Antibody conjugation with trans- Cyclooctene and Desferoxamine to enable in vivo click reaction for pretargeting strategies Irene Feiner, Vanessa Gómez-Vallejo, Javier Calvo, Jordi Llop Irene Feiner ifeiner@cicbiomagune.es Radiochemistry and Nuclear Imaging Group CIC biomaGUNE ySMIN Madrid, 26/02/2018
Pretargeting 2
Pretargeting • Why pretargeting? – Antibody distribution is slow – Radiolabeled antibody leads to a high radiation dose – Functionalized Antibody enables in vivo click reaction – Second component, a small radiolabeled molecule, distributes fast – In vivo coupling to the antibody leads to the high selectivity of antibodies but the low radiation dose of small radiotracers • 2 critical aspects – Find the right time point to inject the second component – Determine the functionalization of antibodies to predict pretargeting efficacy 3
Antibody labeling • To follow biodistribution of monoclonal Antibody (mAb) • To know timepoint for injection of second component 4 M. J. W. D. Vosjan , et al., Nat. Prot. 2010 , 5, 739
The approach • conjugation through lysine residues with – trans -Cyclooctene (TCO) enable for click reaction – p-isothiocyanatobenzyl-desferrioxamine (DFO) enable radiolabeling • determination of the number of functionalities per monoclonal Antibody (mAb) paramount to predict pretargeting efficacy 5
Antibody modification = mAb (1.5 mg/mL) in 0.9 % NaCl, pH 8.7-9.1 Addition of 50 times excess of TCO-NHS (3mg/mL) and 10 times excess of DFO-NCS (3.7 mg/mL) in DMSO Incubation for 45 min at 37ºC Purification by spin filters (50 kDa) with sucrose/NaOAc = buffer TCO-NHS DFO-NCS 6
Average ratio average number of TCO moieties per mAb UV/VIS photospectrometry • Tetrazine-fluorophore (mTzCy3) was attached via click reaction to TCO • resulting spectra could be used to determine the concentrations of mTzCy3, which equals TCO and the concentration of mAb UV/VIS photospectrometry 0.15 absorbance 0.10 0.05 0.00 230 330 430 530 630 wavelength [nm] 7
Average ratio average number of chelators per mAb Titration with spiked Zirconium-oxalate • mAb was labeled with spiked Zr 89 - ox in different ratios of ‘cold Zr- ox’ The different ratios of bound and free Zr 89 were used to calculate the • average number of chelator per mAb 60000.0 Titration 50000.0 40000.0 0 eq cpm 30000.0 1 eq 2 eq 20000.0 3 eq 10000.0 0.0 0 2 4 6 8 10 12 14 fraction 8
Reality • Using lysine residues to conjugate antibodies leads to a high heterogeneity of conjugates TCO-NHS DFO-NCS 9
UPLC/ESI-TOF MS • UPLC/ESI-TOF MS gave a good qualitative insight of the different species present after conjugation, although quantitative data could not be obtained due to poor separation of the different species. Excess chelator/mAb of chelator* (UPLC/ESI-TOF MS) 3 0 – 1 5 0 – 2 10 0 – 3 Excess TCO/mAb of TCO* (UPLC/ESI-TOF MS) 20 0 – 3 30 0 – 3 Mass spectra: 40 0 – 5 unmodified mAb 50 0 – 6 chelator-functionalized mAb 200 0 – 12 * Molar excess in reaction mixture compared to mAb 10
Hydrophobic Interaction Chromatography (HIC) • Improvement of the chromatographic resolution is still ongoing but we already see the great variety of conjugates for the chelator functionalized antibody Mobile phase: A: 125 mM sodium phosphate + 2 M ammonium sulfate B: 125 mM sodium phosphate mAb mAb-chelator 11
Summary • Using lysine residues to conjugate antibodies leads to a high heterogeneity of conjugates • TCO and chelator are very small moieties (152 g/mol and 753 g/mol) compared to the high molecular weight of antibodies (150000 g/mol) which difficult characterization and analysis of the receiving conjugates • Average number of moieties could be obtained for both, TCO and chelator • UPLC/ESI-TOF MS as well as HIC gave insight of the verity of existing conjugates 12
Acknowledgements This project has received funding from the European Union’s Horizon 2020 research and innovationprogramme under the Marie Sklodowska – Curie grant agreement No 675417 13
Acknowledgements Radiochemistry and Nuclear Imaging Group 14
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