Fig 1.
Workflow for Synthesis, Purification, and Characterization of Conjugates.
Mouse monoclonal antibody specific for human CEA was used to prepare four samples for subsequent evaluation and comparison. Two mouse IgG1 isotype control conjugate samples were also prepared as stated in Table 1. The bottom portion of Fig 1 illustrates approximate characterized loading ratios for both drug and dye in various CEA samples.
Table 1.
Analytical Characterization of Antibody Samples and Conjugates.
Fig 2.
Estimation of Physiological Conjugate Stability In Vitro.
The fluorescent ADC and hydrolyzed controls were incubated in PBS at 37°C, and liberated paclitaxel was extracted and quantified. Degree of hydrolysis was defined as the ratio of extracted paclitaxel from test samples compared to hydrolyzed controls. Data points represent triplicate extracted samples (n = 3). Error bars denote standard error.
Fig 3.
Immunofluorescent Staining of Cell lines with Conjugates and Quantification of Signal.
(A) Immunofluorescent images of cell lines stained with various conjugate samples. Two putative CEA-positive cell lines, BxPC-3 and MCF-7, and two control CEA-negative cell lines, HeLa and HepG2, were seeded at 1x104 cells/well in 96-well collagen-coated plates. Cells were immunofluorescently stained with approximately 2ug/mL of α-CEA-680 or α-CEA-680-PTX where noted (red). Nuclei were counterstained with Hoechst dye (blue). Images were obtained on an ArrayScan VTI under 20X magnification and arranged with the HCS View software, Pxlr editor, and Microsoft PowerPoint. (B) Quantification of Immunofluorescent Staining. BxPC-3, MCF-7, HeLa, and HepG2 cell lines were seeded at 1x104 cells/well in 96-well collagen-coated plates. Cell lines were probed with α-CEA-680 (red lines), α-CEA-680-PTX (blue lines), IgG-680 (green lines), or IgG-680-PTX (purple lines) at concentrations of 0 – 60ug/mL. Fluorescent signal intensity was quantified with the ArrayScan VTI and vHCS Scan software. Data points represent average fluorescent intensity (n = 3000 cells), and error bars denote standard error. Error bars are present at every data point, but are very small in some cases.
Fig 4.
Flow Cytometry Analysis of Cell lines Stained with Conjugates.
BxPC-3, MCF-7, HeLa, and HepG2 cells (1x106 in each sample) were probed with either buffer (unstained, black histograms) or a 10ug/mL solution of α-CEA-680 (red histograms), α-CEA-680-PTX (blue histograms), IgG-680 (green histograms), or IgG-680-PTX (purple histograms). Cells were analyzed using the BD Accuri C6 Flow Cytometer and CSampler Workspace software. Each histogram represents 50,000 total events.
Fig 5.
Immunofluorescent Assessment of CEA Internalization.
(A) Quantification of internalized CEA “spots”. BxPC-3 cells were seeded at 1x104 cells/well in 96-well collagen-coated plates. Cells were then treated with 46ng/mL free DyLight™ 680-4xPEG or 2ug/mL of IgG-680, IgG-680-PTX, α-CEA-680, or α-CEA-680-PTX diluted in RPMI completed media (all at 46ng/mL effective DyLight™ 680-4xPEG) for the time points indicated. Cells were then washed, fixed, and counterstained with Hoechst dye. Average internalized spots per cell were obtained with the ArrayScan VTI and vHCS Scan software. Data points represent the average number of internalized spots per cell (n = 10,000 cells), and error bars denote standard error. Error bars are present at every data point, but are very small in some cases. (B) Immunofluorescent images depicting representative cells at each time point. Images (20X magnification) were obtained in concert with spot detection analysis on the ArrayScan VTI, and arranged with the vHCS View software, Pxlr editor, and Microsoft PowerPoint.
Fig 6.
MTT Cytotoxic Assessment of Samples and Conjugates.
BxPC-3 cells (5x103) were plated in 96-well collagen-coated plates, and treated with a range of various test samples as noted in the figure. Connected dashes denote conjugates, and “+” indicates the addition of free, unconjugated compound (i.e. α-CEA-680 is the fluorescent antibody conjugate, where α-CEA + 680 indicates naked antibody plus free fluorescent dye). The sample concentration at each data point is plotted according to the three x-axes. For example, the data point on the far right for α-CEA-680-PTX is comprised of 60ug/mL of antibody, which corresponds to an effective paclitaxel concentration of 400nM, and an effective DyLight 680 concentration of 800nM. Samples are diluted 1:2 serially throughout the rest of the series. Samples that do not contain antibody, paclitaxel, or DyLight 680 are still standardized in this manner for graphical comparison (i.e. the far right data point for naked antibody, α-CEA, is comprised of a 60ug/mL solution and serially diluted 1:2 as before, and α-CEA-PTX starts at 60ug/mL, which corresponds to an effective paclitaxel concentration of 400nM, and is serially diluted identically.) After 24 hours, samples were removed and replaced with fresh media. Cells were allowed to grow for 72 additional hours and then stained with MTT according to the manufacturer’s instructions. The absorbance of each well was measured at 540nm on the Thermo Scientific VarioSkan Flash plate spectrophotometer. Edges of the plate were omitted. Percent viability was defined as the ratio of the A540 of test wells compared to the A540 of untreated control wells. Untreated controls received media and an appropriate vehicle depending on sample. Data points represent the average of three separate experiments performed in triplicate (n = 9 wells). Error bars denote standard error.
Table 2.
IC50 Estimations of Samples and Conjugates.
Fig 7.
Flow Cytometry Cell Cycle Analysis.
BxPC-3 cells were treated with vehicle control, 100nM free paclitaxel, 15ug/mL α-CEA-PTX, or 15ug/mL α-CEA-680-PTX (both at 100nM effective paclitaxel) for 24 hours. Cell cycle analysis was analyzed using the BD Cycletest™ Plus DNA Reagent Kit. All samples were gated identically, and 30,000–40,000 events were collected for each sample.
Fig 8.
In Vivo Tumor Growth Inhibition.
Male nude, athymic mice, 4 weeks of age, were subcutaneously inoculated with BxPC-3 cells (2x106) embedded in 50:50 HBSS:Matrigel. When tumors were palpable (~1 week), mice were tagged and randomly separated into 3 groups. Mice in group 1 (n = 6) received PBS (vehicle). Group 2 (n = 6) received free paclitaxel (6.7uM) in PBS, while group 3 (n = 5) received equimolar amounts of paclitaxel conjugated to antibody (fluorescent ADC, 100ug α-CEA-680-PTX, 6.7uM effective paclitaxel). Each mouse received a total of five doses of the respective treatment, spaced 3 days apart, via retro-orbital injection. Arrows indicate treatment days (days 3, 6, 9, 12, and 15). Tumor size was measured every 3 days using digital calipers, and tumor volume was calculated with the modified ellipsoid formula (v = ½(length × width2)). Each data point represents average tumor volume (n = 5–6 mice). and error bars denote standard error.
Fig 9.
In Vivo Fluorescent Tumor Localization of α-CEA-680-PTX.
Mice in group 3 were imaged 24 hours after every treatment infusion (i.e. treatment on days 3, 6, 9, 12, and 15, imaging on days 4, 7, 10, 13, and 16), and were also imaged once a week after all treatment had stopped (days 23, 30, 37, and 44). At each time point, mice were anesthetized as outlined in the methods section and imaged using the UVP iBox Explorer2 with attached BioLite Xe MultiSpectral Source. Images were obtained with a white light (control) and a NIR excitation filter (600-645nm), an emission filter of 720nm, a magnification of 0.17x, and fixed exposure times as indicated.
Fig 10.
Automatic Exposure Times Throughout the Study.
Mice in group 3 were imaged 24 hours after every treatment infusion (i.e. treatment on days 3, 6, 9, 12, and 15, imaging on days 4, 7, 10, 13, and 16), and were also imaged once a week after all treatment had stopped (days 23, 30, 37, and 44). At each time point, mice were anesthetized as outlined in the methods section and imaged using the UVP iBox Explorer2 with attached BioLite Xe MultiSpectral Source. A variable automatic exposure using the VisionWorks control software was obtained on the days indicated. Treatment infusion days are indicated by arrows (days 3, 6, 9, 12, and 15). Data points represent average automatic exposure times (n = 5 mice). Error bars denote standard error.