CR011 (Celldex Therapeutics) was conjugated via lysine residues to desferrioxamine-p-benzyl-isothiocyanate (Macrocyclics, Inc.) metal chelate (DFO-Bz-NCS) with minor modifications from previous studies [23 (link)]. The conjugation buffer used in the current study was PBS (pH 9) and the radiolabeling buffer was 10 mM sodium citrate buffer (pH 6.8) to reduce antibody aggregation. Zr-89 was produced in house with an effective specific activity of 145 – 656 mCi/μmol. The highest specific activity of [89Zr]DFO-CR011 was achieved at 370 MBq/mg. The ratio of DFO:CR011 was determined using a radioisotopic dilution assay as described previously [30 (link)], except that FeCl3 was used to compete with Zr-89. For cell-binding and animal studies, a specific activity of 185 MBq/mg was prepared to ensure ≥ 95% radiochemical yield. [89Zr]DFO-CR011 was purified by buffer exchanging with 2 mM sodium citrate in saline. Protein aggregation was determined via size exclusion chromatography (Superose 12 10/300 GL) with a flow rate of 0.8 mL/min of 0.1 M sodium phosphate (pH 6.5) containing 0.15 M NaCl buffer. An AKTA fast protein liquid chromatography (FPLC) equipped with radioactivity (Lablogic) and UV (A280nm) detectors was used.
Dfo bz ncs
Manufactured by Macrocyclics
Sourced in United States
DFO-Bz-NCS is a laboratory compound used as a precursor for the synthesis of radiolabeled compounds. It contains a desferrioxamine (DFO) moiety and a benzyl isothiocyanate (Bz-NCS) functional group. This compound can be utilized in the preparation of radiolabeled materials for various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Gentamycin, by Thermo Fisher Scientific (1 mentions)
Herceptin, by Roche (1 mentions)
Perjeta, by Roche (1 mentions)
Skbr3, by American Type Culture Collection (1 mentions)
Bt474, by American Type Culture Collection (1 mentions)
Sephadex g 25m column, by GE Healthcare (1 mentions)
Onartuzumab, by Genentech (1 mentions)
Chelex, by Bio-Rad (1 mentions)
Pd 10 desalting column, by GE Healthcare (1 mentions)
Perjeta, by Genentech (1 mentions)
13 protocols using dfo bz ncs
1
Radiolabeling of Anti-PD-L1 Antibody CR011
2
Zirconium-89 Labeled Trastuzumab Protocol
3
Radiolabeling of Antibody Conjugates
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Trastuzumab or TDM1 were obtained from the MSK Hospital Pharmacy. The pHrodo-TDM1 was obtained by conjugating the free lysine residues of TDM1 with the amine-reactive pH-sensitive pHrodo iFL Red STP ester dye (ThermoFisher Scientific, P36014) according to the manufacturer’s instructions.
To prepare [89Zr]Zr-DFO-antibody, TDM1 or Trastuzumab were first conjugated with the bifunctional chelate p-isothiocyanatobenzyl-desferrioxamine (DFO-Bz-NCS; Macrocyclics, Inc) and then labeled with zirconium-89 (89Zr)59 (link). Radiochemical purity (RCP) was determined by instant thin-layer chromatography. The radiolabeled conjugates used for in vitro and in vivo studies had a RCP of 99%, radiochemical yields ranging from 92 to 97%, specific activities in the range of 21.98–24.73 Mbq/nmol, and immunoreactivities above 90%.
To prepare [89Zr]Zr-DFO-antibody, TDM1 or Trastuzumab were first conjugated with the bifunctional chelate p-isothiocyanatobenzyl-desferrioxamine (DFO-Bz-NCS; Macrocyclics, Inc) and then labeled with zirconium-89 (89Zr)59 (link). Radiochemical purity (RCP) was determined by instant thin-layer chromatography. The radiolabeled conjugates used for in vitro and in vivo studies had a RCP of 99%, radiochemical yields ranging from 92 to 97%, specific activities in the range of 21.98–24.73 Mbq/nmol, and immunoreactivities above 90%.
4
Radiolabeling HER2-Expressing Breast Cancer Cells
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Pertuzumab (Perjeta) and
trastuzumab (Herceptin) were purchased from Roche (South San Francisco,
CA). Desferrioxamine-p-benzyl-isothiocyanate (DFO-Bz-NCS)
was purchased from Macrocyclics (Dallas, TX). The automated production
of 89Zr-oxalate was carried out in house,11 adapted from previously established methods12 (link),13 (link) and neutralized to pH 6.8–7.2 as previously developed for
optimal radiolabeling.14 (link) All other chemicals
were purchased from Sigma-Aldrich (St. Louis, MO) unless stated otherwise.
HER2–expressing breast cancer cell lines BT-474 and SKBR3 as
well as HER2–nonexpressing MDA-MB-231 were purchased from the
American Type Culture Collection (ATCC, Manassas, VA) and cultured
in Iscove’s Modified Dulbecco’s Medium (IMDM) containing
10% fetal bovine serum (FBS) and 50 μg/mL gentamycin (complete
media) in a humidified incubator with 5% CO2 at 37 °C.
Reagents for cell culture were purchased from Life Technologies (Grand
Island, NY) unless stated otherwise.
trastuzumab (Herceptin) were purchased from Roche (South San Francisco,
CA). Desferrioxamine-p-benzyl-isothiocyanate (DFO-Bz-NCS)
was purchased from Macrocyclics (Dallas, TX). The automated production
of 89Zr-oxalate was carried out in house,11 adapted from previously established methods12 (link),13 (link) and neutralized to pH 6.8–7.2 as previously developed for
optimal radiolabeling.14 (link) All other chemicals
were purchased from Sigma-Aldrich (St. Louis, MO) unless stated otherwise.
HER2–expressing breast cancer cell lines BT-474 and SKBR3 as
well as HER2–nonexpressing MDA-MB-231 were purchased from the
American Type Culture Collection (ATCC, Manassas, VA) and cultured
in Iscove’s Modified Dulbecco’s Medium (IMDM) containing
10% fetal bovine serum (FBS) and 50 μg/mL gentamycin (complete
media) in a humidified incubator with 5% CO2 at 37 °C.
Reagents for cell culture were purchased from Life Technologies (Grand
Island, NY) unless stated otherwise.
5
Radiolabeling of aTCRmu-F(ab')2 with 89Zr
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The aTCRmu-F(ab')2, was functionalized by conjugation with the p-isothiocanatobenzyl derivate of desferrioxamine (DFO-Bz-NCS, Macrocyclics Inc., Richardson, TX) for subsequent labeling with zirconium-89 (89Zr; t/2=3.3 days; Emax β+=0.9 MeV). A 3-fold molar excess of the chelator was added to 2-3 mg protein in a total volume of 500 µl followed by incubation at 37 °C for 30 minutes. Purification of the immuno-conjugate from the unbound chelator was performed by size exclusion chromatography (Sephadex G-25 M, PD10 column, cut off >30 kDa, GE Healthcare) according to the protocol described by Perk et al. 9 (link). The immunoconjugate concentration was determined by a Nanophotometer (Implem). The 89Zr-labeling of aTCRmu-F(ab')2 was performed based on the protocol of Vosjan et al. 10 (link) with slight modifications. Briefly, 37.0 to 74.0 MBq of 89Zr in 1 M oxalic acid (BV Cyclotron VU, The Netherland) was adjusted to pH 7.0-7.2 with 2 M sodium carbonate and 0.5 M HEPES (pH 7.0) followed by addition of 100 to 250 µg DFO-aTCRmu-F(ab')2 and 0.5 M HEPES pH 7.0. After incubation of the mixture for 30 min at 37 °C, the 89Zr-Df-immunocomplex was purified using 0.25 M sodium acetate/gentisic acid 5 mg/ml buffer solution (pH 5.5) by size exclusion chromatography (Sephadex G-25M column, GE Healthcare) and radiochemical purity (RCP) was assessed by ITLC and radio-HPLC.
6
Radiolabeling of Antibodies with Zirconium-89
7
Radiolabeling of Panitumumab with Zirconium-89
8
Radiolabeling of BI 754111 with 89Zr
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89Zr was purchased from Perkin-Elmer, Boston, MA, USA, and coupled to BI 754111 via the bifunctional chelator DFO-Bz-NCS (Macrocyclics) [33 (link)]. [89Zr]Zr-BI 754111 was produced in compliance with current Good Manufacturing Practice at the Amsterdam UMC, location VUmc. The procedures for radiolabeling of BI 754111 with 89Zr have been validated with respect to the final quality of the prepared conjugate and the production process. Details can be found in the supplementary information.
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89Zr was purchased from Perkin-Elmer, Boston, MA, USA, and coupled to BI 754111 via the bifunctional chelator DFO-Bz-NCS (Macrocyclics) [33 (link)]. [89Zr]Zr-BI 754111 was produced in compliance with current Good Manufacturing Practice at the Amsterdam UMC, location VUmc. The procedures for radiolabeling of BI 754111 with 89Zr have been validated with respect to the final quality of the prepared conjugate and the production process. Details can be found in the supplementary information.
9
Radiolabeling Antibodies with Zirconium-89
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We adhere to the nomenclature rules for radiopharmaceutical chemistry (30 (link)). Panitumumab, trastuzumab, and cetuximab were obtained from the Memorial Sloan Kettering hospital pharmacy. Onartuzumab was provided by Genentech. The antibodies were conjugated with the bifunctional chelate p-isothiocyanatobenzyl-desferrioxamine (DFO-Bz-NCS; Macrocyclics) and then radiolabeled with 89Zr in accordance with previously reported methods (28 (link)). The antibodies were conjugated with p-SCN-Bn-DFO in a 5:1 DFO:antibody molar ratio at 37°C for 90 min. After reaction, the conjugates were purified via a PD-10 column using Chelex (Bio-Rad) phosphate-buffered saline (0.5 g/L Chelex resin) at pH 7.4. The 89Zr-oxalate (supplied in 1.0 M oxalic acid at Memorial Sloan Kettering Cancer Center (28 (link))) was neutralized to pH 7.0–7.5 with 1.0 M Na2CO3 followed by addition of the corresponding DFO–antibody conjugate in Chelex phosphate-buffered saline (pH 7.4). The mixture was incubated at 37°C for 1 h on an agitating heating block. [89Zr]Zr-DFO-antibody and radiochemical purity was determined by instant thin-layer chromatography, and the product was used for in vivo studies.
10
Pertuzumab Antibody Conjugation for Imaging
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Clinical-grade pertuzumab (Perjeta; Genentech) was conjugated with p-isothiocyanatobenzyl-desferrioxamine (DFO-Bz-NCS) or transcyclooctene (TCO) as described previously (22 (link)). Briefly, a solution of pertuzumab (2.61 mg, 3.26 mg/mL) in phosphate-buffered saline (PBS), pH 7.4, was adjusted to pH 8.4 with 1 M NaHCO3 solution. Thirteen molar equivalents of the bifunctional chelate DFO-Bz-NCS (Macrocyclics, Inc, 10 mg/mL, 13.3 mM) in dimethyl sulfoxide were added. For conjugation with TCO, pertuzumab (3.40 mg, 3.40 mg/mL) in PBS (pH 7.4) was adjusted to pH 8.5 using 0.1 M Na2CO3. Pertuzumab was then reacted with 30 molar equivalents of TCO-N-hydroxysuccinimide (25.0 mg/mL, 94 mM prepared in N,N-dimethylformamide). The DFO or TCO conjugation reactions were prepared fresh before use by incubating the antibody with DFO-Bz-NCS or TCO-N-hydroxysuccinimide at 37°C for 90 min before purification with a PD10 desalting column (GE Healthcare). Antibody–DFO conjugate was used for radiolabeling with 89Zr, and antibody–TCO conjugates were used for pretargeted strategies.
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