Construct design and syntheses
SWCNTs (Nanolab, Newton, MA, USA) were covalently amine functionalized as described previously.3
product was purified from carbonaceous impurities using a C18 Sep-Pak®
(Waters, Milford, MA, USA) and analyzed by high-performance liquid chromatography (HPLC), Raman spectroscopy, and transmission electron microscopy (TEM).3
Amine loading was determined using the Sarin assay, whereas TEM and Raman spectroscopy were performed as described previously.3
provides a list of the key drug constructs, the corresponding nomenclature, studies performed, and the SA values for the therapeutic and imaging studies from this work.
Construct nomenclature, designation, study use, and specific activity
The RIT drug Construct I () was designed to specifically target the tumor vessels and deliver the potent alpha particle-emitting 225
Ac radionuclide generator. 13
The key SWCNT precursor to Construct I was assembled by first converting a fraction of the primary amines on the SWCNT-NH2
construct to reactive hydrazinopyridine (HNH) moieties. Briefly, 0.5 g of SWCNTNH 2
was dissolved in 1 mL of 100 mM sodium phosphate (NaH2
; Sigma-Aldrich, St Louis, MO, USA)/150 mM sodium chloride (NaCl; Sigma-Aldrich), pH 7.8 buffer. Immediately before use, 5.5 mg of succinimidyl 4-hydrazinonicotinate acetone hydrazone (SANH; Solulink Inc, San Diego, CA, USA) was dissolved in 0.2 mL of dry N,N
-dimethylformamide (DMF; Sigma-Aldrich). An aliquot of SANH/DMF solution was added to the SWCNT-NH2
to achieve a 0.25-fold mole ratio of SANH to primary amine. The reaction proceeded at ambient temperature for 2–3 hours at pH 7.6. The product, SWCNT-(HNH)(NH2
), was purified using size exclusion chromatography (SEC) with a 10 DG gel permeation column (Bio-Rad Laboratories, Hercules, CA, USA) as the stationary phase and metal-free water (MFW; Purelab Plus System, US Filter Corp, Lowell, MA, USA) as the mobile phase. The product was lyophilized to yield a solid that was found to be the desired SWCNT-(HNH)(NH2
Figure 1 Graphical representations of the key moieties that were appended to the water-soluble SWCNT-NH2 by covalent-functionalization with radionuclides, DOTA, DFO, and antibodies (Note, not drawn to scale). A) Radioimmunotherapeutic drug Construct I (SWCNT-([ (more ...)
The second step entailed covalently appending multiple copies of 2-(p
-isothiocyanatobenzyl)-DOTA (DOTA-NCS; Macrocyclics, Inc, Dallas, TX, USA) to the remaining amines on the water-soluble SWCNT-(HNH)(NH2
) construct to yield a SWCNT-(DOTA)(HNH) construct in metal-free conditions at pH 9.5 (adjusted with 1 M metal-free carbonate solution) for 40 minutes at room temperature at a stoichiometry of 10:1 (DOTA-NCS to amine).3
The product was purified using a 10 DG gel permeation column with MFW as the mobile phase. The 10 DG column was rendered metal free by washing with 50 mL of 25 mM EDTA (Sigma-Aldrich) followed by rinsing with 250 mL of MFW. The product was lyophilized to yield a solid that was found to be the desired SWCNT-(DOTA)(HNH) construct.
Ac radionuclide was obtained from the US Department of Energy’s Oak Ridge National Laboratory (Oak Ridge, TN, USA). Methods for radiolabeling and purification of a MFW solution of SWCNT-(DOTA)(HNH) with 225
Ac at pH 5 are similar to those described previously.48
Briefly, 0.18 mg of SWCNT-(DOTA)(HNH) in 0.02 mL of MFW was reacted with 0.005 mL of 225
Ac in 50 mM optima grade hydrochloric acid (HCl; Fisher Scientific, Pittsburgh, PA, USA) along with 0.02 mL of 150 g/L l
-ascorbic acid (Sigma-Aldrich) and 0.2 mL of 3 M tetramethylammonium acetate (Fisher Scientific) buffer, pH 5.5, at 60°C for 60 minutes (reaction 1). Purification was accomplished using SEC with a P6 gel stationary phase and a phosphate buffer saline (PBS) mobile phase. Chemicals used in the radiolabeling and purification steps were of American Chemical Society reagent-grade or higher purity. The labeling solutions were prepared and subsequently rendered metal free with Chelex®
100 resin, 200–400 mesh, sodium form (Bio-Rad Laboratories), and sterile filtered through a 0.22 or 0.45 μm filter device. Solutions of 50 mM diethylenetriaminepentaacetic acid (DTPA; Sigma-Aldrich) were sterile filtered and used to quench the labeling reaction prior to SEC. Human serum albumin (HSA; Swiss Red Cross, Bern, Switzerland) and 0.9% NaCl (Abbott Laboratories, North Chicago, IL, USA) were used as received.225
Ac activity was measured with a Squibb CRC-17 Radioisotope Calibrator (or equivalent model; E.R. Squibb and Sons, Inc, Princeton, NJ, USA) set at 775 and multiplying the displayed activity value by 5 to report the activity.
Instant thin layer chromatography using silica gel impregnated paper (ITLC-SG; Gelman Science Inc, Ann Arbor, MI, USA) was used to determine the labeling efficiency of the reaction mixture and the purity of the product. Briefly, a 0.001 mL aliquot was spotted onto the paper strips and developed using 2 different mobile phases.13
Mobile phase 1 was 10 mM EDTA and 2 was 9% NaCl/10 mM sodium hydroxide (NaOH; Sigma- Aldrich). The Rf
of the radiolabeled construct was 0 and any free metal species and metal chelates were characterized by Rf
of 1.0 in mobile phase 1. In mobile phase 2, the radiolabeled construct and free metal species were characterized by Rf
of 0 and the metal chelates by Rf
of 1.0. The strips were counted intact using a System 400 Imaging Scanner (Bioscan Inc, Washington, USA).
The next key step (reaction 2) was the conversion of the VE-cad-specific IgG (E4G10; Imclone Systems, New York, NY, USA) or the isotype control anti-keyhole limpet hemocyanin (anti-KLH) IgG (R&D Systems, Minneapolis, MN, USA) to the reactive arylaldehyde modified–IgG precursors. Briefly, immediately before use, 15 mg of succinimidyl 4-formylbenzoate (SFB; Solulink Inc) was dissolved in 0.5 mL DMF. An aliquot of this modification solution was added to 2.5 mg of E4G10 protein (5 g/L; or the isotype control IgG) to achieve a 10–20 fold molar excess of the reagent. The reaction mixture was incubated at 37°C for 2–3 hours. Purification of the arylaldehyde modified–IgG was performed by SEC as described above. The modified proteins were stored at 4°C. The stoichiometry of substitution (moles of formylbenzoate [FB] per mole IgG) was determined first by assaying the protein concentration using the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA) and the moles of FB per mole IgG using the 2-HNH-dihydrochloride (2-HP; Solulink) quantification assay. Briefly, the addition of a molar excess 2-HP to the FB moiety on E4G10 at pH 4.7 permitted the measurement of the molar substitution ratio by electronic absorption spectroscopy using the arylhydrazone chromophore (maximum absorption [Absmax
] = 350 nm,
= 18,000 M−1
The E4G10-FB antibodies were covalently attached to the SWCNT-([225Ac]DOTA)(HNH) by the reaction of 0.05 mg of SWCNT-([225Ac]DOTA)(HNH) with 0.17 mg of E4G10-FB in 1.2 mL of 100 mM sodium phosphate/150 mM NaCl, pH 5.2 buffer (reaction 3). A similar chemical scheme was used to append several of the anti-KLH-FB moieties to the SWCNT-([225Ac]DOTA)(HNH) precursor to yield the control Construct I. The reaction mixture was incubated at 37°C for several hours, and the high and low SA SWCNT-([225Ac] DOTA)(E4G10) (Construct I) and isotype control Construct I products were stored at 4°C. The products were formulated into 1% HSA for injection.
The radioimmunoimaging (RII) drug Construct II () was designed to specifically target the tumor vessels and deliver the positron-emitting 89Zr radionuclide for PET imaging. The key precursor to Construct II was assembled first by appending multiple copies of the reactive arylaldehyde, SFB. Briefly, the SFB was dissolved in 0.1 mL DMF and a volume of this modification solution was added to 0.5 mg of SWCNT-NH2 (3 g/L) to achieve a ratio of 5 SFB per 100 amines. The reaction mixture was incubated at 37°C for 2–3 hours. Purification of the arylaldehyde modified– SWCNT was performed by SEC as described above. The stoichiometry of substitution was determined (moles of FB per gram SWCNT) using the 2-HP quantification assay as described previously. Then, the remaining amines on the SWCNT-(FB)(NH2) construct were covalently modified by appending the desferrioxamine B (DFO) chelate. The details of preparation of the reactive DFO intermediate reagent are described below.
The synthesis of N-succinylDFO (N-succDFO) was performed by the reaction of DFO mesylate (0.508 g, 0.77 mmol; Calbiochem, Spring Valley, CA, USA) dissolved in 7.5 mL of pyridine (Sigma-Aldrich) with excess (1.704 g, 0.017 mol) succinic anhydride (Sigma-Aldrich) at room temperature for 24 hours. The resulting white suspension was then poured into an aqueous NaOH solution (120 mL, 0.015 M) and stirred at room temperature for 16 hours. The colorless solution was adjusted to pH 2 by the addition of 12 M HCl and cooled with stirring at 4°C for 2 hours. The white precipitate was collected by filtration, washed with copious amounts of 0.01 M HCl and then water, and dried in a vacuum to give the N-succDFO as a white microcrystalline solid (0.306 g, 4.75 × 10−4 mol).
The preparation of ferric DFO-2,3,5,6-tetrafluorophenol (Fe[DFO-TFP]) was performed by reacting the activated ester N
-succDFO (9.0 mg, 0.014 mmol), suspended in 3 mL of 0.9% sterile saline and the pH adjusted to 6.5, with 0.05–0.075 mL of 0.1 M sodium carbonate (Na2
; Sigma-Aldrich). A solution of ferric trichloride hexahydrate (FeCl3
O [4 mg, 0.015 mmol, 0.3 mL of 0.1 M HCl]; Sigma-Aldrich) – was added to this N
-succDFO solution. Upon addition of the FeCl3
, the reaction mixture changed from colorless to deep orange due to the intense electronic absorption band of Fe(DFO) with a peak at 430 nm (430
= 2,216 ± 49 M−1
). After stirring the reaction mixture at room temperature for 1 hour, a 1.2 M solution of TFP (0.3 mL, 0.036 mmol; Sigma-Aldrich) in Chelex-purified acetonitrile (MeCN; Sigma-Aldrich) was added to the reaction followed by the addition of solid N
-ethylcarbodiimide hydrochloride (0120 mg, 0.63 mmol; Sigma Aldrich). The reaction mixture (pH 6.5) was then stirred at room temperature for 1 hour before purifying the Fe(DFO-TFP) product using a C18 Light Sep-Pak cartridge (Waters). The reaction mixture was loaded onto a preactivated (6 mL MeCN and 10 mL H2
O) C18 cartridge, washed with copious amounts of water (>40 mL), and eluted with 1.5 mL MeCN. The final Fe(DFO-TFP) solution had a concentration of ~9.8 mM. The Fe(DFO-TFP) solution was stored at 4ºC.
The Fe(DFO-TFP) reagent was then reacted with the remaining amines on the SWCNT-(FB)(NH2) construct to introduce the DFO chelate onto the SWCNT precursor. The Fe was removed by exposing the metallated precursor to a 10-fold excess of EDTA (0.0674 M, 0.0137 mmol, 0.03 mL) with respect to Fe(N-succDFO-TFP). The reaction was incubated in a water bath at 38°C for 1 hour. The SWCNT-(DFO)(FB) was purified by SEC chromatography to render it Fe free and was ready to be radiolabeled (reaction 4).
Zr was produced via the 89
Zr transmutation reaction on an EBCO TR19/9 variable-beam-energy cyclotron (Ebco Industries Inc, Richmond, BC, Canada) in accordance with previously reported methods.30
Zr-oxalate was isolated in high radionuclidic and radiochemical purity (RCP) >99.9%, with an effective SA of 195–497 TBq/g, (5,280–13,430 Ci/g).30
Methods for radiolabeling and purification of a 10 g/L solution of SWCNT-(DFO)(FB) in MFW with 89
Zr at pH 5 are similar to those described previously.30
Briefly, 0.1 mg of SWCNT-(DFO)(FB) in 0.02 mL of MFW was reacted with 122.5 MBq (3.31 mCi) of 89
Zr in 0.005 mL of 1 M oxalic acid (Sigma-Aldrich), pH 6.5. The pH was adjusted to 8.1 with the addition of 0.17 mL of 1.0 M Na2
. The reaction was heated to 60°C for 60 minutes. Purification was accomplished using SEC with a P6 gel stationary phase and a PBS mobile phase.89
Zr activity was measured with a Squibb CRC-17 Radioisotope Calibrator (or equivalent model) set at 465. ITLC-SG was used to determine the labeling efficiency of the reaction mixture and the purity of the product. The strips were counted intact using a System 400 Imaging Scanner (or equivalent).
The next key step was the conversion of the E4G10 or the isotype control anti-KLH IgGs to the reactive arylhydrazine modified–IgG precursors (reaction 5). Briefly, 1 mg of IgG was dissolved in 0.2 mL of 100 mM sodium phosphate/150 mM NaCl, pH 7.6 buffer. Immediately before use, 2–4 mg of SANH was dissolve in 0.1 mL of dry DMF. A volume of SANH/DMF solution was added to the IgG to achieve a 10–20 fold molar excess of the SANH to antibody. The reaction proceeded at ambient temperature for 2–3 hours at pH 7.6. The product, IgGHNH, was purified using SEC with a 10 DG gel column as the stationary phase and 100 mM 2-(N
-morpholino) ethanesulfonic acid (MES; Sigma-Aldrich)/150 mM NaCl conjugation buffer at pH 5.4 as the mobile phase. The amount of HNH substituent per IgG was determined first by assaying the protein concentration using the BCA protein assay and the moles of arylhydrazine (HNH) per mole IgG using the 4-nitrobenzaldehyde (4-NBA; Solulink) quantification assay. Addition of a molar excess 4-NBA to the HNH moiety on IgG at pH 4.7 permitted the measurement of the molar substitution ratio of the chromophore (Absmax
= 390 nm,
= 24,000 M−1
The E4G10-HNH antibodies (or anti-KLH-HNH) were covalently attached to the SWCNT-([89Zr]DFO)(FB) by the reaction of 0.1 mg of SWCNT-([89Zr]DFO)(FB) with 0.3 mg of E4G10-HNH in 0.25 mL of 100 mM MES/150 mM NaCl conjugation buffer at pH 4.7 (reaction 6). The reaction mixture was incubated at 37°C for several hours. The SWCNT-([89Zr] DFO)(E4G10) (Construct II) and isotype control Construct II products were stored at 4°C and were formulated into 1% HSA for injection.
Amplification of construct specific activity
A fixed mass of SWCNT-(DOTA)(HNH) precursor was radiolabeled with varying amounts of 225Ac activity to determine the reaction yields and specific activities. In each of 5 radiolabeling reactions, the volume, pH, time, temperature, and reagent concentrations were held constant (see specific conditions described above), while only the amount of radionuclide was varied. Briefly, 0.18 mg of SWCNT-(DOTA)(HNH) was 225Ac-radiolabeled in 0.4 mL, at pH 5.5, at 60°C for 60 minutes in 5 different reactions. In amplification reaction 1, the SWCNT-(DOTA) (HNH) was labeled with 0.444 MBq (0.012 mCi); 2, 1.48 MBq (0.04 mCi) was used; 3, 2.26 MBq (0.061 mCi) was used; 4, 21.1 MBq (0.57 mCi) was used; and 5, 193 MBq (5.21 mCi) was used. An aliquot of each reaction was assayed using ITLC-SG (see above), and then, the reaction was quenched with the addition of DTPA. The reaction mixture was then purified by SEC (see above) and the purified product assayed by ITLC-SG and the recovered activity measured.
Data for numerous preclinical radiolabeling preparations of 225
Ac-E4G10 were also compiled for comparison. The radiolabeling data using our published methods48
from 11-dose preparations that used 0.75 ± 0.13 mg (mean ± standard deviation) of E4G10 and 93.6 ± 51.1 MBq (2.53 ± 1.38 mCi) of 225
Ac per dose were used as comparison to the results from the SWCNT-(DOTA)(HNH) labeling study.
As a further demonstration of the consistency of our published 2-step IgG radiolabeling methodology, lintuzumab (Protein Design Labs, Inc, Mountain View, CA, USA), a monoclonal IgG that targets CD33 on leukemia cells, was routinely radiolabeled with 225Ac for a Phase I clinical trial to produce 225Ac-lintuzumab. The radiolabeling data from 17 clinical dose preparations that used 1.4 ± 0.5 mg (mean ± standard deviation) of lintuzumab and 91 ± 55.1 MBq (2.46 ± 1.49 mCi) of 225Ac were also included as comparison to the results from the SWCNT-(DOTA)(HNH) labeling study.
Three-dimensional fluorescent-mediated tomography imaging study to assess the PK of E4G10 and determine the number of VE-cad monomer epitopes per cell in vivo
Three-dimensional fluorescent-mediated tomography (FMT) experiments were performed by using the FMT-2500 (VisEn Medical, Boston, MA, USA) to determine the PK profile of the of E4G10 (and anti-KLH isotype control) IgGs and the number of binding sites per newly formed vascular endothelial cell in the LS174T xenograft model.
The E4G10 and anti-KLH antibodies were reacted with the succinimidyl ester of Alexa Fluor® 680 carboxylic acid (AF680; Invitrogen, Carlsbad, CA, USA) per the manufacturer’s instructions to prepare 2 antibody constructs for an in vivo near-infrared (NIR) FMT imaging study. Briefly, the constructs were prepared by the reaction of a 10–20 fold mole excess of the succinimidyl ester of the AF680 dye per milligram of IgG at pH 8 for 2 hours at ambient temperature. The dye-labeled constructs were purified by SEC chromatography as described above and characterized by UV-visible spectroscopy (measured the absorbance at 280 and 679 nm per the manufacturer’s instructions) and SEC HPLC. The HPLC system used a Beckman Coulter System Gold Bioessential 125/168 diode array detection system (Beckman Coulter, Fullerton, CA, USA) equipped with an in-line Jasco FP-2020 fluorescence detector (Tokyo, Japan). The stationary phase was a Tosoh Science G3000SWXL column (300 mm × 7.8 mm, 5 μm; Fisher Scientific) and a 20 mM sodium acetate (Sigma-Aldrich), 150 mM NaCl, pH 6.4, mobile phase at 1 mL/min at ambient temperature.
Two groups of 5 nude mice with the LS174T tumor were randomly assembled, and each mouse received 0.03 mg of the construct in 0.1 mL in 1% HSA via intravenous (IV) retro-orbital sinus injection. NIR FMT imaging was performed every 24–48 hours over a 7-day time period by using the specific 680 channel (excitation/emission [Ex/Em]: 680 nm/700 nm). The volume of interest (VOI) was drawn over the whole tumor (as visualized by the 3-dimensional photographic image acquisition), and fluorescence uptake was quantified. Mice were maintained on a diet of low-fluorescence chow (AIN76A; Harlan Teklad, Wisconsin) to minimize background noise. The FMT device was calibrated for use with sample standards of the E4G10-AF680 and anti-KLH-AF680 constructs in accordance with the manufacturer’s guidelines. The values obtained from the measurements of these standards of the injected dose were entered into the TrueQuant software (VisEn Medical) to allow for quantification.
To determine the number of VE-cad epitopes in these tumors, we employed the data obtained from Hilmas and Gillette49
that reported a morphometric analyses of tumor microvasculature during growth. Their data described changes in the tumor vascular volume, vessel diameter, mean vessel length, and surface area per unit volume of tumor tissue. Further, it was assumed that a VE cell has an area50
of 1E-3 mm2
(0.141 mm × 0.007 mm) and that there were 1E9 cells per gram of tumor. The data of Hilmas and Gillette49
reported that a 500 mm3
tumor had a vascular surface area per tumor volume of 13 mm2
and as the tumor volume increased (up to 1,500 mm3
), the ratio of vascular surface area per tumor volume decreased and leveled at 12 mm2
RII study of tumor vasculature
A RII study was performed in the LS174T xenograft tumor model with SWCNT-([89Zr]DFO)(E4G10) vs appropriate controls. Briefly, tumor cells were xenografted 13 days before treatment (the mean ± standard deviation tumor volumes for the animals in this study were 558 ± 413 mm3 at the time RII commenced). Mice were randomly separated into 3 groups before treatment, and all mice received a single IV dose of drug via the lateral tail vein. All the SWCNT-([89Zr]DFO)(IgG) constructs were labeled to high SA (592 GBq/g SWCNT [16 Ci/g]). Group 1 mice (n = 4) received a single dose of Construct II containing 4.18 MBq (0.113 mCi) 89Zr, 7,000 ng SWCNT, and 15,700 ng E4G10. Group 2 mice (n = 3) received a single IV 0.8 mg dose of unlabeled E4G10 (50-fold excess relative to the construct-associated E4G10) 30 minutes before the single dose of Construct II containing 4.18 MBq 89Zr, 7,000 ng SWCNT, and 15,700 ng E4G10. This group served as a blocking control. Group 3 mice (n = 3) received a single dose of the isotype control Construct II containing 3.08 MBq (0.083 mCi) 89Zr, 5,200 ng SWCNT, and 12,100 ng anti-KLH.
The PET study was performed with a microPET FocusTM
120 (CTI Molecular Imaging, Knoxville, TN, USA). Mice were maintained under 2% isoflurane/oxygen anesthesia during the scanning. Images were recorded at various time points between 0–96 hours after injection. The list-mode data were acquired for between 10 and 30 minutes using a γ-ray energy window of 350–750 keV and a coincidence timing window of 6 ns. For all static images, scan time was adjusted to ensure a minimum of 20-million coincident events recorded. Data were sorted into 2-dimensional histograms by Fourier rebinning, and transverse images were reconstructed by filtered back-projection into a 128 × 128 × 63 (0.72 × 0.72 × 1.3 mm) matrix. The reconstructed spatial resolution for 89
Zr was 1.9 mm full width at half maximum at the center of the field of view. The image data were normalized to correct for nonuniformity of response of the PET, dead-time count losses, positron-branching ratio, and physical decay at the time of injection but no attenuation, scatter, or partial volume-averaging correction was applied. An empirically determined system calibration factor (in units of [mCi/mL]/[cps/voxel]) for mice was used to convert voxel count rates to activity concentrations. The resulting image data were then normalized to the administered activity to parameterize images in terms of %ID/g. Manually drawn 2-dimensional region of interest (ROI) or 3-dimensional VOI were used to determined the maximum and mean % ID/g (decay corrected to the time of injection) in various tissues.6
Images were analyzed by using ASIPro VM 5.0 software (Concorde Microsystems, Knoxville, TN, USA).