Supporting Information MANOTA: A PROMISING BIFUNCTIONAL CHELATING AGENT FOR COPPER-64 IMMUNOPET

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1 Electronic Supplementary Material (ESI) for Dalton Transactions. This journal is The Royal Society of Chemistry 2017 Supporting Information MANOTA: A PROMISING BIFUNCTIONAL CHELATING AGENT FOR COPPER-64 IMMUNOPET Mathieu Moreau, a Sophie Poty, a Jean-Marc Vrigneaud, b Paul Walker, c Mélanie Guillemin, b Olivier Raguin, d Alexandra Oudot, b Claire Bernhard, a Christine Goze, a Frédéric Boschetti, e Bertrand Collin, a,b François Brunotte, b,c and Franck Denat a a ICMUB, UMR CNRS 6302, Univ. Bourgogne Franche-Comté, Dijon Cedex, France b Service de médecine nucléaire, centre Georges-François Leclerc, Dijon, France c Le2i, UMR CNRS 6306, Univ. Bourgogne Franche-Comté, Dijon Cedex, France d Oncodesign, Dijon Cedex, France e Chematech, Dijon, France. *Corresponding Author: Institut de Chimie Moléculaire de l Université de Bourgogne, UMR CNRS 6302, Univ. Bourgogne Franche-Comté, 9 avenue Alain Savary, BP 47870, Dijon Cedex, France Phone: +33 (0) ; franck.denat@u-bourgogne.fr

2 Figure S1. General synthetic route from Fab fragmentation to radiolabelling

3 Figure S2. Preparation and characterization of Fab-trastuzumab fragment. (A) Purification process of Fab-trastuzumab. After a first purification by ion exchange chromatography (IEX), the pure Fab was obtained after gel filtration (GF). Collected fraction is indicated by red markers. (B) Purity was assessed by SDS-PAGE and confirmed by LC-ESI- MS (C). Deconvoluted mass spectrum of Fab-trastuzumab, the inset shows the raw mass spectrum of the analyte.

4 100 % incorporated * t (h) 64 Cu-DOTA-Fab-trastuzumab 64 Cu-DOTAGA-Fab-trastuzumab 64 Cu-NODAGA-Fab-trastuzumab 64 Cu-MANOTA-Fab-trastuzumab Figure S3. Stability test at room temperature. Stability of the four 64 Cu-BFC-Fabtrastuzumab in competition with 2000 equivalents of EDTA at room temperature over 48 h (mean ± SD, n = 2) determined by ITLC-SG. * indicates significant difference (P < 0.05, Newman-Keuls correction) compared with the other chelates.

5 Figure S4. Representative ITLC radiochromatogram of Stability test at 37 C after 0, 24 and 48 h of incubation 64 CuCl 2 p-ncs-bz-dota-fab-trastuzumab p-ncs-bz-dotaga-fab-trastuzumab

6 p-ncs-bz-nodaga-fab-trastuzumab p-ncs-bz-manota-fab-trastuzumab

7 Figure S5. Representative ITLC radiochromatogram of Plasma stability at 37 C after 0, 24 and 48 h of incubation 64 CuCl 2 p-ncs-bz-dota-fab-trastuzumab p-ncs-bz-dotaga-fab-trastuzumab

8 p-ncs-bz-nodaga-fab-trastuzumab p-ncs-bz-manota-fab-trastuzumab

9 Figure S6. Immunoreactivity of Fab-trastuzumab conjugates. Determination of the immunoreactivity of (A) 64 Cu-DOTA-Fab-trastuzumab, (B) 64 Cu-DOTAGA-Fab-trastuzumab, (C) 64 Cu-NODAGA-Fab-trastuzumab and (D) 64 Cu-MANOTA-Fab-trastuzumab ( : total binding, : non specific binding). Trace amounts of 64 Cu-BFC-Fab-trastuzumab were incubated with increasing concentration of HCC1954 tumour cells. Non-specific binding was evaluated with excess unlabelled trastuzumab. The fraction of radioactivity bound to cells (B/T%) was counted. The means of duplicate samples are plotted ± standard deviation (unless smaller than the point as plotted).

10 Figure S7. Affinity of Fab-trastuzumab conjugates. Binding curve obtained in saturation assays for (A) 64 Cu-DOTA-Fab-trastuzumab, (B) 64 Cu-DOTAGA-Fab-trastuzumab, (C) 64 Cu- NODAGA-Fab-trastuzumab and (D) 64 Cu-MANOTA-Fab-trastuzumab in order to determine binding dissociation constant (, total binding;, nonspecific binding; solid line, curvefitting). Briefly, HCC1954 tumour cells were incubated with increasing concentration of the radiolabelled conjugate. Nonspecific binding was evaluated with excess unlabelled trastuzumab. The radioactivity associated to cells (expressed in counts per minute, cpm) was counted. The means of duplicate samples are plotted ± standard deviation (unless smaller than the point as plotted).

11 Figure S8. PET-MR images 4h post-injection. PET-MR images (PET maximum intensity projection and representative coronal MR slice) of Balb/C nude mice bearing BT-474 xenografts at 4 h postinjection with 64 Cu-BFC-Fab-trastuzumab (upper row) and 64 Cu-BFC- Fab-rituximab (lower row) conjugated with DOTA (A), DOTAGA (B), NODAGA (C) and MANOTA (D). Tumour location is indicated by a white arrow and liver is indicated by a red arrow.

12 Table S1. 24 h ex vivo biodistribution data and results of statistical analysis a Fab-trastuzumab Fab-rituximab 64 CuCl 2 DOTA DOTAGA NODAGA MANOTA DOTA DOTAGA NODAGA MANOTA tissue Blood (0.55) (0.08) (0.14) (0.02) (0.30) (0.16) (0.19) (0.01) Heart Lung Kidneys 2.31 (0.66) 4.05 (0.40) (27.12) , 2, , 6, (0.29) 1.79 (0.28) 0.73 (0.06) 3.31 (0.60) 2.87 (0.42) 2.26 (0.34) 1.31 (0.11) , 2, , (0.27) (10.94) 5.30 (4.41) (12.39) 0.93 (0.05) 5.06 (0.63) 4.13 (0.57) 4.18 (0.56) 1.73 (1.14) , 6, (9.76) (26.23) (11.82) (9.61) (2.62) 3.28 (1.01) 4.56 (1.75) (2.50) 9.50 (0.92) Spleen Liver Intestines Carcass Tumour 4.61 (1.24) 9.81 (1.59) 4.12 (0.59) 1.71 (0.29) 3.90 (0.67) 2.92 (0.37) 1.93 (0.37) (2.28) 6.41 (1.05) 5.35 (0.62) 4.12 (0.28) 4 4 1, 2 6, 7, (0.51) 8.39 (1.26) 3.16 (0.48) (2.38) (0.79) (2.17) 5.47 (0.34) , 2, 3 6, 7, 8 5, 8 5, 8 5, 6, (0.42) 3.95 (0.73) 1.20 (0.12) 5.83 (0.97) 5.05 ( (0.94) 1.42 (0.08) , 2, , 6, (0.11) 1.25 (0.08) 0.82 (0.07) 2.84 (0.55) 1.81 (0.23) 1.62 (0.33) 1.16 (0.08) 2, 3, 4 1, 4 1, 4 1, 2, 3 6, 7, (2.53) (4.40) 8.75 (1.42) (2.31) 4.93 (1.14) 4.94 (0.14) 2.85 (0.61) 2.09 (0.31) 7, 8 7, 8 5, 6 5, (2.76) (4.17) 7.98 (1.88) 2.23 (0.44) 5.39 (0.54) n a Biodistribution data is given as % ID/g mean, with SD in parentheses. Statistical analysis was performed by a one-way ANOVA. Numbers in italic font indicate significant difference (P < 0.05, Newman-Keuls correction) compared with the indicated chelate. n indicates the group size.