Study On The Preparation Of Affibody Molecular Probes And Their Application In Tumor Imaging

Affibody molecules have been demonstrated to be a promising generalizableplatform for developing imaging or therapeutic agents for different molecular targets.In this dissertation, the chemical synthesis of anti-EGFR and anti-HER2Affibodymolecules, the preparation of Affibody based molecular probes and the application ofthe probes in tumor imaging were studied.The anti-EGFR Affibody molecule, Ac-Cys-ZEGFR:1907, and three anti-HER2Affibody molecules, Ac-Cys-Z_HER2:342， Ac-Z_HER2:342(Cys³⁹) orAc-Z_HER2:342-Cys,were synthesized using conventional solid phase peptide synthesismethod. The purities of four Affibody molecules were all over95%by HPLC analysisand the peptides were then characterized by MALDI-TOF-MS. The effect of couplingreagents, cleavage reagents and purification on the synthetic yields and efficiency ofAffibody molecules was investigated intensively.Four Affibody based fluorescent probes were prepared by fluorescence labelingCys-Z_EGFR:1907with four commercial near-infrared fluorescent dyes, Cy5.5-ZEGFR:1907，Alex680-Z_EGFR:1907，SR680-Z_EGFR:1907or800CW-Z_EGFR:1907, labeling yields were allover95%. The purities of four fluorescent probes were all over95%by HPLCanalysis and the probes were then characterized by MALDI-TOF-MS. Threeconjugates were synthesized with DOTA and three anti-HER2Affibody molecules,DOTA-Cys-Z_HER2:342, DOTA-Z_HER2:342(Cys³⁹) or DOTA-Z_HER2:342-Cys, yields wereall over95%. The purities of three conjugates were all over95%by HPLC analysisand the conjugates were then characterized by MALDI-TOF-MS. Three radio-probeswere prepared by radio-labeling three anti-HER2Affibody moleculeswith（64）^Cu,（64）^Cu-DOTA-Cys-Z_HER2:342，（64）^Cu-DOTA-ZHER2:342(Cys³⁹)or（64）^Cu-DOTA-Z_HER2:342-Cys, labeling yields were all over62%.The EGFR binding property and specificity of the four NIR fluorescent Affibodyprobes were studied by fluorescence microscopy using high EGFR expressing A431cells and low EGFR expressing MCF7cells, Cy5.5-Z_EGFR:1907，Alex680-Z_EGFR:1907，SR680-Z_EGFR:1907or800CW-Z_EGFR:1907. The binding affinities of the probes （KD） toEGFR were further determined by flow cytometry. All four probes exhibited high EGFR binding affinities in nM range. In vivo optical imaging of the four probes wasperformed in the mice bearing subcutaneous A431tumors. In vivo optical imaging ofthe four probes revealed that they all showed fast tumor targeting ability and goodtumor-to-normal tissue contrast as early as0.5h post-injection （p.i.）. Thetumor-to-normal tissue ratio reached a peak at2to4h p.i. by regional of interest （ROI）analysis of images. Ex vivo studies further demonstrated that the four probes had hightumor uptakes. Cy5.5-ZEGFR:1907seems to be the most promising probe among themfor further tumor imaging applications and clinical translations.Cell uptake assays of three anti-HER2Affibody radio-probes were carried usinghigh HER2expressing SKOV3cells,（64）^Cu-DOTA-Cys-Z_HER2:342，（64）^Cu-DOTA-Z_HER2:342(Cys³⁹)or（64）^Cu-DOTA-Z_HER2:342-Cys. The binding affinities of the probes （KD） to HER2astested by cell saturation analysis were in nM range. The serum stabilities of the probeswere studied using mouse serum and the metabolic stabilities of three probes werethen conducted using tumor-bearing mice. PET imaging of three probes wasperformed in the mice bearing subcutaneous SKOV3tumors. PET imaging of theprobes revealed that they all showed fast tumor targeting ability，high tumoraccumulation, and good tumor-to-normal tissue contrast as early as0.5hpost-injection （p.i.）. The%ID/g values of the probes in tumor reached a peak at2to4h p.i.. Co-injection of the probe with anti-HER2Affibody proteins reduced the tumoruptake of three probes, biodistribution studies further demonstrated that the probeshad high tumor uptakes. This work demonstrated that there was no significantdifference among the three probes in the in vitro and in vivo properties.