In vivo imaging of the voltage-gated potassium channel Kv10.1 utilizing SPECT in combination with radiolabeled antibodies
by Thomas Krüwel
Date of Examination:2015-11-17
Date of issue:2015-11-19
Advisor:Prof. Dr. Frauke Alves
Referee:Prof. Dr. Frauke Alves
Referee:Prof. Dr. Walter Stühmer
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Abstract
English
The detection of tumors in an early phase of tumor development is an important achievement to improve the overall prognosis of the patient. Besides accurate information of tumor load and spread, the retrieval of the expression of biomarkers on the tumor cell surface at the earliest time point is a prerequisite for a successful targeted therapeutic approach. In order to acquire information on expression of tumor associated proteins in vivo, functional imaging with specific probes targeting tumor biomarkers such as human voltage-gated potassium channel Kv10.1 or epidermal growth factor receptor 1 (EGFR) is a promising approach. In this study, 10 novel anti-Kv10.1 nanobodies were generated by phage display and characterized in vitro, in order to be able to visualize tumor lesions in vivo in future by applying multi-pinhole SPECT targeting Kv10.1. Molecular characterization of the binding properties of the anti-Kv10.1 nanobodies using ELISA, immunoprecipitation, Western blotting, flow cytometry and surface plasmon resonance identified two promising clones, C4 and D9, specifically detecting Kv10.1 transfected HEK cells and the fusion protein H1X. Since the SPECT system was not used for imaging of small animals before, the system and the imaging procedure was first set up and validated by using the clinically approved full IgG1 antibody 99mTc-Cetuximab as well as nanobody 99mTc-D10, both targeting EGFR in human MDA-MB-231 and MDA-MB-468 mammary carcinoma as well as in A431 epidermoid tumor bearing mice. The anti-EGFR nanobody 99mTc-D10 was efficiently labeled with [99mTc(CO)3(OH2)3]+ yielding a specific activity of 183 MBq/nmol ± 35 and a radiochemical purity of 97.7% ± 1.2 whereas the Hynic-derivatized antibody 99mTc-Cetuximab yielded a specific activity of 1700 MBq/nmol ± 105 and a purity of 97.3% ± 0.4. In vivo, MDA-MB-468 and MDA-MB-231 mammary tumors were visualized by SPECT applying 98 pmol of the anti-EGFR antibody 99mTc-Cetuximab with tumor uptakes of 5.49% ID/cm3 ± 2.2 and 2.13% ID/cm3 ± 0.37, respectively, 24 h post i.v. injection. Mice bearing MDA-MB-468 tumors that received the isotype control 99mTc-IgG1 antibody (98 pmol) showed a significant uptake of 2.1% ID/cm3 ± 0.1 to the tumor after 24 h post i.v. injection proving a high unspecific tumor uptake of the antibody. Biodistribution analysis of mice receiving 99mTc-Cetuximab revealed high tumor uptakes of 14.6% ID/g ± 6.89 and 6.19% ID/g ± 2.71 for MDA-MB-468 and MDA-MB-231 tumors, respectively, compared to a remaining activity in the blood of approx. 5.5% ID/g in both cohorts resulting in tumor-to-blood ratios of 3.1 and 1.2, respectively. Since 99mTc-Cetuximab is cleared via hepatic excretion from the body, a high liver uptake of approx. 20% ID/g was determined. Visualization of very small EGFR positive epidermoid A431 and mammary MDA-MB-468 tumors with nanobody 99mTc-D10 by SPECT imaging already 45 min post i.v. administration resulted in tumor uptakes of 1.0% ID/cm3 ± 0.6 and 0.6% ID/cm3 ± 0.2, respectively, with excellent in vivo contrast and ex vivo tumor to blood and tissue ratios due to the fast blood clearance with a serum half-life of 4.9 min. The use of control nanobody 99mTc-F5 showed no significant tumor uptake. No accumulation of 99mTc-D10 was observed in MDA-MB-231 tumors characterized by a very low EGFR expression. Biodistribution analysis of 99mTc-D10 revealed A431 and MDA-MB-468 tumor uptakes of 2.3% ID/g ± 0. 7 and 1.3% ID/g ± 0.3, respectively, compared to a remaining activity in the blood of approx. 0.2% ID/g. This resulted in tumor-to-blood ratios of 12.1 and 5.4 for A431 and MDA-MB-468 tumors, respectively. Since the nanobody 99mTc-D10 is cleared via renal excretion from the body, high uptake was determined in the kidneys and the urine (214 ± 30%ID/g and 69 ± 31%ID/g, respectively). The here presented specific and high contrast in vivo visualization of small human tumors overexpressing EGFR by preclinical multi-pinhole SPECT already 45 min after administration of anti-EGFR nanobody 99mTc-D10 provides the basis for a possible future in vivo use of anti-Kv10.1 nanobodies for tumor visualization by SPECT.
Keywords: In vivo imaging; SPECT; tumor visualization; Kv10.1; EGFR; nanobody; single-domain antibody; Phage display; Technetium-99m