levels, AR, and FOXA1 expression and their clinical significance in TNBC patients. Further, we investigated their interaction with other biomarkers like programmed cell death ligand-1 (PD-L1), breast cancer type 1 susceptibility protein (BRCA1), poly (ADP-Ribose) polymerase 1 (PARP1), and Na+/H+ exchanger regulatory factor 1 (NHERF1). The expression of the proteins was evaluated by immunohistochemistry in 124 TNBC samples. TILs were performed adhering to International TILs Working Group 2014 criteria. Cox proportional hazards models were also used to identify risk factors associated with poor prognosis. Multivariate analysis identified TILs as independent prognostic factor of disease free survival (DFS; p = 0.045). A Kaplan–Meyer analysis revealed that the patients with high TILs had a better DFS compared to patients with low TILs (p = 0.037), and the phenotypes TILs−/AR+ and TILs−/FOXA1− had a worse DFS (p = 0.032, p = 0.001 respectively). AR was associated with FOXA1 expression (p = 0.007), and the tumors FOXA1+ presented low levels of TILs (p = 0.028). A poor DFS was observed for AR+/FOXA1+ tumors compared to other TNBCs (p = 0.0117). Low TILs score was associated with poor patients’ survival, and TILs level in combination with AR or FOXA1 expression affected patient’s clinical outcome. In addition, AR+/FOXA1+ phenotype identified a specific subgroup of TNBC patients with poor prognosis. These data may suggest new ways of therapeutic intervention to support current treatments.