Robert Briddell

Modulation of protein aggregation by polyethylene glycol conjugation: GCSF as a case study

Polyethylene glycol (PEG) conjugation to proteins has emerged as an important technology to produce drug molecules with sustained duration in the body. However, the implications of PEG conjugation to protein aggregation have not been well understood. In this study, conducted under physiological pH and temperature, N‐terminal attachment of a 20 kDa PEG moiety to GCSF had the ability to (1) prevent protein precipitation by rendering the aggregates soluble, and (2) slow the rate of aggregation relative to GCSF. Our data suggest that PEG‐GCSF solubility was mediated by favorable solvation of water molecules around the PEG group. PEG‐GCSF appeared to aggregate on the same pathway as that of GCSF, as evidenced by (a) almost identical secondary structural transitions accompanying aggregation, (b) almost identical covalent character in the aggregates, and (c) the ability of PEG‐GCSF to rescue GCSF precipitation. To understand the role of PEG length, the aggregation properties of free GCSF were compared to 5kPEG‐GCSF and 20kPEG‐GCSF. It was observed that even 5kPEG‐GCSF avoided precipitation by forming soluble aggregates, and the stability toward aggregation was vastly improved compared to GCSF, but only marginally less stable than the 20kPEG‐GCSF. Biological activity measurements demonstrated that both 5kPEG‐GCSF and 20kPEG‐GCSF retained greater activity after incubation at physiological conditions than free GCSF, consistent with the stability measurements. The data is most compatible with a model where PEG conjugation preserves the mechanism underlying protein aggregation in GCSF, steric hindrance by PEG influences aggregation rate, while aqueous solubility is mediated by polar PEG groups on the aggregate surface.

Ex vivo expansion of hematopoietic progenitor cells and mature cells

Hematopoietic cells have the potential for providing benefit in a variety of clinical settings. These include cells for support of patients undergoing high-dose chemotherapy, as a target for replacement gene therapy, and as a source of cells for immunotherapy. The limitation to many of these applications has been the total absolute number of defined target cells. Therefore many investigators have explored methods to culture hematopoietic cells in vitro to increase the numbers of these cells. Studies attempting to expand hematopoietic stem cells, progenitor cells, and mature cells in vitro have become possible over the past decade due to the availability of recombinant growth factors and cell selection technologies. To date, no studies have demonstrated convincing data on the expansion of true stem cells, and so the focus of this review is the expansion of committed progenitor cells and mature cells. A number of clinical studies have been preformed using a variety of culture conditions, and several studies are currently in progress that explore the use of ex vivo expanded cells. These studies will be discussed in this review. There are evolving data that suggest that there are real clinical benefits associated with the use of the expanded cells; however, we are still at the early stages of understanding how to optimally culture different cell populations. The next decade should determine what culture conditions and what cell populations are needed for a range of clinical applications.

Engraftment of Primates with G‐CSF Mobilized Peripheral Blood CD34+ Progenitor Cells Expanded in G‐CSF, SCF and MGDF Decreases the Duration and Severity of Neutropenia

We used a primate model of autologous peripheral blood progenitor cell (PBPC) transplantation to study the effect of in vitro expansion on committed progenitor cell engraftment and marrow recovery after transplantation. Four groups of baboons were transplanted with enriched autologous CD34+ PBPC collected by apheresis after five days of G‐CSF administration (100 μg/kg/day). Groups I and III were transplanted with cryopreserved CD34+ PBPC and Groups II and IV were transplanted with CD34+ PBPC that had been cultured for 10 days in Amgen‐defined (serum free) medium and stimulated with G‐CSF, megakaryocyte growth and development factor (MGDF), and stem cell factor each at 100 ηg/ml. Group III and IV animals were administered G‐CSF (100 μg/kg/day) and MGDF (25 μg/kg/day) after transplant, while animals in Groups I and II were not. For the cultured CD34+ PBPC from groups II and IV, the total cell numbers expanded 14.4 ± 8.3 and 4.0 ± 0.7‐fold, respectively, and CFU‐GM expanded 7.2 ± 0.3 and 8.0 ± 0.4‐fold, respectively. All animals engrafted. If no growth factor support was given after transplant (Groups II and I), the recovery of WBC and platelet production after transplant was prolonged if cells had been cultured prior to transplant (Group II). Administration of post‐transplant G‐CSF and MGDF shortened the period of neutropenia (ANC < 500/μL) from 13 ± 4 (Group I) to 10 ± 4 (Group III) days for animals transplanted with non‐expanded CD34+ PBPC. For animals transplanted with ex vivo‐expanded CD34+ PBPC, post‐transplant administration of G‐CSF and MGDF shortened the duration of neutropenia from 14 ± 2 (Group II) to 3 ± 4 (Group IV) days. Recovery of platelet production was slower in all animals transplanted with expanded CD34+ PBPC regardless of post‐transplant growth factor administration. Progenitor cells generated in vitro can contribute to early engraftment and mitigate neutropenia when growth factor support is administered post‐transplant. Thrombocytopenia was not decreased despite evidence of expansion of megakaryocytes in cultured CD34+ populations.

Recombinant Human Ligand for MPL, Megakaryocyte Growth and Development Factor (MGDF), Stimulates Thrombopoiesis in Vivo in Normal and Myelosuppressed Baboons

Megakaryocyte growth and development factor (MGDF) is a ligand for c‐mpl and a member of the hematopoietic growth factor superfamily. Recombinant murine MGDF specifically stimulates thrombopoiesis in mice. Recombinant human (rHu) MGDF stimulates megakaryocytic differentiation of baboon CD 34+ marrow cells in vitro. Therefore, we determined the in vivo biological effects of rHuMGDF administered to normal baboons in the absence and presence of myelosuppression with 5‐fluorouracil (5‐FU). rHuMGDF was administered to normal baboons as single s.c. injection at doses of 1, 10, 25 and 50 μg/kg/day for 10 days and, as a control, heat‐inactivated MGDF was administered at a dose of 10 μg/kg/day. Platelet counts were markedly increased in all animals administered native rHuMGDF but not in animals given heat‐inactivated rHuMGDF. Platelet counts began to increase between three and six days after starting rHuMGDF administration and the maximum average increases were 1.7‐, 3.4‐, 5.1‐ and 4.0‐fold above baseline in animals administered 1, 10, 25 and 50 μg/kg/day, respectively. Maximum platelet counts were reached between 7 and 10 days after starting rHuMGDF and maintained for four days after the last dose. Thereafter, platelet counts decreased, reaching stable pretreatment values between 11 and 14 days after the last dose of rHuMGDF. No changes in red cell mass, peripheral blood white blood cell counts or differentials were observed during rHuMGDF treatment. For animals administered 10, 25 and 50 μg/kg/day of rHuMGDF, megakaryocytes increased more than threefold in marrow, were markedly enlarged, and had increased numbers of lobes. Overall marrow cellularity remained unchanged, as did red cell and white cell morphology. No marrow fibrosis was detected. Progenitor cells were not increased in marrow but did increase modestly in the peripheral blood, associated with increased numbers of CD34+ cells in circulation.

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.

CHAPTER 43 – Stem cell factor

This chapter deals with stem cell factor (SCF), which has been shown to be an essential growth factor in development, and maintenance of normal blood cell production. It synergizes with other hematopoietic growth factors to stimulate stem cells, primitive progenitor cells, and committed progenitor cells. Its clinical utility has been minimized by dose-limiting anaphylactoid reactions, however, clinical studies have demonstrated that in combination with granulocyte macrophage colony-stimulating factor (G-CSF), SCF can stimulate mobilization of increased numbers of CD34 + cells in peripheral blood progenitor cells (PBPC) products. If predictors of poor be a key growth factor for ex vivo manipulation of hematopoietic stem cells to produce regenerative tissues for various diseases mobilization can be developed. However, with the recent demonstration of plasticity of hematopoietic stem cells, generating cells of various tissues including, cardiac, neural, liver, lung, and endothelial cells. SCF may be a key growth factor for ex vivo manipulation of hematopoietic stem cells to produce regenerative tissues for various diseases.

A method for the determination of the number of reticulated platelets from whole blood

Abstract Platelets still containing RNA and DNA remnants are considered to be the immediate progeny of megakaryocytes which have matured beyond the proplatelet formation stage. Numerous investigators have shown that platelets which stain with thiazole orange, a nucleic acid dye, have increased RNA content, and therefore are the youngest platelets in the circulation. These immature, or reticulated platelets, are analogous to red blood cell reticulocytes in their level of differentiation, but not their function. Platelet analysis using thiazole orange dye, in combination with flow cytometry, has been recently shown by a number of investigators to be an optimal method for detection of this immature platelet population. However, the many variations in protocols cause results to greatly differ between laboratories. In our studies, we used a similar whole blood analysis approach developed by Matic and colleagues to quantitate the amount of dye needed for optimal staining. Therefore, we used a dose-escalation of thiazole orange (0.25 μg–50 μg) in combination with CD61 conjugated with phycoerythrin without any wash steps, which assures minimal platelet loss. CD61 was used in this setting in order to set an initial gate on the platelet population. There was a direct, dose-response relationship between the amount of thiazole orange added and the number of reticulated platelets observed. A plateau was reached with 5 μg thiazole orange per 5 μL of whole blood, while 12.5 μg was found to be optimal. Increasing amounts of thiazole orange produced a population shift, or mean channel shift, that needed to be accounted for when setting gates for thiazole orange positive events. We observed that nearly 7% of the platelets in normal resting BDF 1 mice were reticulated. This compares to a reticulated population in humans of less than 10%. This protocol combines the ease of sample preparation with stability and accuracy for determining the number of reticulated platelets, and should be a useful method in efforts to standardize the enumeration of reticulated platelets from whole blood. Furthermore, the use of thiazole orange for determining reticulated platelet numbers provides a reproducible and validated method for the analysis of platelet precursors, and, thus, may be a useful tool for predicting platelet recovery post-chemotherapy.

Cholesterol effects megakaryocytopoiesis

Abstract Platelet and lipid profiles are elevated in individuals with atherosclerosis. We examined the effects of cholesterol on megakaryocytopoiesis, since megakaryocytes (MK) give rise to the platelets. Individuals with slightly elevated cholesterol (>190mg/dL; N=22) had lower mean platelet volumes, but higher platelet counts resulting in a higher platelet mass than individuals with lower cholesterol (