Active EMMPRIN Vaccination Does Not Affect Healthy Tissues Despite Selectively Reducing Tumor Growth and Metastasis

Introduction: EMMPRIN is a multifunctional, pro-angiogenic protein that mediates leukocyte activation and interactions with epithelial cells. We have previously shown that actively vaccinating s.c. CT26 tumor-bearing mice with a novel EMMPRIN epitope synthesized as a multiple antigenic peptide (designated 161-MAP) significantly reduced, and even regressed some of the tumors, reduced metastasis when these cells were injected intravenously to form lung metastases, and prevented recurrence (Simanovich et al, OncoImmunology, 2016, 6:e1261778). Mice vaccinated with 161-MAP exhibited reduced angiogenesis, their microenvironment was immune modulated to alleviate immune suppression allowing increased infiltration of cytotoxic T cells, and their tumor cells underwent enhanced apoptosis, compared to mice vaccinated with a scrambled peptide (SCR-MAP), activating all arms of the immune response. We now show that this 161-MAP vaccination and its systemic immune response had no effects on the kidney and colon tissues that express high levels of EMMPRIN.

Methods and Results: To assess kidney functions we measured the serum levels of creatinine and blood urea nitrogen (BUN) by ELISA, finding no difference between the 161-MAP and SCR-MAP vaccinated mice. EMMPRIN mRNA expression was reduced by 2-folds (p=0.03) only in the tumor tissue, but remained unaffected in the colon and kidney tissues of 161-MAP (vs. SCR-MAP) vaccinated mice, as evaluated by qPCR. Using both immunohistochemistry and ELISA we could detect no difference in EMMPRIN protein expression in the colon and kidney tissues between the two groups, although their levels were comparable to the protein levels in the tumor tissues that were lowered in the 161-MAP (vs. SCR-MAP) by 40%, p=0.0179. As EMMPRIN induces VEGF and MMP-9, we determined their levels in the tissues, and found no difference between the two vaccinated groups in the colon and kidney, but reduction in their levels (by 1.72, p=0.0093 and 2.7-folds, p=0.0041, respectively) in the tumor tissues of the 161-MAP vaccinated mice vs. the SCR-MAP ones.

Conclusions: Active vaccination with 161-MAP specifically targets tumor tissues, and inhibits tumor growth and metastases. However, although EMMPRIN is highly expressed in healthy tissues such as the colon and kidney, the immune response was limited to the tumor, and did not affect the colon and kidney. This suggests that presence of the antigen and the full activation of all arms of the immune response are not sufficient, and additional factors, perhaps emanating from the inflamed tissue, are necessary. The mechanisms that render such selectivity of the immune response to one location merit more investigation.