Preclinical Assessment of Immunogenicity and Protectivity of Novel ROR1 Fusion Proteins in a Mouse Tumor Model

SIMPLE SUMMARY: Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a tumor-associated antigen reported to be overexpressed in a variety of malignancies. The aim of this study is preclinical evaluation of various ROR1 fusion proteins as novel cancer vaccines in a fully syngeneic mouse tumor model. Our results showed that a fusion protein containing mouse ROR1, IgG Fc, and a universal tetanus toxin (TT) helper T-cell carrier could break tolerance against mouse ROR1 autoantigen and completely inhibit the growth of syngeneic ROR1+ tumor cells. ABSTRACT: The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a new tumor associated antigen (TAA) which is overexpressed in several hematopoietic and solid malignancies. The present study aimed to produce and evaluate different fusion proteins of mouse ROR1 (mROR1) to enhance immunogenicity and protective efficacy of ROR1. Four ROR1 fusion proteins composed of extracellular region of mROR1, immunogenic fragments of TT as well as Fc region of mouse IgG2a were produced and employed to immunize Balb/C mice. Humoral and cellular immune responses and anti-tumor effects of these fusion proteins were evaluated using two different syngeneic murine ROR1+ tumor models. ROR1-specific antibodies were induced in all groups of mice. The levels of IFN-γ, IL-17 and IL-22 cytokines in culture supernatants of stimulated splenocytes were increased in all groups of immunized mice, particularly mice immunized with TT-mROR1-Fc fusion proteins. The frequency of ROR1-specific CTLs was higher in mice immunized with TT-mROR1-Fc fusion proteins. Finally, results of tumor challenge in immunized mice showed that immunization with TT-mROR1-Fc fusion proteins completely inhibited ROR1+ tumor cells growth in two different syngeneic tumor models until day 120 post tumor challenge. Our preclinical findings, for the first time, showed that our fusion proteins could be considered as a potential candidate vaccine for active immunotherapy of ROR1-expressing malignancies.