Construction of a macromolecular recombinant drug for the targeted therapy of hematological malignancies

The use of cytotoxic agents capable to selectively target surface molecules on a malignant cell is a promising approach for the treatment of cancer, especially hematologic malignacies. Immunotoxins, in particular, are polypeptides comprising an antibody-derived domain and a toxic portion, usually represented by a bacterial or plant toxin: the antibody specifically binds a target antigen and, following endocytosis, delivers the toxic payload to the interior of the cell, interfering with fundamental metabolic pathways. Among several leukemia/lymphoma-associated surface antigens, one of the most attractive molecular targets for this kind of therapeutic strategy is CD22, a membrane glycoprotein with coreceptor functions, whose expression is restricted to B lymphocytes. The present thesis describes the construction and characterization of a recombinant immunotoxin in which the binding domain is represented by a CD22-specific single-chain antibody fragment (scFv), while the cytotoxic activity is carried out by a truncated version of Pseudomonas aeruginosa Exotoxin A, a bacterial toxin that inhibits the mechanism of protein synthesis in eukaryotes. Through the molecular cloning of sequences coding for the variable domains of heavy and light chains (VH and VL, respectively) of an anti-CD22 murine monoclonal antibody, it was possible to assemble a plasmid construct coding for a scFv that was eventually produced in a bacterial expression system. Immunofluorecence analysis on CD22-positive cells and immunoenzymatic assays on the purified antigen proved that the antibody fragment maintains the binding specificity of the parental monoclonal antibody, exhibiting a fairly good affinity for CD22. The scFv was later genetically fused to the enzymatic domain of a bacterial toxin. The resulting immunotoxin was expressed in Escherichia coli and recovered from insoluble cytoplasmic aggregates after purification by affinity chromatography, with yields of 1-2 mg from a 1 litre culture. The binding properties of the recombinant immunotoxin are comparable to those of the scFv; V moreover, as ascertained through cell-proliferation assays, it can selectively poison CD22- expressing cells with an IC50 (i.e. concentration inhibiting 50% of the maximal cell proliferation) around 1-10 nM. It can be concluded that our anti-CD22 immunotoxin combines the binding qualities of the scFv antibody and the potent enzymatic activity of the bacterial toxin. After further characterization we will explore the opportunity to start a process of molecular optimization, aiming at the construction of a novel biotechnological drug for the treatment of hematological malignancies in humans.