Abstract
Thrombopoietin (TPO, the ligand for c-mpl) is a key factor for megakaryopoiesis. Several clinical trials of TPO have been conducted for thrombocytopenia without much success due to, in part, the production of neutralized antibodies against endogenous TPO, which causes thrombocytopenia. To overcome this problem, we previously demonstrated that mouse type minibody against c-mpl, with an amino acid sequence totally different from TPO, showed megakaryopoiesis and increased platelet numbers in monkey. This time, using CDR grafting, we generated a humanized sc(Fv)2VB22B minibody (huVB22B) against c-mpl for therapeutic use. The new minibody showed almost the same activity in vitro as TPO and the mouse type minibody, confirmed by both a human megakaryocyte cell (CD41+) differentiation assay and a proliferation assay with TPO-dependent cell line, M-07e. Single sc or iv administration of huVB22B to cynomolgus monkeys showed a dose-dependent increase in platelet numbers. Pharmacokinetic analysis showed that the plasma half-life (T1/2) of huVB22B at iv and sc administration to cynomolgus monkeys was 7–8 h and 11–16 h, respectively. After administration of huVB22B, the platelets of these monkeys increased and showed functional aggregation in response to ADP in vitro. Repeated administration of huVB22B (0.2, 2 and 20mg/kg/week) revealed that the increase in platelet level in cynomolgus monkeys was maintained for a month. Very slight reticular fibers in bone marrow were detected in a high dose group (20mg/ kg). No overt changes were detected by toxicity evaluations including clinical pathology and histopathology in 0.2 and 2mg/kg groups. No neutralized activities in plasma were observed during these experiments. Next, we examined the activities of huVB22B on human bone marrow-derived CD34-positive cells (BM-CD34+) and umbilical cord blood-derived CD34-positive cells (UCB-CD34+) in vitro. BM-CD34+ and UCB-CD34+ cells were cultured with huVB22B in serum free medium. HuVB22B induced differentiation of CD41+ cells from BM-CD34+ or UCB-CD34+ cells in a similar dose-dependent manner. However, UCB-CD34+ cells showed greater proliferation in response to huVB22B compared to BM-CD34+ cells. We then examined the in vivo activities of huVB22B on UCB CD34+ cells by treating NOD/SCID mice transplanted with human UCB-CD34+ cells with huVB22B and examining the bone marrow cells of the mice. The results showed that, compared with the control, administration of huVB22B showed an increase in the number of human hematopoietic progenitor cells (CD34+), lymphoid lineage cells (CD19+), and myeloid lineage cells (CD33+) in addition to human CFU-Meg cells (CD41+). These results suggest that c-mpl stimulation in vivo after transplantation might increase engraftment of progenitor cells in the bone marrow microenvironment and subsequently induce differentiation to multilineage cells. Umbilical cord blood transplantation faces frequent complications including a low-level stem/progenitor cell engraftment and delayed platelet recovery. Our results suggest that c-mpl stimulation might be used to increase the engraftment of UCB stem/progenitor cells and shorten the time of platelet recovery following UCB transplantation.
Transplantation Induces Profound Changes in the Transcriptional Asset of Hematopoietic Stem Cells: Identification of Specific Signatures Using Machine Learning Techniques