UPSNs were surface modified with radioisotope chelators (CHX); 2-S-(4-Isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA; Macrocyclics Inc, Dallas, TX) or deferroxamine (p-SCN-Bn-DFO, Macrocyclics Inc, Dallas, TX) or fluorescent dye, fluorescein isothiocyanate (FITC) for further in vitro and in vivo studies, following procedures described previously.[17 (link)] Briefly, aqueous solution of UPSN-NH2 was reacted with CHX-SCN or FITC-SCN in a 1:10 molar ratio. The pH of the solution was tuned to 8–9 and the reaction was carried out at room temperature with vigorous stirring for 2 h. As-synthesized UPSN-CHX or UPSN-FITC were purified on PD-10 columns (GE Healthcare) with PBS as the mobile phase to remove unreacted molecules.
P scn bn dfo
Manufactured by Macrocyclics
Sourced in United States
P-SCN-Bn-DFO is a bifunctional chelating agent used for the conjugation of biomolecules to radionuclides. It contains a p-isothiocyanatobenzyl group for covalent coupling to biomolecules and a desferrioxamine (DFO) group for chelation of radiometals.
Automatically generated - may contain errors
Lab products found in correlation
Pd 10 column, by GE Healthcare (1 mentions)
P scn bn nota, by Macrocyclics (1 mentions)
P scn bn dota, by Macrocyclics (1 mentions)
Glass microfiber chromatography paper, by Agilent Technologies (1 mentions)
Ph indicator paper, by Merck Group (1 mentions)
Barnstead nanopure purification system, by Thermo Fisher Scientific (1 mentions)
Crc 25r dose calibrator, by Mirion Technologies (1 mentions)
Nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
P scn bn dtpa, by Macrocyclics (1 mentions)
Anti pd1 clone rpm1 14, by BioXCell (1 mentions)
12 protocols using p scn bn dfo
1
Surface Modification of UPSNs
2
Demetallation and Conjugation of αGPC3 Antibody
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To demetallate the αGPC3 antibody for labeling with radiometals, it was concentrated to 6 mg/mL and then dialyzed against metal-free saline (150 mM NaCl, and 1 mM EDTA adjusted to pH 7 and passed over a Chelex 100 column) for 3 days at 4 °C with a minimum of three buffer changes a day. The day before the conjugation reaction, the antibody was dialyzed for an additional two buffer changes to replace the saline with demetallated HEPES buffer (50 mM HEPES (N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid, 150 mM NaCl, and 1 mM EDTA adjusted to pH 8.5 and passed over a Chelex 100 column). In acid washed microcentrifuge tubes, the demetallated αGPC3 antibody was conjugated with 10 equivalents of either p-SCN-Bn-DFO or p-SCN-Bn-DOTA (both from Macrocyclics) as a solution at 10 mg/mL in DMSO. These reactions were allowed to run overnight at room temperature with gentle mixing. The reaction mixtures were then dialyzed against a metal-free citrate buffer (50 mM sodium citrate and 150 mM NaCl with pH 5.5) over 3 days at 4 °C followed by dialysis against 150 mM saline (pH 7.0) for another 3 days. Each buffer change contained Chelex resin to scavenge metals. The final conjugates were then stored in acid washed tubes at 4 °C.
3
Synthesis of PODS-Fe(III) Complex
4
Radiolabeling Reagents Characterization
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All reagents were purchased from Sigma-Aldrich unless otherwise stated and were used without further purification. The chelating agents p-SCN-Bn-DFO and p-SCN-Bn-DTPA were purchased from Macrocyclics Inc. (Dallas, TX). Water was deionised using a Barnstead NANOpure purification system (Thermo Scientific) and had a resistance of >18.2 MΩ cm−1 at 25 °C. Protein concentration measurements were made on a ND-1000 spectrophotometer (NanoDrop Technologies, Inc.). Instant thin-layer chromatography (iTLC) was performed on glass microfiber chromatography paper (Agilent Technologies) and strips were analysed with a Bioscan AR-2000 radio-TLC scanner (Eckert & Ziegler). pH was determined using pH indicator paper (Merck Millipore). Radioactivity measurements were made using a CRC-25R dose calibrator (Capintec, Inc.).
5
Radiolabeling of huA33 Antibody
6
Preparation of DFO-Trastuzumab-BOD665 Conjugates
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A 2 mg/mL p-SCN-Bn-DFO (1-(4-isothiocyanatophenyl)-3-[6,17-dihydroxy-7,10,18,21-tetraoxo-27-(N-acetylhydroxylamino)-6,11,17,22-tetraazaheptaeicosine]thiourea)
(Macrocyclics) stock solution in DMSO was prepared, and a 10-fold
molar excess was added to 100 μL of 5 mg/mL trastuzumab-BOD665
in 0.1 M NaHCO3 (pH 8.9–9.0). The reaction mixture
was covered to protect it from light and incubated at 37 °C for
1 h at 450 rpm. The resulting DFO-trastuzumab-BOD665 conjugates were
purified using centrifugal filters as per the previously described
method, and the buffer was exchanged to PBS (pH 7.2).
(Macrocyclics) stock solution in DMSO was prepared, and a 10-fold
molar excess was added to 100 μL of 5 mg/mL trastuzumab-BOD665
in 0.1 M NaHCO3 (pH 8.9–9.0). The reaction mixture
was covered to protect it from light and incubated at 37 °C for
1 h at 450 rpm. The resulting DFO-trastuzumab-BOD665 conjugates were
purified using centrifugal filters as per the previously described
method, and the buffer was exchanged to PBS (pH 7.2).
7
In Vivo Immune Checkpoint Blockade
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InVivoMAb anti-mouse CD69 (clone CD69.2.2), anti–CTLA-4 (clone 9H10), and anti–PD-1 (clone RPM1–14) were purchased from BioXCell. The deferoxamine (DFO) bifunctional chelator (p-SCN-Bn-DFO) was purchased from Macrocyclics. Zirconium-89 (89Zr) was purchased from the Washington University (St. Louis, MO).
8
Zirconium-89 Labeling of Her-TAT Antibody
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A 10-fold molar excess of p-SCN–Bn–deferoxamine (p-SCN–Bn–DFO, Macrocyclics) from a 2 mg mL−1 stock solution in DMSO was added to Her–TAT(0–5) (500 μg) in 0.1 M NaHCO3 (pH 8.5, 100 μL). The reaction mixture was incubated at 37 °C for 1 h with gentle shaking (450 rpm). The DFO-modified antibody conjugates were then purified by centrifugal filtration as previously described and adjusted to 2 mg mL−1 with PBS (pH 7.2) in preparation for radiolabelling with zirconium-89 (89Zr).
9
Zirconium-89 Chelation Protocol
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The heterobifunctional chelate, p-SCN-Bn-DFO, was from Macrocyclics (Dallas, TX). Reagents and chemicals were from Sigma-Aldrich (St. Louis, MO) unless otherwise stated. Zirconium-89 was from the Mallinckrodt Institute of Radiology (Washington University Medical School, St. Louis, MO).
10
Radiolabeling α-CD133 for Cancer Imaging
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All reagents
were purchased from Fisher Scientific
(Thermo Fisher Scientific; Waltham, MA, USA) unless otherwise noted.
αCD133 was provided by the Paul Lampe Laboratory at the Fred
Hutchinson Cancer Center. Protein concentrations were determined via
UV–vis spectroscopy using a molar absorptivity at 280 nm of
2.1 × 105 M–1 cm–1 and a molecular weight of 1.5 × 105. All the water
used was ultrapure (>18.2 MΩ·cm at 25 °C). p-SCN-Bn-DFO and p-SCN-Bn-CHX-A″-DTPA
were purchased from Macrocyclics, Inc. (Plano, TX, USA). MALDI mass
spectrometry was performed by the Alberta Proteomics and Mass Spectrometry
Facility (University of Alberta; Edmonton, AB, Canada). 89Zr was provided by 3D Imaging (Little Rock, AR, USA), and 177Lu was provided by ITM Radiopharma (Munich, Germany).
were purchased from Fisher Scientific
(Thermo Fisher Scientific; Waltham, MA, USA) unless otherwise noted.
αCD133 was provided by the Paul Lampe Laboratory at the Fred
Hutchinson Cancer Center. Protein concentrations were determined via
UV–vis spectroscopy using a molar absorptivity at 280 nm of
2.1 × 105 M–1 cm–1 and a molecular weight of 1.5 × 105. All the water
used was ultrapure (>18.2 MΩ·cm at 25 °C). p-SCN-Bn-DFO and p-SCN-Bn-CHX-A″-DTPA
were purchased from Macrocyclics, Inc. (Plano, TX, USA). MALDI mass
spectrometry was performed by the Alberta Proteomics and Mass Spectrometry
Facility (University of Alberta; Edmonton, AB, Canada). 89Zr was provided by 3D Imaging (Little Rock, AR, USA), and 177Lu was provided by ITM Radiopharma (Munich, Germany).
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