Targeting CD38 antigen as a therapeutic strategy for hematological malignancies

The success of conventional chemotherapy and radiotherapy for the treatment of cancer has been limited due to several factors like chemoresistance to drugs and peripheral toxicity caused by the lack of specificity of these approaches. For this reason the interest in targeted therapies using immunotoxins (ITs) especially for the treatment of hematological malignancies is increasing. Immunotoxins are chimeric proteins with a cell-selective ligand (antibody-derived domain, cytokine or growth factor) which drives the binding and internalization of a chemically linked or genetically fused toxic portion, generally represented by a plant or bacterial toxin which acts by interfering with protein synthesis. Here we report on the construction of novel therapeutic fusion proteins designed to induce target antigen-restricted apoptosis in human B-cell neoplasias and the evaluation of the potentiating effect obtained by the association of the ITs with drugs involved in intracellular metabolic pathways. The binding portion of our ITs is represented by a single-chain antibody fragment (scFv) directed against CD38 antigen, a surface molecule highly expressed by B lymphocytes of a particularly aggressive sub-group of Chronic Lymphocytic Leukemia (CLL) leading to the prognostically unfavorable Richter’s Syndrome and by the neoplastic immature plasma cells in Multiple Myeloma (MM). The scFv is fused to a toxic portion which acts by inhibiting the mechanism of protein synthesis in eukaryotes and in our ITs is represented by a truncated version of the bacterial toxin Pseudomonas aeruginosa Exotoxin A (PE40) or alternatively by the plant toxin saporin. We firstly designed a PE40- and a saporin-based IT comprising a scFv derived from a monoclonal antibody (mAb) developed and characterized in our laboratory. All the recombinant constructs were produced in the bacterial expression system E. coli and purified from inclusion bodies by IMAC. However, the scFv format (1E8) did not allow to preserve the binding efficiency of the parental monoclonal. Moreover, the recombinant ITs created by the fusion of 1E8 scFv with PE40 or saporin showed a low binding affinity to the CD38 target cells and, as a consequence, only negligible citotoxic activity was detected. With the creation of the divalent form of the 1E8 scFv, our purpose was to increase the binding affinity of the constructs. Despite the discouraging results of the flow-cytometric binding assay, DIV1E8-SAP demonstrated to inhibit protein synthesis of CD38-positive cells with an IC50 in the sub-nanomolar range. Then we designed two anti-CD38 recombinant ITs whose binding portion was a scFv derived from a mAb with an epitope specificity different from that of the previously described 1E8. AT13/5-PE and AT13/5-SAP showed good binding properties with a high affinity and specificity for CD38 antigen expressed on the surface of Burkitt’s lymphoma cells and myeloma cells. We proved the ability of these ITs to inhibit protein synthesis in the cell lines studied and we clearly demonstrated a dose-response effect of the ITs. The arrest of protein synthesis caused by the AT13/5-derived ITs finally leads to the triggering of the apoptotic cascade and to cell death. By using apoptosis assays we demonstrated the capability of AT13/5-PE and AT13/5-SAP to induce apoptosis of Daudi and RPMI8226 cells. Then we proved that the association of our ITs with therapeutic molecules acting on different targets of the signal transduction cascade involved in cell growth, survival and proliferation, could be synergistic in some cell lines. In particular we observed that drugs involved in the Bcl-2, Bcl-xL and Bcl-w inhibition (BH3-mimetics) can increase the potency of our ITs. Finally we demonstrated a first proof of concept about the efficacy of AT13/5-derived ITs on B-lymphocytes derived from CLL patient, but this study needs to be implemented with a wider number of cases.