Structural Superimposition

Abstact

BACKGROUND: Tumor heterogeneity has been identified as a major roadblock for cancer immunotherapy. To overcome this, universal effector cell engagers with interchangeable tumor-targeting adaptors have been developed. Current concepts in this field consist of antibody-based adaptors. Small-molecule (SM) ligands, on the other hand, infiltrate tissues rapidly, have short half-lives, and are potentially orally available. Therefore, we hypothesized that utilizing SM adaptors, combined with effector antibodies, could represent an attractive off-the-shelf therapy. METHODS: Here, we introduce the development of target-agnostic, small-molecule-guided hapten- and T cell-bispecific (TCB) antibodies with high affinity between the adaptor-effector pair. Specifically, we designed SM adaptors based on known tumor-targeting ligands with specificity to the antigens folate receptor 1 (FOLR1), prostate-specific membrane antigen (PSMA) and carbonic anhydrase IX (CAIX), and conjugated them to Ca(2+)-loaded 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM) as a hapten. As an effector antibody, we designed DOTAM-specific T cell bispecific antibodies (DOTAM-TCB) with femtomolar (fM) affinity for the hapten. RESULTS: In vitro experiments showed in-solution assembly of stable adaptor-effector complexes. Additionally, T cell-mediated tumor killing was initiated by the universal DOTAM-TCB when combined with adaptors across various tumor cells. Ex vivo study of CAIX-directed Ca-DOTAM-acetazolamide (Ca-DOTAM-AAZ) with DOTAM-TCB showed potent activation of T cells against murine-engrafted human HT-29 tumor. CONCLUSIONS: The studies described here demonstrate proof-of-concept for using hapten-containing small molecules as adaptors for effective universal T cell engager-based cancer immunotherapy.