Formulation and In Vitro Characterization of PLGA/PLGA-PEG Nanoparticles Loaded with Murine Granulocyte-Macrophage Colony-Stimulating Factor.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has demonstrated notable clinical activity in cancer immunotherapy, but it is limited by systemic toxicities, poor bioavailability, rapid clearance, and instability in vivo. Nanoparticles (NPs) may overcome these limitations and provide a mechanism for passive targeting of tumors. This study aimed to develop GM-CSF-loaded PLGA/PLGA-PEG NPs and evaluate them in vitro as a potential candidate for in vivo administration. NPs were created by a phase-separation technique that did not require toxic/protein-denaturing solvents or harsh agitation techniques and encapsulated GM-CSF in a more stable precipitated form. NP sizes were within 200 nm for enhanced permeability and retention (EPR) effect with negative zeta potentials, spherical morphology, and high entrapment efficiencies. The optimal formulation was identified by sustained release of approximately 70% of loaded GM-CSF over 24 h, alongside an average size of 143 ± 35 nm and entrapment efficiency of 84 ± 5%. These NPs were successfully freeze-dried in 5% (w/v) hydroxypropyl-ß-cyclodextrin for long-term storage and further characterized. Bioactivity of released GM-CSF was determined by observing GM-CSF receptor activation on murine monocytes and remained fully intact. NPs were not cytotoxic to murine bone marrow-derived macrophages (BMDMs) at concentrations up to 1 mg/mL as determined by MTT and trypan blue exclusion assays. Lastly, NP components generated no significant transcription of inflammation-regulating genes from BMDMs compared to IFNγ+LPS "M1" controls. This report lays the preliminary groundwork to validate in vivo studies with GM-CSF-loaded PLGA/PEG-PLGA NPs for tumor immunomodulation. Overall, these data suggest that in vivo delivery will be well tolerated.

Synthesis of granulocyte-macrophage colony-stimulating factor as homogeneous glycoforms and early comparisons with yeast cell-derived material.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a medicinally important glycoprotein, used as an immunostimulant following bone-marrow transplant. On the basis of reports of its potential utility as an anticancer vaccine adjuvant, we undertook to develop a synthetic route toward single-glycoform GM-CSF. We describe herein a convergent total synthesis of GM-CSF aglycone and two homogeneous glycoforms. Analytical and biological studies confirm the structure and activity of these synthetic congeners.

Cost-effective manufacture of an allogeneic GM-CSF-secreting breast tumor vaccine in an academic cGMP facility.

BACKGROUND: GM-CSF-secreting, allogeneic cell-based cancer vaccines have shown promise for the treatment of a variety of solid tumors. We have now applied this approach to breast cancer. The aim of these studies was to optimize expansion parameters, qualify the manufacturing process, and establish expected outcomes for cGMP-compliant manufacturing of two GM-CSF-secreting breast tumor cell lines. METHODS: The variables affecting the efficiency of expanding and formulating two allogeneic GM-CSF-secreting cell lines, 2T47D-V and 3SKBR3-7, were systematically evaluated. Production criteria investigated included alternative cell culture vessels (flasks vs. cell factories), centrifugation time and speed variables for large volume cell concentration, cell seeding density, the minimal concentration of FBS required for maximal cell expansion, and the dose and timing of irradiation in relation to cryopreservation. RESULTS: These studies demonstrate that, in comparison with standard 150-cm2 tissue culture flasks, Nunc 10-Stack Cell Factories are a more efficient and practical cell culture vessel for vaccine cell line manufacture. Centrifugation optimization studies using the COBE 2991 Cell Processor established that a speed of 2000 r.p.m. (450 g) for 2 min reliably concentrated the cells while maintaining acceptable viability and bioactivity. Radiation studies established that lethal irradiation prior to cryopreservation does not compromise the quality of the product, as measured by post-thaw cell viability and GM-CSF cell line-specific secretion levels. Finally, studies aimed at optimizing the production of one vaccine cell line, 3SKBR3-7, demonstrated that seeding the cells at a higher density and maintaining them in half the initial concentration of FBS maximized the yield of bioactive cells, resulting in significant cost savings. DISCUSSION: A manufacturing process that simultaneously maximizes cell yield, minimizes cell manipulation and maintains vaccine cell potency is critical for producing cell-based cancer vaccines in an academic setting. These studies define a feasible, reproducible and cost-effective methodology for production of a GM-CSF-secreting breast cancer vaccine that is cGMP compliant.

Design, synthesis, and conformational studies of the hGM-CSF derived peptide (13-27)-Gly-(75-87).

An analogue of the human granulocyte-macrophage colony-stimulating factor (hGM-CSF), hGM-CSF(13-27)-Gly-(75-87) was synthesized by solid phase methodology. This analogue was designed to comprise helices A and C of the native growth factor, linked by a glycine bridge. Helices A and C form half of a four-helix bundle motif in the crystal structure of the native factor and are involved in the interaction with alpha- and beta-chains of the heterodimeric receptor. A conformational analysis of the synthetic analogue by CD, two-dimensional nmr spectroscopy, and molecular dynamics calculations is reported. The analogue is in a random structure in water and assumes a partially alpha-helical conformation in a 1 : 1 trifluoroethanol/water mixture. The helix content in this medium is approximately 70%. By 2D-nmr spectroscopy, two helical segments were identified in the sequences corresponding to helices A and C. In addition to medium- and short-range NOESY connectivities, a long-range cross peak was found between the Cbeta proton of Val(16) and NH proton of His(87) (using the numbering of the native protein). Experimentally derived interproton distances were used as restraints in molecular dynamics calculations, utilizing the x-ray coordinates as the initial structure. The final structure is characterized by two helical segments in close spatial proximity, connected by a loop region. This structure is similar to that of the corresponding domain in the x-ray structure of the native growth factor in which helices A and C are oriented in an antiparallel fashion. The N-terminal residues Gly-Pro of helix C are involved in an irregular turn connecting the two helical segments. As a consequence, helix C is appreciably shifted and slightly rotated with respect to helix A compared to the x-ray structure of the native growth factor. These small differences in the topology of the two helices could explain the lower biological activity of this analogue with respect to that of the native growth factor.