Development of growth factor-cytokine fusion proteins with increased hematopoietic activity: Physiological and cellular effects.

摘要

Hematopoietic precursor cells express cell surface receptors for a variety of cytokines and growth factors. These hematopoietic secreted humoral factors can influence the proliferation, differentiation, survival and mobilisation of blood stem cells. Moreover, simultaneous exposure to different combinations of hematopoietic hormones can synergistically affect one or more of these cellular responses. Our laboratory has been involved in studying the role played by insulin-like growth factors (IGFs) in hematopoiesis. IGFs are known to stimulate the expansion of precursor cells from various hematopoietic lineages when administered in vivo. However, difficulties in producing recombinant IGFs with conventional bacterial or yeast expression systems make the use of IGFs as therapeutic hematopoietic agents a cost prohibitive concept. A baculovirus based expression system was developed for the production of large amounts of properly folded and biologically active secreted IGF analogues (BOMIGFs). This system was later adapted for the synthesis of a fusion protein consisting of BOMIGF and intedeukin-3 (BOMIGF-IL-3). The in vitro and in vivo activities of this chimeric molecule were subsequently studied.; The BOMIGF-IL-3 chimera promoted greater thymidine incorporation activity into TF-1 and bovine fetal erythroid cells than observed with the combined administration of BOMIGF and IL-3. This chimera also stimulated the formation of BFU-Es, CFU-GMs and, in particular, the highly proliferative macroscopic colonies in peripheral blood cell hematopoietic colony formation assays. These effects were reproduced in colony formation assays from bone marrow- and spleen-derived colony-forming cells from mice treated with BOMIGF-IL-3. This chimera also helped in the recovery of weight loss, anemia and neutropenia in an AZT-mediated myelossuppression mouse model. The chimera was shown to be significantly better than the corresponding equimolar mixture of the single factors at promoting the survival of TF-1 cells. This effect is associated with a sustained activation of PI-3 kinase, STAT5 phosphorylation and Bclx_L expression. The chimera was also better than the co-addition of BOMIGF and IL-3 at stimulating the migration of TF-1 cells across Transwell plates. The chimera-dependent potentiation of migration appears to be mediated by at the very least, an enhancement of PI-3 kinase activity.; This recombinant BOMIGF-IL-3 fusion molecule could prove useful for the therapeutic treatment of conditions with decreased production of blood cells such as in AZT- or chemotherapy-induced anemia. Other useful applications may include the mobilisation of stem cells and their ex vivo expansion for the purpose of stem cell transplantation.