Introduction: Clinical data from diverse cell therapy trials highlight two critical issues. The first is resistance to, and relapse from, therapy due to antigen loss. In solid tumors antigen heterogeneity is common and cell therapies targeting a single antigen routinely fail as target antigen-negative tumor cells escape detection and emerge to drive tumor resistance. In hematologic malignancies, antigen loss after treatment drives relapses – up to 50% in B cell malignancies and near 100% in multiple myeloma. A related issue is that most cell therapeutics fail to persist after an initial expansion phase in the patient. This is particularly true in the case of solid tumors. This failure to persist may be due to unproductive engagement of the cell therapeutic with the target tumor cell. Here we describe the use of CAR-CD19 T cells (CAR19s) secreting bridging proteins to overcome these two issues. We leverage the intrinsic persistence properties of CAR19s and use these CARs to target multiple tumor antigens simultaneously using bridging proteins that link the CAR19s to multi-functional binding domains. Experimental Procedures: A stabilized form of the CD19 extracellular domain (ECD) (see Klesmith et al 2019) was cloned in frame with antigen binding domains, eg. scFv or VHH. We designed a core “bridging block” that consisted of an anti-Her2 scFv linked to the stabilized CD19 ECD to create a CD19-ECD-anti-Her2 bridging protein. This bridging protein sequence was then cloned into a lentiviral vector downstream of a CAR19 domain and a P2A cleavage site. Donor primary T cells transduced with the lentiviral particles expressed the CAR19 and secreted the bridging protein. The activities of the stabilized CD19-anti-Her2 bridging protein and the CAR19 T cells secreting that bridging protein were assessed using standard cytotoxicity assays in vitro and in vivo. Finally, we added an anti-EGFR scFv, thus creating a multi-antigen targeting bridging protein. We also created a version having a protease-sensitive mask to reduce EGFR binding outside of the acidic tumor microenvironment. Results: Use of the stabilized CD19 ECD in bridging proteins improves protein expression. Here we show for the first time that this improvement extends to bridging protein expression from CAR19 T cells. CAR19 T cells that secrete the stabilized CD19-anti-Her2 bridging protein are highly potent in vitro and in vivo. The CD19-ECD-anti-Her2 bridging block was further modified with an anti-EGFR scFv. This novel multi-antigen targeting bridging protein can bind either or both targeted antigens and supports highly potent cytotoxicity against dual positive tumor cells thus addressing the issue of solid tumor antigen heterogeneity. Ongoing programs add a third domain in an indication-specific manner, eg. an anti-IL-13Ra2 binding domain. In all cases the intrinsic anti-CD19 CAR activity is preserved, providing these unique CARs with a tumor-independent and self-renewing antigen depot in CD19-positive normal B cells. Conclusions: Here we have created a robust system for targeting multiple tumor antigens simultaneously. Further the use of CAR19s supports CAR-T cell persistence independently of tumor antigen expression. This unique technology addresses critical issues in cell therapy using a potent technology whose modular nature allows for rapid program and pipeline development.

Citation Format: Paul Rennert, Christine Ambrose, Lihe Su, Fay Dufort, Alyssa Birt, Tom Sanford, Lan Wu, Roy Lobb. CAR-CD19 T cells secreting multi-antigen bridging proteins eliminate solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B020. doi:10.1158/1535-7163.TARG-19-B020