Cell culture and patient-derived HNSCC xenografts
MDA-MB-468 human breast cancer cells (1.3 × 106 EGFR/cell) (Reilly and Gariepy, 1998) were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA) and 1% penicillin streptomycin (Sigma-Aldrich). A primary tumour specimen (#391) was surgically obtained from a patient with HNSCC under a protocol approved by the Research Ethics Board at the University Health Network (Protocol No. 12–5639). This tumour was dissected into small fragments (~ 1 mm3) and engrafted subcutaneously (s.c.) on the right flank of NOD-Rag1null IL2rgnull (NRG) mice. These patient-derived tumour xenografts (PDX) were serially propagated in NRG mice following an Animal Care Protocol (No. 1542.28) approved by the Animal Care Committee at the University Health Network and following Canadian Council on Animal Care guidelines. The PDX used in this study were between the 3rd to 5th passage from the initial engraftment of the HNSCC tumour in NRG mice.
Panitumumab and trastuzumab F(ab´)2
Panitumumab F(ab´)2 (MW ~ 110 kDa) were produced by proteolytic digestion of panitumumab IgG (Vectibix®, Amgen, Thousand Oaks, CA) using immobilized pepsin (Pierce Biotechnology, Rockford, IL) as reported with minor modifications (Boyle et al., 2015). Briefly, panitumumab IgG was buffer-exchanged into 20 mM sodium acetate buffer (pH 4.5) by ultrafiltration on an Amicon ultracentrifugal unit (Millipore, Burlington, MA; MWCO = 30 kDa). Panitumumab IgG was then incubated at a ratio of 4 mg of IgG per 0.25 mg of immobilized pepsin resin in 0.25 mL slurry at 37 °C for 5 h in a Excella E24 Incubator Shaker (New Brunswick Scientific, Edison, NJ) at 300 rpm. Following digestion, the resin suspension was rinsed with ice cold phosphate buffered saline (PBS), pH 7.4, and centrifuged at 1000×g for 5 min and the supernatant collected. This was repeated 2 times and the pooled supernatants were filtered through a Millex®-GV PDVF 0.22 μm filter to remove residual resin. F(ab´)2 were re-concentrated to 20.0–24.5 mg/mL and buffer-exchanged into 100 mM NaHCO3 buffer, pH 8.2 on an Amicon ultracentrifugal unit (MWCO = 30 kDa). The 100 mM NaHCO3 buffer, pH 8.2 buffer was purified from trace metals by passage through a column of Chelex-100 cation exchange resin (BioRad, Mississauga, ON, Canada). The F(ab´)2 concentration was measured spectrophotometrically at 280 nm (A280 of a 1 mg/mL solution = 1.40). The purity and homogeneity of F(ab´)2 were assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on a 7.5% Mini-Protean Tris/glycine mini-gel (BioRad) under reducing (2-mercaptoethanol) and non-reducing conditions with bands stained with Biosafe Coomassie Blue G-250 (BioRad). Anti-human epidermal growth factor receptor-2 (HER2) trastuzumab F(ab´)2 was prepared by proteolytic digestion of trastuzumab IgG (Herceptin, Roche, Mississauga, ON, Canada).
Labeling of F(ab´)2 with 64Cu or 177Lu
Panitumumab F(ab´)2 (2 mg; 20.0–24.5 mg/mL) in 100 mM NaHCO3 buffer, pH 8.2 were conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) by reaction with a 20-fold molar excess of the N-hydroxysuccinimide ester (DOTA-NHS; Macrocyclics, Dallas, TX) for 2 h at room temperature (RT) on a nutating mixer (VWR, Mississauga, ON, Canada). DOTA-panitumumab F(ab´)2 were purified from excess DOTA and buffer-exchanged into 100 mM HEPES, pH 5.5 and re-concentrated to 20–28.5 mg/mL using an Amicon ultracentrifugal unit (MWCO = 10 kDa) centrifuged at 7500×g for 5 mins. This was repeated 8 times to ensure complete removal of excess DOTA. The DOTA substitution level [moles DOTA/mole F(ab´)2] was determined by trace-labeling an aliquot (100 μg; 5 μL) of the impure conjugation mixture with [64Cu]CuCl2 (Washington University, St. Louis, MO; 40 MBq/μL) and measuring the proportion of [64Cu]Cu-DOTA-panitumumab F(ab´)2 and [64Cu]Cu-DOTA by instant thin layer-silica gel chromatography (ITLC-SG; Agilent Technologies, Santa Clara, CA) developed in 100 mM sodium citrate, pH 5.5, then multiplying this proportion by the 20:1 M ratio of NHS-DOTA:F(ab´)2 in the reaction (Reilly, 2007). The Rf values of [64Cu]Cu-DOTA-panitumumab F(ab´)2 and free [64Cu]Cu-DOTA (or 64Cu) were 0.0 and 1.0, respectively.
Purified DOTA-panitumumab F(ab´)2 (~ 600 μg in 30 μL) were labeled with 64Cu to a specific activity of 0.074–1.7 MBq/μg by incubation with [64Cu]CuCl2 (40 MBq/μL; Washington University) for 1 h at 42 °C. No post-labeling purification was performed. The radiochemical purity (RCP) of [64Cu]Cu-DOTA-panitumumab F(ab´)2 was confirmed by ITLC and size exclusion (SE)-HPLC on an Agilent 1260 Infinity II HPLC system fitted with a BioSep SEC-S4000 column (300 × 7.8 mm; Phenomenex, Torrance, CA) eluted with NaH2PO4 buffer, pH 7.0 at a flow rate of 0.50 mL/min with UV detection (λ = 280 nm) and radioactivity detection by a Flowstar LB514 radioactivity detector fitted with a BGO-X flow cell (Berthold Technologies, Bad Wildbad, Germany). DOTA-panitumumab F(ab´)2 (~ 600 μg in 30 μL) were labeled at a SA = 0.07–0.2 MBq/μg with 177Lu by incubation wih [177Lu]LuCl3 (McMaster University, Hamilton, ON, Canada) at 42 °C for 3 h. The RCP of [177Lu]Lu-DOTA-panitumumab F(ab´)2 was determined by ITLC as described for [64Cu]Cu-DOTA-panitumumab F(ab´)2. Trastuzumab F(ab´)2 were similarly conjugated to DOTA and labeled with 177Lu.
A saturation radioligand binding assay was performed to measure the dissociation constant (Kd) and maximum number of binding sites (Bmax) for binding of [177Lu]Lu-DOTA-panitumumab F(ab´)2 to EGFR on MDA-MB-468 human breast cancer cells (1.3 × 106 EGFR/cell) (Reilly and Gariepy, 1998). Increasing concentrations of [177Lu]Lu-DOTA-panitumumab F(ab´)2 (0.098–200 nmoles/L) were incubated with 1 × 106 MDA-MB-468 cells in 200 μL of PBS, pH = 7.4 in 1.5 mL Eppendorf tubes at 4 °C for 3 h with gentle shaking every 30 mins. The samples were then centrifuged at 1000×g for 2 mins on an Eppendorf Centrifuge 5424 (Thermo Fisher Scientific, Waltham, MA) and the supernatant containing unbound 177Lu collected. The cell pellets were rinsed with ice cold PBS, pH 7.4, centrifuged again and the supernatant was collected and pooled with the previously collected supernatant. This was repeated twice. Total bound 177Lu (TB) in the cell pellets and unbound 177Lu in the supernatant were measured in a γ-counter (Model 1480; PerkinElmer, Waltham, MA). Non-specifically bound 177Lu (NSB) was assessed by repeating the assay in the presence of 50-fold molar excess of panitumumab IgG. Specifically bound 177Lu (SB) was calculated by subtracting NSB from TB. [177Lu]Lu-DOTA-panitumumab F(ab´)2 bound to MDA-MB-468 cells (pmoles) was plotted vs. the concentration of free (unbound) [177Lu]Lu-DOTA-panitumumab F(ab´)2 (nmoles/L) and the curve was fitted to a one-site-receptor-binding model using Prism Ver. 4.0 software (GraphPad, San Diego, CA).
Biodistribution (BOD) studies and microPET/CT
The tumour and normal tissue uptake of [64Cu]Cu-DOTA-panitumumab F(ab´)2 were determined by BOD and microPET/CT studies in NRG mice with subcutaneous (s.c.) HNSCC PDX. Groups of 3–4 tumour-bearing NRG mice were injected i.v. (tail vein) with [64Cu]Cu-DOTA-panitumumab F(ab´)2 (5.5–14.0 MBq; 50 μg) and sacrificed at 6 h, 24 or 48 h post-injection (p.i.). The tumour and samples of blood and normal tissues were obtained, weighed and the radioactivity in each measured in a γ-counter using a window (425–640 keV) to include the 511 keV annihilation γ-photon of 64Cu. Tumour and normal organ uptake were expressed as percent injected dose/g (%ID/g). MicroPET/CT was performed after i.v. injection (tail vein) of 37 MBq (50 μg) of [64Cu]Cu-DOTA-panitumumab F(ab´)2 in a group of 4 NRG mice with s.c. HNSCC PDX. Mice were anaesthetized using 2% isoflurane in O2 and were imaged in a supine position on a NanoScan® SPECT/CT/PET system (Mediso, Budapest, Hungary). PET images were acquired for 10, 20 and 40 mins at 6, 24 and 48 h p.i. of [64Cu]Cu-DOTA-panitumumab F(ab´)2, respectively. Images were reconstructed by an ordered subset expectation maximization (OSEM) algorithm and consisted of 4 subsets and 4 iterations with attenuation and scatter correction supported by an isotropic voxel size of 300 μm. Prior to PET, CT images were acquired with 50 kVp X-rays, 980 μA and 300 msec exposure time. CT scans were reconstructed using the medium voxel and slice thickness settings, resulting in an isotropic voxel size of 250 μm. PET and CT images were co-registered by the Mediso Nucline NanoScan 3.00.020.0000 software. PET and CT DICOM files were exported using Mediso’s Nucline Acquisition/Reconstruction Software to the Inveon Research Workplace Software 4.0 (Siemens) for analysis and quantification of 64Cu uptake (%ID/g) as well as estimation of the volume of the liver and tumour. Regions of interest (ROI) were drawn around the tumour and liver on the PET images aided by delineation of the anatomy on the CT images to compare the accuracy of PET for quantifying the tumour and liver uptake compared to BOD studies. The ROI for each organ was drawn two-dimensionally on axial slices of the image. To ensure the coverage of the entire organ, > 5 ROIs were drawn before the slices were convoluted to form a 3D volume that encompassed the entire organ.
Biodistribution (BOD) studies and microSPECT/CT
The tumour and normal tissue uptake of [177Lu]Lu-DOTA-panitumumab F(ab´)2 was determined by BOD and microSPECT/CT studies. [177Lu]Lu-DOTA-panitumumab F(ab´)2 (6.5 MBq; 50 μg) were injected i.v. (tail vein) in NRG mice with s.c. HNSCC PDX and groups (n = 3–4) of mice were sacrificed at 6 h, 24 h or 48 h p.i. The tumour, and samples of blood and other tnormal issues were obtained, weighed and counted in a γ-counter using a window (130–470 kev) to include the γ-photons of 177Lu [Eγ = 113 keV (6.6%) and Eγ = 208 keV (11%)]. Tumour and normal organ uptake was expressed as %ID/g. The excised tumours were subjected to immunohistochemical (IHC) staining with anti-human EGFR antibodies (Invitrogen, Carlsbad, CA; Cat. No. 28–8763) to confirm EGFR positivity. In addition, the tumour and normal tissue BOD of irrelevant [177Lu]Lu-DOTA-trastuzumab F(ab´)2 (2.90 MBq; 50 μg) were determined in a separate group of 5 NRG mice bearing HNSCC PDX at 24 h p.i. The excised tumours in these mice were stained for HER2 using anti-human HER2 antibodies (Invitrogen Cat. No. MA5–14509). Representative mice were anaesthetized using 2% isoflurane in O2 and microSPECT/CT images were acquired in a supine position, at 6 h, 24 h and 48 h p.i. of [177Lu]Lu-DOTA-panitumumab F(ab´)2 on a NanoScan® SPECT/CT/PET system (Mediso). Images were acquired in a 256 × 256 matrix. A window (± 10%) was set around each of the γ-photopeaks (208.4 keV; 112.9 keV; 56.1 keV) of 177Lu. A Mediso APT62 collimator (WB-HS standard) was affixed to each of the 4 detector NaI (TI) detector heads. Images were reconstructed by Monte Carlo methods with three subsets of data undergoing 48 iterations using the Mediso Nucline NanoScan acquisition and reconstruction software (ver 3.00.020.0000). Prior to SPECT, imaging CT images were acquired with 50 kVp X-rays, 980 μA and a 300 msec exposure time. CT scans were reconstructed using the medium voxel and slice thickness settings resulting in an isotropic voxel size of 250 μm. SPECT and CT were co-registered by the Mediso Nucline acquisition/reconstruction software. MicroSPECT/CT images were similarly obtained for NRG mice with HNSCC PDX at 24 h p.i. of [177Lu]Lu-DOTA-trastuzumab F(ab´)2. All animal studies were conducted under a protocol (AUP 2843.8) approved by the Animal Care Committee at the University Health Network following Canadian Council on Animal Care guidelines.
Tumour and normal organ dosimetry
The radiation equivalent doses in NRG mice with s.c. HNSCC PDX after i.v. injection of [64Cu]Cu-DOTA-panitumumab F(ab´)2 or [177Lu]Lu-DOTA-panitumumab F(ab´)2 were estimated from the tumour and normal organ uptake of activity in source organs measured in BOD studies. The mean equivalent dose (D) was calculated as D = Ãs × S × WR, where Ãs is the time-integrated activity in the source organs or tumour and S are the Snyder values for mice (Bitar et al., 2007; Xie and Zaidi, 2013) and WR is the radiation weighting factor. WR is 1 for x-rays, γ rays and β-particles, thus taken as 1 in this study. Ãs in the source organs or in the tumour were estimated using Prism Ver. 4.0 software (GraphPad) from the area-under-the-curve (AUC) up to 48 h p.i. of [64Cu]Cu-DOTA-panitumumab F(ab´)2 or [177Lu]Lu-DOTA-panitumumab F(ab´)2 (AUC0–48 h; Bq × sec) derived from the activity vs. time curves. The activity/source organ at each time point t (s) was calculated using the formula %ID/g (t) × ID/100 × organ weight × exp.(−kt), where %ID/g was obtained from the BOD studies, ID was the injected dose in Bq, and k was the decay constant for 64Cu (1.52 × 10− 5 s− 1) or 177Lu (1.21 × 10− 6 s − 1). The time integrated activity from 48 h p.i. to infinity (A48 h – ∞; Bq × sec) was calculated by dividing the activity at 48 h p.i. by the decay constant for 64Cu or 177Lu, assuming further elimination of activity from source organs only by radioactive decay. The S-value for the tumour was estimated using the sphere model in OLINDA/EXM software based on the measured tumour mass (Stabin et al., 2005). This was repeated with the estimated uptake in the tumour and liver determined by ROI analysis of the microPET/CT images at 6 h, 24 h and 48 h p.i. of [64Cu]Cu-DOTA-panitumumab F(ab´)2. To predict the doses in normal organs in humans, the activity in source organs of a human adult female with a 2 cm diameter spherical tumour in the neck at the different times up to 48 h p.i. of the RICs were estimated based on proportional extrapolation of the activities in mice using the % kg/g method, i.e. (%ID/organ)human = [(%ID/organ)mouse × (mouse body weight/human body weight)] (Kirschner et al., 1973). Mouse and human body weight used in the calculation were set to 30 g and 56,900 g, respectively. The time-integrated activity from 0 to 48 h p.i. (AUC0–48 h; Bq × sec) and from 48 h p.i. to infinity (A48 h – ∞; Bq × sec) were calculated as described earlier for mice, and the combined A0h to ∞ for each human source organ was used to predict the equivalent doses in human female adults (mSv/Bq) using OLINDA/EXM 1.0 software (Stabin et al., 2005).
All results were reported as mean ± SEM. Statistical comparisons for normal organ and tumour uptake of [64Cu]Cu-DOTA-panitumumab F(ab´)2 and [177Lu]Lu-DOTA-panitumumab F(ab´)2 were made using a Mann-Whitney U test (P < 0.05) and Prism 4.0 software (GraphPad). Statistical comparisons for radiation equivalent dose were made using a two-tailed unpaired Student’s t-test (P < 0.05) using PrismVersion 4.0 software (GraphPad).