Proteolytic Activation of T Cell Engagers And Bispecific Proteins Using Modular Coiled-Coil Peptides

Abstract

Abstract Bispecific T cell engagers (BiTEs) are engineered immunotherapeutic molecules designed to direct the body’s immune system against malignant cells by physically connecting T cells to target cells, triggering cytotoxic responses. BiTEs have typically engaged T cells via αCD3 domains, such as OKT3 or UCHT1, which are linked to a second binding domain specific for a tumor-associated antigen. The risk of on-target off-tumor toxicity is a serious concern for BiTE immunotherapy due to the expression of tumor-associated antigen targets on healthy tissues. To address this concern, we have developed protease-responsive masked-BiTEs using OKT3 and UCHT1 mAb clones, that activate T cells only after exposure to tumor micro-environment-associated proteases and stimulate the T cell activation by their tumor-antigenic targets. Our protease-responsive masked BiTEs (mBiTEs) are comprised of αCD3 Fab proteins that are sterically prevented from binding CD3 via N’ terminal fusion of coiled-coil leucine zipper dimerization domains to each Fab chain, with further C’ terminal linkage to a tumoral-antigen targeting nanobody (either αPSMA or αHER2). The peptide linking each Fab chain and its coiled-coil masking zipper harbors a protease cleavable site, such that engagement of either recombinant or T cell displayed CD3 is significantly reduced until mBiTE protease activation. OKT3 and UCHT1-based masked T cell engagers (mTEs) showed ∼400-fold and ∼800-fold reduced affinity for CD3 protein compared to unmodified T cell engagers (TEs), with ∼222-fold and ∼457-fold restoration of binding upon proteolytic unmasking, respectively. mBiTEs did not activate Jurkat T cells while proteolytically unmasked proteins did, and mBiTEs harboring linkers cleavable by the cancer associated matric metalloprotease 2 (MMP2) enzyme enabled killing of PSMA + and HER2 + cancer cells in an MMP2-dependent manner in PBMC co-culture assays. The mBiTEs we describe here could be of broad utility towards helping constrain BiTE-mediated immune-activation to protease-high environments, such as the solid tumor microenvironment.

The authors' abstract, as published at the source. ACS Chemical Biology, 2026 · DOI ↗