Cynthia Hartley

A new form of Filgrastim with sustained duration in vivo and enhanced ability to mobilize PBPC in both mice and humans

Granulocyte colony-stimulating factor (G-CSF) has proven effective in the prophylaxis of chemotherapy-induced neutropenia and as a mobilizer of peripheral blood progenitor cells. The longevity of G-CSF action is limited by its removal from the body by two mechanisms. The first is thought to be mediated via receptors (receptor mediated clearance [RMC]) predominantly on neutrophils, the second process is likely the result of renal clearance. With the intention of developing a novel form of Filgrastim (r-met HuG-CSF) with a sustained duration of action in vivo, a new derivative named SD/01 has been made by association of Filgrastim with poly(ethylene glycol). The desired properties of this new agent would include a prolonged duration of action sufficient to cover a complete single course of chemotherapy. SD/01 is shown here to sustain significantly elevated neutrophil counts in hematopoietically normal mice for 5 days. In neutropenic mice effects were noted for at least 9 days, accompanying a significant reduction in the duration of chemotherapy induced neutropenia. Normal human volunteers showed higher than baseline ANC for around 9 to 10 days after a single injection of SD/01. Data from these normal volunteers also indicate that mobilization of CD34+ cells and progenitors may occur in a more timely manner and to around the same absolute numbers as with repeated daily injections of unmodified Filgrastim. These data indicate that SD/01 represents an efficacious novel form of Filgrastim with actions sustained for between one and two weeks from a single injection.

Kinetics of haematopoietic recovery after dose-intensive chemo/radiotherapy in mice: optimized erythroid support with darbepoetin alpha

Summary. Despite its frequency and impact on clinical outcomes, anaemia in cancer patients remains poorly understood and suboptimally treated. The definition of optimum treatment schedules with erythropoietic agents requires a suitable model of chemotherapy‐induced progressive anaemia. This study investigated novel strategies such as once‐per‐chemotherapy‐cycle dosing, synchronization between erythroid supportive care and chemotherapy, and definition of the optimum timing of erythroid support. A murine model of carboplatin chemotherapy/radiotherapy (CRT)‐induced anaemia was used, which caused progressive anaemia across multiple cycles. Weekly administration of recombinant human erythropoietin (rHuEPO) was effective, but the longer‐acting darbepoetin alpha resulted in superior responses. In all animals, anaemia became progressive and more refractory across cycles because of accumulated bone marrow damage. Exploiting a specific enzyme‐linked immunosorbent assay, which could distinguish between darbepoetin alpha and endogenous erythropoietin, the effect of CRT upon the pharmacokinetics of darbepoetin alpha showed that clearance of darbepoetin alpha, and presumably erythropoietin, was at least partially dependent on a chemotherapy‐sensitive pathway. Scheduling data suggested that administration of erythropoietic agents prior to chemotherapy was more effective than administration after chemotherapy. There was no evidence that erythropoietic agents exacerbated anaemia, even when administered immediately prior to CRT in an attempt to ‘prime’ erythroid cells for the effects of CRT.

Comparison of Epoetin Alfa and Darbepoetin Alfa Biological Activity under Different Administration Schedules in Normal Mice

The unit of erythropoietic activity has long been the standard by which erythropoietic agents are judged, but the development of long-acting agents such as darbepoetin alfa has highlighted the shortcomings of this approach. To this point, we compared the in vivo activity of Epoetin alfa and darbepoetin alfa per microgram of protein core. Using the established mass-to-unit conversion for Epoetin alfa (1 µg ≅ 200 U), we then calculated darbepoetin alfa activity in units. Activity varied with treatment regimen (1 µg darbepoetin alfa ≅ 800 U for 3 times weekly dosing to 8,000 U for a single injection). This analysis reveals the inadequacy of evaluating darbepoetin alfa activity in terms of standard erythropoietic units. We therefore propose that for molecules with heightened biological activity, a more legitimate basis for comparison is the protein mass.

The role of stem cell factor in mobilization of peripheral blood progenitor cells

Stem cell factor (SCF) is a hematopoietic growth factor which acts on both primitive and mature progenitors cells. In animals, high doses of SCF alone stimulate increases in cells of multiple lineages and mobilize peripheral blood progenitor cells (PBPC). Phase I studies of rhSCF have demonstrated dose related side effects which are consistent with mast cell activation. Based upon in vitro synergy between SCF and G-CSF we have demonstrated the potential of low doses of SCF to synergize with G-CSF to give enhanced mobilization of PBPC. These PBPC have increased potential for both short and long term engraftment in lethally irradiated mice and lead to more rapid recovery of platelets. On going Phase I/II studies with rhSCF plus rhG-CSF for mobilization of PBPC, demonstrated similar increases in PBPC compared to rhG-CSF alone. These data suggest a clinical role of rhSCF in combination with rhG-CSF for optimal mobilization of PBPC.

An analysis of the effects of combined treatment with rmGM-CSF and PEG- rHuMGDF in murine bone marrow transplant recipients

We have studied the potential of combination growth factor treatment with GM‐CSF and megakaryocyte growth and development factor (MGDF) to stimulate hematopoietic recovery in mice following bone marrow transplantation. More rapid recovery of neutrophils occurred in mice treated with recombinant murine (rm)GM‐CSF plus pegylated recombinant human (PEG‐rHu)MGDF than carrier treated controls, however this recovery was equivalent to the effect of treatment with rmGM‐CSF alone. PEG‐rHuMGDF stimulated a more rapid recovery of platelets with no effect on neutrophil recovery. At the two tested doses of rmGM‐CSF (72 and 200 μg/kg/day) the platelet recovery was inferior to that in carrier treated mice. Also, the addition of rmGM‐CSF to PEG‐rHuMGDF had a dose‐related negative impact on platelet recovery compared to PEG‐rHuMGDF alone.

PEG-rHuMGDF promotes multilineage hematopoietic recovery in myelosuppressed mice

PEG-rHuMGDF administered to normal mice is a lineage-specific growth factor for megakaryocytes and platelets as judged by morphologic examination of hematologic cells in marrow and peripheral blood smears. The purpose of this study was to document that PEG-rHuMGDF in myelosuppressed mice promotes multilineage hematopoietic recovery. High-dose 5-fluorouracil (5-FU) in mice results in profound myelosuppression and 0-30% survival. Mice receiving a single dose of PEG-rHuMGDF (1000 microg/kg) 1 day after 5-FU (225 mg/kg) demonstrate an increased survival (76% vs 27% in control mice at 14 days). Compared to surviving controls, PEG-rHuMGDF-treated mice not only show the expected higher platelet counts, but also increased marrow colony-forming unit granulocyte-macrophage, increased multilineage marrow cellularity, and increased neutrophil, monocyte, and lymphocyte counts in peripheral blood. PEG-rHuMGDF- and vehicle-treated mice both develop hepatic abscesses after 5-FU treatment, but the abscesses in the PEG-rHuMGDF-treated mice contain more neutrophils, suggesting that myeloid reconstitution contributes to their survival. Furthermore, survival in 5-FU-treated mice is significantly improved by granulocyte colony-stimulating factor and antibiotics, suggesting that infection rather than thrombocytopenia is the predominant cause of death. PEG-rHuMGDF after 5-FU promotes survival accompanied by accelerated lymphohematopoietic repopulation, suggesting that PEG-rHuMGDF, a lineage-specific thrombopoietic factor in normal mice, promotes multilineage hematopoietic recovery in myelosuppressed mice.

Most purported antibodies to the human granulocyte colony-stimulating factor receptor are not specific

OBJECTIVE Antibodies to human granulocyte colony-stimulating factor receptor (HuG-CSFR) are widely available and have been used in numerous studies to evaluate the expression of this protein on normal and malignant cells of hematopoietic and nonhematopoietic origin. Spurred by recent studies that demonstrated that two commonly used antibodies against the erythropoietin and thrombopoietin receptors can in fact bind to completely unrelated and more broadly expressed proteins, we screened 27 commercially available monoclonal and polyclonal antibodies with claimed specificity to HuG-CSFR to determine if they are specific to this receptor. MATERIALS AND METHODS Antibodies were evaluated by Western blotting, flow cytometry, and immunohistochemistry using 293T cells engineered to overexpress HuG-CSFR protein, immortalized human hematopoietic cell lines expressing endogenous G-CSFR, and purified human neutrophils. RESULTS Only two monoclonal antibodies and one polyclonal antibody could be employed using defined Western blotting or flow cytometry protocols to detect G-CSFR protein in cell lysates or on the surface of cells that express G-CSFR messenger RNA with no binding to cells that did not express the gene. None of the antibodies were suitable for immunohistochemistry. Competitive inhibition with soluble G-CSFR extracellular domain and small inhibitory RNA-mediated knock-down of G-CSFR messenger RNA further demonstrated the limited specificity of these antibodies for HuG-CSFR expressed on the cell surface. CONCLUSIONS Most commercially available anti-HuG-CSFR antibodies do not bind specifically to this protein. These studies highlight the need for investigators to validate antibodies in their own systems to avoid the inadvertent use of nonspecifically binding antibodies that could lead, as exemplified in this case with a hematopoietic growth factor receptor, to erroneous conclusions about protein expression.

Prolonged neutropenia in a novel mouse granulocyte colony-stimulating factor neutralizing auto-immunoglobulin G mouse model

Therapeutic use of recombinant human cytokines in humans can result in the generation of drug-specific antibodies. To predetermine the maximum potential effects of a granulocyte colony-stimulating factor (G-CSF) neutralizing auto-immunoglobulin G (auto-IgG) response during recombinant human G-CSF therapy, we developed a mouse model of mouse G-CSF (mG-CSF) neutralizing auto-IgG response. Mice were immunized and boosted with mG-CSF chemically conjugated to either keyhole limpet hemocyanin or ovalbumin on an alternating schedule. Sera were analyzed for mG-CSF-specific titers and full blood counts were performed on a Technicon H-1E. On day 252, tissues were collected for histology. IgG was protein A affinity purified from pooled mG-CSF autoimmune sera. Mice immunized with mG-CSF conjugates produced mG-CSF-specific auto-IgG responses that lasted for the length of the study. Significant neutropenia (p(max) < 0.004) was concurrent with the rise in mG-CSF-specific IgG titers. However, neutrophil counts remained at approximately 20% of preimmunization levels through day 252. Endogenous mG-CSF neutralizing auto-IgG had no significant effect on hemoglobin, erythrocyte, lymphocyte, eosinophil, basophil, and platelet counts, and had minor, transient, or no effects on monocyte counts. Bone marrow colony assays from mG-CSF autoimmune mice demonstrated no significant effect of G-CSF neutralization on the numbers or proliferative capacity of preneutrophil lineage progenitors. Purified IgG from mG-CSF autoimmune mice neutralized mG-CSF in vitro. High-titer G-CSF neutralizing auto-IgG in adult mice partially inhibited steady-state granulopoiesis and had little or no effect on steady-state levels of other hematopoietic cells.

Neutropenia in a novel anti-mg-csf autoantibody mouse model

Abstract Therapeutic use of cytokines in humans can result in the generation of antibodies that bind to the drug. In order to predict the potential effects of an autoantibody response to recombinant human G-CSF therapy, we developed a novel, reproducible mouse model of anti-G-CSF specific humoral immunity. Two groups of mice were immunized and boosted with mouse G-CSF (mG-CSF) chemically conjugated to either KLH or OVA on an alternating schedule. All mice immunized with the mG-CSF conjugates produced high titer, mG-CSF specific IgG responses that lasted for the length of the study (252 days). Significant neutropenia was concurrent with the rise in anti-mG-CSF IgG titers (p

Treatment with sd/01 relative to chemotherapy in mice

Abstract The effectiveness of Filgrastim SD/01 (SD/01) has been demonstrated in the relief of neutropenia associated with cancer chemotherapy. The studies reported to date administered SD/01 primarily on the day after chemotherapy in a similar manner to the injection of conventional Filgrastim. It was not clear whether the same dosing paradigm would be optimum for an engineered form of Filgrastim with substantially altered pharmacokinetics. In order to examine the optimum timing of SD/01 relative to chemotherapy, mice were treated with 325 mg/kg cyclophosphamide (CY) on study day 0. They were also treated with a bolus dose of 1000 g/kg SD/01 on days −5, −4, −3, −2, −1, 1, 2, 3, 4 or 5. Blood was collected for complete blood counts on study days −1, +1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12. Normal untreated mice have an ANC of 0.75 to 1.25 × 10 3 /μL. Following CY treatment ANC fell to a nadir of .01 × 10 3 /μL on day 4 then quickly rebounded to a high of 3.5 × 10 3 /μL on day 8, returning to normal by day 10. Post-CY treatment with SD/01 on days 1, 2 or 3 improved the ANC nadir and brought counts back to baseline between days 4 and 5 (one day earlier). Delaying the SD/01 to days 4 and 5 after CY did not accelerate the return to baseline ANC. Pretreatment with SD/01 did not impact the depth or timing of the nadir or the day ANC passed the baseline count during recovery. Pre-treatment with SD/01 raised the ANC depending on the interval between treatment and CY exposure. Thus, 5 days pretreatment yielded an ANC of 14 × 10 3 /μL, 4 days 11 × 10 3 /μL, 3 days 8 × 10 3 /μL, 2 days 3 × 10 3 /μL, and 0.53 × 10 3 /μL with 1 day pre-treatment. Pre-treatment with SD/01 (ie before chemo) is relatively ineffective compared to post-chemo treatment. Day 1 post-chemotherapy treatment appears to be the most effective timing for decreasing the severity and duration of neutropenia—delaying administration makes it less effective, and pre-treating does not appear to improve or worsen ANC recovery.