靶向ENPP3的双特异性T细胞衔接器前药的发现与开发

T cell engager (TCE) antibodies have been proven to be effective for a variety of hematological malignancies, with at least 10 marketed drugs to date; however, their efficacy in many solid tumors is still very limited due to a number of challenges. One of the leading challenges is the lack of tumor-specific targets, leading to an increased risk of on-target, off-tumor toxicity. An approach to address this challenge is to build a “prodrug” TCE that is conditionally activated in the tumor microenvironment (TME), taking advantages of various masking technology platforms that block the release of active TCE drug outside of the TME. To develop such a novel TCE molecule with improved tumor selectivity, potency, and safety profile, we first discovered a masking peptide specific to a CD3 VHH antibody that we previously developed, and demonstrated that the masked CD3 VHH can effectively reduce T cell activation several hundred-fold. Next, we fused this masking peptide to the CD3 VHH arm via a proteolytically-labile linker containing sequence motifs sensitive to proteases enriched in the TME, and showed that upon cleavage by proteases, the T cell-activating potency of such a masked CD3 VHH can be effectively recovered. Finally, with such a CD3 VHH armed with cleavable linker and masking peptide, we built a series of prodrug TCE molecules targeting ENPP3 (ectonucleotide pyrophosphatase/phosphodiesterase family member 3), a tumor associated antigen with good tumor selectivity for renal cell carcinoma. Our in vitro validation studies demonstrated that the potency of these TCE prodrugs in killing ENPP3+ tumor cells can be restored to a similar level as the unmasked, active TCE molecule upon proteolytic cleavage. Our in vivo studies using an ENPP3+ CDX model with transplanted human PBMCs demonstrated two important points: (i) the prodrug/masked ENPP3-targeted TCE can potently inhibit tumor growth to a comparable level as the unmasked version and clinical benchmarks, and (ii) the prodrug TCE can be dosed at least 100-fold higher than the active drugs without any observed toxicity, suggesting a greatly improved safety profile. Further preclinical development of these ENPP3-targeted prodrug TCE molecules, including pharmacokinetics and toxicity studies in non-human primates, is ongoing.