An Optimized Method for Manufacturing a Clinical Scale Dendritic Cell-Based Vaccine for the Treatment of Glioblastoma

<div><p>Immune-based treatments represent a promising new class of therapy designed to boost the immune system to specifically eradicate malignant cells. Immunotherapy may generate specific anti-tumor immune responses, and dendritic cells (DC), professional antigen-presenting cells, are widely used in experimental cancer immunotherapy. Several reports describe methods for the generation of mature, antigen-pulsed DC for clinical use. Improved quality and standardization are desirable to obtain GMP-compliant protocols. In this study we describe the generation of DC from 31 Glioblastoma (GB) patients starting from their monocytes isolated by immunomagnetic CD14 selection using the CliniMACS® device. Upon differentiation of CD14+ with IL-4 and GM-CSF, DC were induced to maturation with TNF-α, PGE₂, IL-1β, and IL-6. Whole tumor lysate was obtained, for the first time, in a closed system using the semi-automated dissociator GentleMACS®. The yield of proteins improved by 130% compared to the manual dissociation method. Interestingly the Mean Fluorescence Intensity for CD83 increased significantly in DC pulsed with “new method” lysate compared to DC pulsed with “classical method” lysate. Our results indicate that immunomagnetic isolation of CD14⁺ monocytes using the CliniMACS® device and their pulsing with whole tumor lysate proteins is a suitable method for clinical-scale generation of high quality, functional DC under GMP-grade conditions.</p> </div

Summary of DC production data.

(a)<p>number of WBC present in the starting leucapheresis.</p>(b)<p>number of CD14⁺ cells obtained after CliniMACs selection.</p>(c)<p>number of mDC obtained at the end of culture.</p>(d)<p>yield of mDC respect to the starting WBC.</p>(e)<p>yield of mDC respect to the cultured CD14⁺.</p>(f)<p>ns = Not significant (p>0.05) for all the parameter evaluated.</p

Functional evaluation of DC.

<p>The ability of iDC versus mDC in the antigen presentation was compared evaluating the potency of DC as their <i>in vitro</i> allo-stimulatory capacity of PBMC from healthy volunteers. Final product (mDC, black bar, 31 experiments) resulted more potent than its immature counterpart (iDC, empty bar, 18 experiments) in the MLR induction. We compared new lysate activated and old lysate activated cells for their ability in the induction of allogeneic MLR functional assay. “New method-tumor lysate” (19 productions) activation seem to slightly improve the functional ability of mDC in their allo-stimulatory ability respect to “classical method-tumor lysate” (12 productions) activation but this difference is not statistically significant. By contrast no proliferative responses were induced by antigen-loaded mDC in the lymphocyte of GB patients prior to vaccination (red bar, 18 experiments). Data are expressed as Stimulation Index (SI); statistical bars on graph indicate the SD value.</p

Morphology of microglia/macrophages (red).

<p>CD11b immunopositive cells at day 1 after ischemia and NC(4 h) infusion display a round, phagocytic morphology in the injected (A) but not in the contralateral side were no NC are present (B). Microglia/macrophages appear to surround NC as evidenced in C were NC are marked with Hoechst (blue). A further detail of the interaction between these cells is provided in D where one microglia/macrophage appears to cap a NC (marked with 5-CFDA, green). Bar (A–C): 20 µm; bar (D): 7 µm.</p

Box-plot of neuronal count performed in ischemic mice 7 and 14 days after ischemia with the optical dissector method.

<p>Differences of neuronal density between contralateral and ipsilateral striata are sorted by treatment and time. The box represents the median and 25^th–75^th percentiles, whiskers represent the range, + represents the mean of the group. The hierarchical ANOVA showed a significant effect for the treatment (P = 0.02). The Bonferroni post-hoc test showed significant differences (P<0.05) among mean density differences of the isch/NC(4 h) group and the isch/PBS, isch/fibr and isch/NC(7 d) groups. Data plotted have been multiplied by 1×10⁴.</p

Confocal analysis of immunoreactivity of NC(4 h) - green - and nestin, GFAP or NG-2 - red - at day 1.

<p>Colocalization (arrows) can be observed between NC(4 h) and nestin, NC(4 h) and GFAP, NC(4 h) and NG-2. Images are taken in striatum, in proximity of the ventricular wall where most cells can be found at this time point. Bar: 25 µm.</p

Experimental protocols.

<p>Ischemia was induced for 30 minutes, 2 hours after the <i>in vitro</i> placement of the isolated brain. NC were perfused for 1 h immediately either after the reopening of the vessel or 1 our later (protocol 2). The perfusion was followed by a wash-out period with a solution without NCs. At 5 hours <i>in vitro</i> the brains were fixed for immunohistochemistry. The bottom of the panel shows an example of simultaneous DC recordings from 4 different sites in an isolated guinea pig brain. Hypoxic depressions (HD) were recorded in the electrodes located in the regions vascularized by the occluded MCA. Potentials evoked by LOT stimulation before and during the first part of ischemia (arrowhead) disappeared when HD occurred, and recovered during MCA reperfusion. Evoked potentials in the hemisphere contralateral to MCA occlusion were not altered.</p

Primers used for quantitative real-time polymerase chain reaction analysis of SDF-1α, BDNF, IGF-1, VEGF-A, TGF-β1, β-actin transcript levels.

a<p>forward</p>b<p>reverse</p

Quantification of microglia/macrophage activation.

<p>The area occupied by CD11b immunopositive cells was measured in striatum of sham-operated or ischemic mice receiving PBS, NC(4 h) or NC(7 d) at different time points (see Experimental design, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000373#pone-0000373-g001" target="_blank">Fig. 1A and B</a>). Data are expressed as mean±SEM (n = 4–5). Two-way ANOVA: (A) F: 58.05, p<0.0001; (B) F: 10.19, p = 0.0002. <i>Posthoc</i> Bonferroni test: *p<0.05, **p<0.01, ***p<0.001 vs sham/PBS; °°p<0.01, °°°p<0.001 vs isch/PBS, ##p<0.01, ###p<0.001 vs sham/NC.</p

Figure 2

<p>A. Recostrution of the distribution of NCs superimposed on a coronal section immunoreacted with anti-MAP-2 antibody (immunoperoxydase staining) to identify the ischemic region. Green fluorescent CFDA-stained stem cells were counted and plotted on the adjacent section stained with the MAP-2 fluorescent antibody (calibration bar = 1 cm). In B and C, microphotographs of green fluorescent CFDA-stained stem cells on sections counterstained with the fluorescent anti-MAP-2 antibody are shown at ×10 (B) and ×40 (C) magnifications. The pictures were taken in the transition region between the MAP-2⁺ and the MAP-2⁻ areas in the olfactory region. Calibration bars: 150 µm and 16 µm for B and C, respectively.</p