Effective cancer immunotherapy combining mRNA-encoded bispecific antibodies that induce polyclonal T cell engagement and PD-L1-dependent 4-1BB costimulation
Oana Hangiu, Rocío Navarro, Susana Frago, Laura Rubio‐Pérez, Antonio Tapia‐Galisteo, Laura Díez-Alonso, Marina Gómez-Rosel, Noelia Silva-Pilipich, Lucía Vanrell, Cristian Smerdou, Kenneth A. Howard, Laura Sanz, Luis Álvarez‐Vallina, Marta Compte
Abstract
Background Immune checkpoint inhibitors have revolutionized cancer therapy, but many patients fail to respond or develop resistance, often due to reduced T cell activity. Costimulation via 4-1BB has emerged as a promising approach to enhance the effector function of antigen-primed T cells. Bispecific T cell-engaging (TCE) antibodies are an effective way to provide tumor-specific T cell receptor-mediated signaling to tumor-infiltrating lymphocytes. mRNA-based delivery of bispecific antibodies, offer a novel approach to enhance tumor-specific immune responses while minimizing adverse effects. Methods Two bispecific antibodies were generated: the EGFR x CD3 TCE antibody (LiTE) and the PD-L1 x 4-1BB costimulatory antibody (LiTCo), which was further fused to a high FcRn albumin variant (Albu-LiTCo). The mRNA encoding these bispecific antibodies contains an N1-methylpseudouridine modified nucleoside and regulatory sequences to ensure proper expression and stability. A series of in vitro assays and cell-based analyses were performed to characterize both antibodies. The in vivo efficacy of the mRNA-encoded bispecific antibodies was evaluated in xenograft tumor models expressing EGFR. Results We investigated the combined effect of two mRNA-encoded Fc-free bispecific antibodies with complementary mechanisms of action: an EGFR-targeting TCE and a half-life extended PD-L1 x 4-1BB costimulatory antibody. The mRNAs encoding both bispecific LiTE RNA and Albu-LiTCo RNA , showed similar binding specificity and in vitro function to their protein analogues. Pharmacokinetic studies demonstrated sustained expression of both bispecific antibodies following intravenous administration of the mRNAs formulated using a polymer/lipid-based nanoparticle (LNP) but different pharmacokinetic profiles, shorter for the TCE and longer for the PD-L1 x 4-1BB. When administered as a mRNA-LNP combination (Combo RNA ), the growth of EGFR-positive tumors in immunocompetent mice was significantly inhibited, resulting in tumor regression in 20% of cases with no associated toxicity. Histological analysis confirmed increased T cell infiltration in the tumors treated with LITE RNA and Combo RNA . Repeated administration resulted in sustained production of bispecific antibodies with different exposure cycles and potent antitumor activity with a favorable safety profile. Conclusions These results highlight the potential of combining two mRNA-encoded bispecific antibodies with different mechanisms of action and programmable half-life for cancer immunotherapy.