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Antibody conjugation with trans- Cyclooctene and Desferoxamine to - - PowerPoint PPT Presentation
Antibody conjugation with trans- Cyclooctene and Desferoxamine to - - PowerPoint PPT Presentation
Antibody conjugation with trans- Cyclooctene and Desferoxamine to enable in vivo click reaction for pretargeting strategies Irene Feiner, Vanessa Gmez-Vallejo, Javier Calvo, Jordi Llop Irene Feiner ifeiner@cicbiomagune.es Radiochemistry and
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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
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Antibody labeling
- M. J. W. D. Vosjan, et al., Nat. Prot. 2010, 5, 739
- To follow
biodistribution of monoclonal Antibody (mAb)
- To know timepoint for
injection of second component
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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
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Antibody modification
DFO-NCS TCO-NHS
= =
- 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
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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
0.00 0.05 0.10 0.15
230 330 430 530 630
absorbance wavelength [nm]
UV/VIS photospectrometry
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Average ratio
average number of chelators per mAb Titration with spiked Zirconium-oxalate
- mAb was labeled with spiked Zr89-ox in different ratios of ‘cold Zr-ox’
- The different ratios of bound and free Zr89 were used to calculate the
average number of chelator per mAb
0.0 10000.0 20000.0 30000.0 40000.0 50000.0 60000.0 2 4 6 8 10 12 14 cpm fraction
Titration
0 eq 1 eq 2 eq 3 eq
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Reality
DFO-NCS TCO-NHS
- Using lysine residues to conjugate antibodies leads to a high heterogeneity
- f conjugates
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UPLC/ESI-TOF MS
Excess
- f chelator*
chelator/mAb (UPLC/ESI-TOF MS) 3 0 – 1 5 0 – 2 10 0 – 3 Excess
- f TCO*
TCO/mAb (UPLC/ESI-TOF MS) 20 0 – 3 30 0 – 3 40 0 – 5 50 0 – 6 200 0 – 12 Mass spectra:
- unmodified mAb
- chelator-functionalized mAb
- UPLC/ESI-TOF MS gave a good qualitative insight of the different species
present after conjugation, although quantitative data could not be
- btained due to poor separation of the different species.
10 * Molar excess in reaction mixture compared to mAb
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Hydrophobic Interaction Chromatography (HIC)
Mobile phase: A: 125 mM sodium phosphate + 2 M ammonium sulfate B: 125 mM sodium phosphate
mAb mAb-chelator
- Improvement of the chromatographic resolution is still ongoing but we
already see the great variety of conjugates for the chelator functionalized antibody
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Summary
- Using lysine residues to conjugate antibodies leads to a high heterogeneity
- f 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
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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
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