Browsing by Author "Koumarianou, Eftychia"

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    Astatine-211 labeled anti-HER2 5F7 single domain antibody fragment conjugates: radiolabeling and preliminary evaluation.
    (Nucl Med Biol, 2017-09-19) Choi, Jaeyeon; Vaidyanathan, Ganesan; Koumarianou, Eftychia; Kang, Choong Mo; Zalutsky, Michael R
    INTRODUCTION: Derived from heavy chain only camelid antibodies, ~15-kDa single-domain antibody fragments (sdAbs) are an attractive platform for developing molecularly specific imaging probes and targeted radiotherapeutics. The rapid tumor accumulation and normal tissue clearance of sdAbs might be ideal for use with (211)At, a 7.2-h half-life α-emitter, if appropriate labeling chemistry can be devised to trap (211)At in cancer cells after sdAb binding. This study evaluated two reagents, [(211)At]SAGMB and iso-[(211)At]SAGMB, for this purpose. METHODS: [(211)At]SAGMB and iso-[(211)At]SAGMB, and their radioiodinated analogues [(131)I]SGMIB and iso-[(131)I]SGMIB, were synthesized by halodestannylation and reacted with the anti-HER2 sdAb 5F7. Radiochemical purity, immunoreactivity and binding affinity were determined. Paired-label internalization assays on HER2-expressing BT474M1 breast carcinoma cells directly compared [(131)I]SGMIB-5F7/[(211)At]SAGMB-5F7 and iso-[(131)I]SGMIB-5F7/iso-[(211)At]SAGMB-5F7 tandems. The biodistribution of the two tandems was evaluated in SCID mice with subcutaneous BT474M1 xenografts. RESULTS: Radiochemical yields for Boc2-iso-[(211)At]SAGMB and Boc2-[(211)At]SAGMB synthesis, and efficiencies for coupling of iso-[(211)At]SAGMB and [(211)At]SAGMB to 5F7 were similar, with radiochemical purities of [(211)At]SAGMB-5F7 and iso-[(211)At]SAGMB-5F7 >98%. iso-[(211)At]SAGMB-5F7 and [(211)At]SAGMB-5F7 had immunoreactive fractions >80% and HER2 binding affinities of less than 5 nM. Internalization assays demonstrated high intracellular trapping of radioactivity, with little difference observed between corresponding (211)At- and (131)I-labeled 5F7 conjugates. Higher BT474M1 intracellular retention was observed from 1-6 h for the iso-conjugates (iso-[(211)At]SAGMB-5F7, 74.3 ± 2.8%, vs. [(211)At]SAGMB-5F7, 63.7 ± 0.4% at 2 h) with the opposite behavior observed at 24 h. Peak tumor uptake for iso-[(211)At]SAGMB-5F7 was 23.4 ± 2.2% ID/g at 4 h, slightly lower than its radioiodinated counterpart, but significantly higher than observed with [(211)At]SAGMB-5F7. Except in kidneys and lungs, tumor-to-normal organ ratios for iso-[(211)At]SAGMB-5F7 were greater than 10:1 by 2 h, and significantly higher than those for [(211)At]SAGMB-5F7. CONCLUSION: These (211)At-labeled sdAb conjugates, particularly iso-[(211)At]SAGMB-5F7, warrant further evaluation for targeted α-particle radiotherapy of HER2-expressing cancers.
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    D-Amino acid peptide residualizing agents bearing N-hydroxysuccinimido- and maleimido-functional groups and their application for trastuzumab radioiodination.
    (Nucl Med Biol, 2015-01) Pruszynski, Marek; Koumarianou, Eftychia; Vaidyanathan, Ganesan; Chitneni, Satish; Zalutsky, Michael R
    INTRODUCTION: Proteins that undergo receptor-mediated endocytosis are subject to lysosomal degradation, requiring radioiodination methods that minimize loss of radioactivity from tumor cells after this process occurs. To accomplish this, we developed the residualizing radioiodination agent N(ϵ)-(3-[(*)I]iodobenzoyl)-Lys(5)-N(α)-maleimido-Gly(1)-D-GEEEK (Mal-D-GEEEK-[(*)I]IB), which enhanced tumor uptake but also increased kidney activity and necessitates generation of sulfhydryl moieties on the protein. The purpose of the current study was to synthesize and evaluate a new D-amino acid based agent that might avoid these potential problems. METHODS: N(α)-(3-iodobenzoyl)-(5-succinimidyloxycarbonyl)-D-EEEG (NHS-IB-D-EEEG), which contains 3 D-glutamates to provide negative charge and a N-hydroxysuccinimide function to permit conjugation to unmodified proteins, and the corresponding tin precursor were produced by solid phase peptide synthesis and subsequent conjugation with appropriate reagents. Radioiodination of the anti-HER2 antibody trastuzumab using NHS-IB-D-EEEG and Mal-D-GEEEK-IB was compared. Paired-label internalization assays on BT474 breast carcinoma cells and biodistribution studies in athymic mice bearing BT474M1 xenografts were performed to evaluate the two radioiodinated D-peptide trastuzumab conjugates. RESULTS: NHS-[(131)I]IB-D-EEEG was produced in 53.8%±13.4% and conjugated to trastuzumab in 39.5%±7.6% yield. Paired-label internalization assays with trastuzumab-NHS-[(131)I]IB-D-EEEG and trastuzumab-Mal-D-GEEEK-[(125)I]IB demonstrated similar intracellular trapping for both conjugates at 1h ((131)I, 84.4%±6.1%; (125)I, 88.6%±5.2%) through 24h ((131)I, 60.7%±6.8%; (125)I, 64.9%±6.9%). In the biodistribution experiment, tumor uptake peaked at 48 h (trastuzumab-NHS-[(131)I]IB-D-EEEG, 29.8%±3.6%ID/g; trastuzumab-Mal-D-GEEEK-[(125)I]IB, 45.3%±5.3%ID/g) and was significantly higher for (125)I at all time points. In general, normal tissue levels were lower for trastuzumab-NHS-[(131)I]IB-D-EEEG, with the differences being greatest in kidneys ((131)I, 2.2%±0.4%ID/g; (125)I, 16.9%±2.8%ID/g at 144 h). CONCLUSION: NHS-[(131)I]IB-D-EEEG warrants further evaluation as a residualizing radioiodination agent for labeling internalizing antibodies/fragments, particularly for applications where excessive renal accumulation could be problematic.
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    Modular nanotransporters: a versatile approach for enhancing nuclear delivery and cytotoxicity of Auger electron-emitting 125I.
    (EJNMMI Res, 2012-10-29) Slastnikova, Tatiana A; Koumarianou, Eftychia; Rosenkranz, Andrey A; Vaidyanathan, Ganesan; Lupanova, Tatiana N; Sobolev, Alexander S; Zalutsky, Michael R
    UNLABELLED: BACKGROUND: This study evaluates the potential utility of a modular nanotransporter (MNT) for enhancing the nuclear delivery and cytotoxicity of the Auger electron emitter 125I in cancer cells that overexpress the epidermal growth factor receptor (EGFR). METHODS: MNTs are recombinant multifunctional polypeptides that we have developed for achieving selective delivery of short-range therapeutics into cancer cells. MNTs contain functional modules for receptor binding, internalization, endosomal escape and nuclear translocation, thereby facilitating the transport of drugs from the cell surface to the nucleus. The MNT described herein utilized EGF as the targeting ligand and was labeled with 125I using N-succinimidyl-4-guanidinomethyl-3-[125I]iodobenzoate (SGMIB). Membrane binding, intracellular and nuclear accumulation kinetics, and clonogenic survival assays were performed using the EGFR-expressing A431 epidermoid carcinoma and D247 MG glioma cell lines. RESULTS: [125I]SGMIB-MNT bound to A431 and D247 MG cells with an affinity comparable to that of native EGF. More than 60% of internalized [125I]SGMIB-MNT radioactivity accumulated in the cell nuclei after a 1-h incubation. The cytotoxic effectiveness of [125I]SGMIB-MNT compared with 125I-labeled bovine serum albumin control was enhanced by a factor of 60 for D247 MG cells and more than 1,000-fold for A431 cells, which express higher levels of EGFR. CONCLUSIONS: MNT can be utilized to deliver 125I into the nuclei of cancer cells overexpressing EGFR, significantly enhancing cytotoxicity. Further evaluation of [125I]SGMIB-MNT as a targeted radiotherapeutic for EGFR-expressing cancer cells appears warranted.