A Targeted Catalytic Nanobody (T-CAN) with Asparaginolytic Activity

SIMPLE SUMMARY: The therapy of Acute Lymphoblastic Leukemia (ALL) is based on Escherichia coli (E. coli) L-asparaginase, which is a very effective drug in most cases. However, its side effects sometimes prevent its usage or impose its interruption. The main issues derive from its bacterial origin, which elicits a strong immune response in the patient, and from its generalized action on all body compartments. In this work, we describe how we generated a fully active and miniaturized form of L-asparaginase starting from a camel single domain antibody, a class of antibodies known to have a very limited immunogenicity in humans. We also targeted it onto tumor cells by attaching it to an antibody fragment directed onto the CD19 B-cell surface receptor, expressed on ALL cells. We named this new molecule “Targeted Catalytic Nanobody” (T-CAN). The T-CAN is active and successfully binds to CD19 expressing cells in vitro. Thanks to its reduced immunogenic potential, it represents a new tool which deserves further development. ABSTRACT: E. coli L-asparaginase is an amidohydrolase (EC 3.5.1.1) which has been successfully used for the treatment of Acute Lymphoblastic Leukemia for over 50 years. Despite its efficacy, its side effects, and especially its intrinsic immunogenicity, hamper its usage in a significant subset of cases, thus limiting therapeutic options. Innovative solutions to improve on these drawbacks have been attempted, but none of them have been truly successful so far. In this work, we fully replaced the enzyme scaffold, generating an active, miniaturized form of L-asparaginase by protein engineering of a camel single domain antibody, a class of antibodies known to have a limited immunogenicity in humans. We then targeted it onto tumor cells by an antibody scFv fragment directed onto the CD19 B-cell surface receptor expressed on ALL cells. We named this new type of nanobody-based antibody-drug conjugate “Targeted Catalytic Nanobody” (T-CAN). The new molecule retains the catalytic activity and the binding capability of the original modules and successfully targets CD19 expressing cells in vitro. Thanks to its theoretically reduced immunogenic potential compared to the original molecule, the T-CAN can represent a novel approach to tackle current limitations in L-asparaginase usage.