Abstract
Simple SummaryThe 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.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.
Highlights
The asparaginolytic activity is typical of a class of amidohydrolases, namely Asparaginases (EC 3.5.1.1, ASNases), found in bacteria, fungi and plants [1]
The crystallographic structure of this single domain antibodies (sdAbs) was used as a template to model a novel engineered protein containing the main catalytic residues of E. coli type II
In the work here presented, a camelid single domain antibody was chosen as the minimal scaffold and engineered in order to provide it with asparaginolytic activity by the transplantation of specific key residues selected from E. coli ASNase
Summary
The asparaginolytic activity is typical of a class of amidohydrolases, namely Asparaginases (EC 3.5.1.1, ASNases), found in bacteria, fungi and plants [1]. ASNases are of great clinical interest since they inhibit the growth of some tumor types by removing the 4.0/). Asparagine and, to a lesser extent, the glutamine supply that they need to proliferate. Pharmaceutical grade ASNases are derived from E. coli or Erwinia chrysanthemi (E. chrysanthemi) and have been used in the clinics since the 1950s for the treatment of Acute Lymphoblastic. ASNase-based therapy for ALL has a 90% success rate in pediatric patients, but it is still associated with several side effects and lower efficacy in adult patients [3]. One of the most limiting is its immunogenicity, more remarkable in adults than in children and dependent on the enzyme bacterial origin. A molecule recognized as “non-self” (antigen) by the immune system of the host, triggers a complex network of cells and molecules leading to the production of specific antibodies
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