Ex-vivo expansion of hematopoietic progenitor cells: Preliminary results in breast cancer

Ex-vivo expanded progenitor cells have been proposed as a source of cells to support high-dose chemotherapy and to decrease or eliminate the period of neutropenia following transplantation. To date, no clinical studies using ex vivo expanded cells, have demonstrated any decrease in the time to neutrophil or platelet recovery, although a number of clinical studies have been performed using a variety of growth factor cocktails and culture conditions. Over the past 6 years we have developed a static culture system that results in optimal expansion of myeloid progenitor cells. We have initiated a clinical study to evaluate this culture system in breast cancer patients receiving peripheral blood progenitor cells (PBPC) to support high- dose chemotherapy. CD34 selected cells were cultured for 10 days in 800 ml of defined media (Amgen Inc.) containing 100 ng/ml each of rhSCF, rhG-CSF and rhMGDF in 1L teflon bags (American Fluoroseal) at 20,000 to 50,000 cells per ml. After culture the cells were washed with 3 volumes of PBS to remove all media and growth factors and reinfused on day 0 of transplant followed by daily administration of rhG-CSF. On day +1 the patients received an unexpanded PBPC product to ensure the durability of the graft. Patients transplanted with expanded PBPC cells recovered neutrophil counts (ANC > 500/μl) as early as day 4 post transplant with a median of 6 days (range 4 to 14 days). In comparison, our historical control group of patients (N = 175) had a median time to neutrophil engraftment of 9 days (range 7 to 24 days). A second cohort of patients were transplanted with expanded cells alone and a similar rapid engraftment was obtained. The first patients are now over 70 days post transplant with durable engraftment. No effect on platelet recovery has been observed in any patients to date. These data demonstrate that PBPC expanded under the conditions defined can significantly shorten the time to engraftment of neutrophils. The marrow is the principal site for blood cell formation in humans. In normal adults the body produces about 2.5 billion red blood cells (RBC), 2.5 billion platelets (plts) and 10 billion granulocytes per kilogram of body weight per day. The production of mature blood cells is a continual process that is the result of proliferation and differentiation of stem cells, committed progenitor cells and differentiated cells. Within these three stages extensive expansion of cell numbers occurs through cell division. A single stem cell has been proposed to be capable of 50 cell divisions or doublings and has the capacity to generate up to 10¹⁵ cells, or sufficient cells for up to 27 years. The proliferation and differentiation of cells is controlled by a group of proteins called hematopoietic growth factors (HGFs). Many of the HGFs have been isolated and cloned and are large amounts of recombinant proteins are available for clinical use or ex vivo manipulations of cells. If we could replicate this cell amplification in vitro with the HGFs, it may be possible to generate large numbers of cells that could be used for a variety of clinical applications. These include: Supplementing stem cell grafts with more mature precursors to shorten or potentially prevent chemotherapy-induced pancytopenia. Increasing the number of primitive progenitors to ensure hematopoietic support for multiple cycles of high-dose therapy. Obtaining a sufficient number of stem cells from a single marrow aspirate or pheresis procedure, thus reducing the need for large-scale harvesting of marrow or multiple leukaphereses. Generating sufficient cells from a single cord blood unit to reconstitute an adult following high-dose chemotherapy. Purging stem cell products of contaminating tumor cells. Generating large volumes of immunologically active cells with antitumor activity to be used in immunotherapeutic regimens. Increasing the pool of stem cells which could be targets for the delivery of gene therapy. It is important therefore, to consider the clinical application when discussing ex vivo expansion approaches, and define culture conditions that are relevant to the particular cell type being expanded. The relevant properties of the cells in each of the different hematopoietic compartments are summarized below: - Stem Cells, Long term engraftment, Targets for gene replacement therapy; - Committed Progenitor Cells, Intermediate and short term engraftment; - Mature Cells Short term engraftment, Immune Therapy. The focus of many groups has been to expand stem cells, however, to date no data has been published demonstrating significant expansion of stem cells. Also, gene transduction approaches have been limited in the transduction efficiencies achieved with human stem cells. Therefore, we have focused our studies on developing culture conditions that result in the expansion of committed progenitor cells and mature cells. The first demonstration of the use of growth factors to generate increased numbers of specific cell population was performed by Bradley in the early 1980's using crude conditioned media as sources of growth factors. In these studies it was shown that incubation of post FU mouse bone marrow cells in WEHI-3 CM for 7 days, resulted in a 60 fold increase of CFU-S₁₃, and a 53 fold increase in GM-CFC. In subsequent studies from this group, it was shown that pre-incubation with HGFs (also crude CM) could expand primitive murine progenitor cells called high proliferative potential colony forming cells (HPP-CFC) and cells with in vivo marrow repopulating ability. Using a similar culture system of human bone marrow cells, in teflon bottles, it was shown in 1988 that the combination of rhGM-CSF plus rhIL-3 could generate a 7 fold increase in committed progenitor cells (granulocyte macrophage colony forming cells -GM- CFC). In 1991, Bernstein et al showed that incubation of single CD34+Lin- cells in the combination of interleukin-3 (IL-3), granulocyte colony stimulating factor (G-CSF) and stem cell factor (SCF) gave rise to an increase of 10-fold of colonies in vitro. The use of ex vivo expansion to generate mature neutrophil precursors was proposed in 1992 by Haylock et al. These authors demonstrated that the combination of IL-1, IL-3, IL-6, GM-CSF and SCF could generate a 1,324-fold increase in nucleated cells and a 66- fold increase in GM-CFC. The cells produced under these conditions were predominantly neutrophil precursors. The culture conditions used were static cultures and utilized CD34+ cells as the starting population. Several Investigators have demonstrated the requirement for CD34 selection of the starting cells for optimal expansion. Subsequent studies were performed at a clinical scale using optimal culture conditions in teflon bags and with fully defined media appropriate for clinical applications. This work utilized the growth factor cocktail comprising of SCF, G-CSF and MGDF. Other cocktails of growth factors are effective in expanding CD34+ cells, however the availability of clinical grade growth factors has been limited due to commercial considerations. The in vivo potential of ex vivo expanded cells was first reported in murine studies by Muench et al. This study demonstrated that bone marrow cells expanded in SCF plus IL-1 engrafted lethally irradiated mice and were capable of sustaining hematopoiesis long term in these animals. In addition, the bone marrow from these engrafted mice could repopulate secondary recipients. The authors concluded that the expansion of mouse bone marrow cells did not adversely effect the proliferative capacity and lineage potential of the stem cell compartment. Recent studies in normal baboons, have demonstrated the potential clinical benefit of ex vivo expanded cells. Andrews and colleagues harvested PBPC from G-CSF mobilized normal baboons and expanded the CD34+ cells for 10 day in SCF plus G-CSF plus MGDF. After the culture period the cells were washed and infused into the baboons after lethal irradiation. The fold-expansion obtained was low compared to human CD34+ cells and probably due to species variations of the growth factors and cell behaviour in culture conditions developed for expansion of human cells. GM-CFC were expanded 7 to 8 fold. Table 1 summarizes the engraftment characteristics of the different treatment groups in these studies. Group IV, transplanted with expanded CD34+ cells and given post transplant G-CSF and MGDF, had a significantly shorter duration of neutropenia and significantly higher WBC and PMN nadirs compared to animals in the other groups. In fact 2 of the 3 animals had no days with neutrophils below 500/μl, a clinical endpoint used for neutrophil engraftment. In these studies, in vitro expansion did not influence platelet recovery despite the use of MGDF in both cultures and after transplantation. Further studies will be needed to determine the culture conditions that will enhance platelet recovery of PBPC.