Imaging using Affibody molecules

Transcription

1 Imaging using Affibody molecules Structure and selection of Vladimir olmachev Biomedical Radiation Sciences, Uppsala University Protein A-domain scaffold Randomization of 13 selected positions 2x Affibody library members 8 amino acids (~ 7 kda) Nygren: FEBS J. 28: Löfblom: FEBS Lett. 2; Antibody kda Features of Affibody Molecule 6-7 kda High (picomolar) affinity Small size Robustness Both recombinant and synthetic production with sitespecific labelling argets for HER2 (.22 nm) rlova: Cancer Res. 26; EGFR (.9 nm) olmachev: EJNMMI. 2; IGF-1R (. nm) olmachev: J Nucl Med. 212; HER3 (.7 nm) ronqvist: PEDS. 2; PDGFR (.4 nm) Lindborg: J Mol Biol. 211; Imaging of these tyrosine kinase receptors can be used for predicting or monitoring responce to specific anti-cancer therapy Features of have been labelled for. Robustness: cysteine-independent folding; re-folding within 3 µs in physiological conditions. Permits the use of: extreme ph (3.-11.), high temperature (up to 9 o C), high concentration of a reductant, lipophilic solvents, high molarity buffers SPEC PE herapy c 18 F 131 I 111 In 76 Br 211 At I 12 I 9 Y 68 Ga 114m In 64 Cu 177 Lu 7() Co 186 Re

2 Site-specific labeling of synthetic Site-specific incorporation of chelators during peptide synthesis: DA, NA, NDAGA maggg, masss, maeee, maese [DA-A1] Z HER2:S1 [DA-] Z HER2:S1 [DA-8] Z HER2:S1 Well-defined conjugates with reproducible biodistribution umor-to-organ ratio 8 A1 bone Site-specific labeling of recombinant Site-specific coupling of chelators and prosthetic groups to an unique engineered thiol: maleimido-derivatives of DA, NA, NDAGA ; 18 F-FBEM; 76 Br/ 131 I-HPEM; Mal-A + 18 F-FBA. Site-specific incorporation cysteine-containing peptide-based chelators for c and Re; Histidine-tag based chelators for c and Re; Well-defined conjugates with reproducible biodistribution Features of Rapid inetics: 111 In-DA-Z HER2:342 Small size ( ca. 7 kda) in combination with high affinity rapid extravasation; rapid tissue penetration; strong binding to molecular target; rapid clearance of unbound tracer; no EPR effect, highly specific uptake High-contrast imaging shortly after injection % IA/g ime pi, h tumor 1 h p.i. 2 h p.i. 4 h p.i. rlova: Cancer Research, 27: idney umor 111 In-DA-Z HER2:342 / SV3 Rapid inetics: I-PIB-Z HER2:342 vs I-trastuzumab Rapid inetics: I-PIB-Z HER2:342 vs I-trastuzumab I-trastzumab I-Affibody molecule rlova: Journal of Nuclear Medicine, 29; I-trastzumab 24 h p.i. I-Affibody molecule rlova: Journal of Nuclear Medicine, 29;

3 Rapid inetics: I-PIB-Z HER2:342 vs I-trastuzumab I-trastzumab 72 h p.i. I-Affibody molecule rlova: Journal of Nuclear Medicine, 29; Specific 111 In-DA-Z HER2:342 No EPR effect, highly specific tumor uptake Unspecific 111 specific unspecific In-DA-Z aq rlova: Cancer Research, 27: Uptake, % IA/g Uptake in SV-3 xenografts 4 h p.i %IA/g.13.1 %IA/g ptimising biodistribution of c-labeled Engfeldt : Eur J Nucl Med; 27:722 N S N Me N maggg Comparative biodistribution in NMRI mice, 4 h pi maggg magsg masss 12 maggg mageg maeee kidney GI tract * kidney GI tract * Engfeldt Eur J Nucl Med 27:1843 ran. Bioconjugate Chem, 27: maeee masee maese kidney GI tract * masss mass ma kidney GI tract * Ekblad. Eur J Nucl Med, 28:224 ran. Bioconjugate Chem, 28:268 Does it work at C-terminus? Ahlgren J Nucl Med. 2: Comparative biodistribution in NMRI mice, 4 h pi -GGGC -GGSC -GGEC -GGC -GSEC kidney intestines* ZV1 AENFNEMRNAYWEIALLPNLNQQRAFIRSLYDDPSQSANLLAEALNDAQGSEC ZV2 AENFNEMRNAYWEIALLPNLNQQRAFIRSLYDDPSQSANLLAEALNDAQGGGC ZV3 AENFNEMRNAYWEIALLPNLNQQRAFIRSLYDDPSQSANLLAEALNDAQGGSC ZV4 AENFNEMRNAYWEIALLPNLNQQRAFIRSLYDDPSQSANLLAEALNDAQGGEC ZV AENFNEMRNAYWEIALLPNLNQQRAFIRSLYDDPSQSANLLAEALNDAQGGC Wållberg. J Nucl Med. 211;2:461.

4 c Gamma-camera imaging, 4 h pi (SV-3 xenografts) c-zv2 c-zv1 Imaging of HER2-expressing SV-3 xenografts using c-labeled Z HER2:342 Wållberg. J Nucl Med. 211;2:461. tumor caecum kidneys 1. Engfeldt et al., Eur J Nucl Med 27, 34:722; 2. ran et al., Bioconjugate Chem, 27, 18:196; 3. Ekblad et al., Eur J Nucl Med, 28, 3:224; 4. Ahlgren et al. J Nucl Med. 29, :781; -6. Wållberg et al. J Nucl Med 211;2:461. Chemical properties and position of chelator/linker for labelling of influence: Predominant excretion pathway; Uptake and retention in excretory organs; Blood clearance rate (tracer and radiocatabolites) ptimization of purification tags can improve biodistribution L influencing imaging contrast. Careful studies concerning influence of labeling chemistry on biodistribution and targeting may make a difference between success and failure! c(c) 3 - His 6 -Z HER2:342 (IsoLink) c-maese- Z HER2:342 Influence of N-terminal His 6 -tag on biodistribution of site-specifically labelled at C- terminus, 4 h pi c-h 6 -Z HER2:342 (-C) c-z HER2:239 (-C) kidney GI tract * Ahlgren. J Nucl Med. 29 :781. Ahlgren. Bioconjug Chem. 28 : In-H 6 -Z HER2:342 -C-mal-DA 111 In-Z HER2:342 -C-mal-DA kidney GI tract * Hypotheses 1. Placement of His-tag on C-terminus could reduce uptake of c(c) 3 -labelled Affibody molecules; 2. Incorporation of negatively charged residues into His-tag could reduce uptake even if Histag is placed at N-terminus. 3. Despite changes is position and composition of histidine tag, - IMAC purification would be possible - c(c) 3 -labelling would be possible. NH 2 N C NH C H 2 X N C NH X N NH

5 Biodistribution of c(c) 3 -labelled in NMRI mice 4h pi. GSSHHHHHHLQ GSHHHHHH 12 8 His6-ZHER2:342 ZHER2:342-His6 HEHEHE-ZHER2:342 stomach kidney saliv thyroid GI tract carcass MHEHEHE olmachev. Bioconjug Chem. 2:213. Biodistribution in NMRI mice 4h pi. Site-specific 111 In-labeling using MMA-DA Liver uptake and hepatobiliary excretion of c(c) 3 -labeled in mice Hofstrom: J Med Chem. 211:3817. olmachev (unpublished data) Clinical proof-of-concept. SPEC using 111 In-DA-Z HER2:342 (3 h p.i) Purification tags can be used for modification of biodistribution of targeting radiolabeled scaffold proteins 18 F-FDG PE scan HER2-SPEC scan horacic Wall Metastasis CRNAL horacic Wall Metastasis CRNAL Baum: J Nucl Med, 2; SAGIAL

6 Summary offer advantages of: Fast tumor uptake and rapid clearance from High affinity High specificity Robustness Defined structure is a promising technology platform for development of tracers for molecular imaging. Acknowledgements Amelie E arlström Anders Wennborg Anna rlova Anna Sjöberg Daniel Rosik Camilla Hofström Charles Widström Fredrik Y Freijd Hans Lundqvist Hadis Honarvar Helena Wållberg Joachim Feldwisch Jennie Malmberg Joanna Strand Jörgen Carlsson Lars Abrahmsen Mattias Sandström Magnus Hjertman Mikaela Magnusson Mohamed Altai Sara Ahlgren Stefan Ståhl Seyed Hosseinimehr orun Ekblad orbjörn Gräslund huy ran Zohreh Varasteh and many other