Fluorine-18 Labeling of the HER2-Targeting Single-Domain Antibody 2Rs15d Using a Residualizing Label and Preclinical Evaluation

  • Zhengyuan Zhou
  • Ganesan Vaidyanathan
  • Darryl McDougald
  • Choong Mo Kang
  • Irina Balyasnikova
  • Nick Devoogdt
  • Angeline N. Ta
  • Brian R. McNaughton
  • Michael R. Zalutsky
Research Article

Abstract

Purpose

Our previous studies with F-18-labeled anti-HER2 single-domain antibodies (sdAbs) utilized 5F7, which binds to the same epitope on HER2 as trastuzumab, complicating its use for positron emission tomography (PET) imaging of patients undergoing trastuzumab therapy. On the other hand, sdAb 2Rs15d binds to a different epitope on HER2 and thus might be a preferable vector for imaging in these patients. The aim of this study was to evaluate the tumor targeting of F-18 -labeled 2Rs15d in HER2-expressing breast carcinoma cells and xenografts.

Procedures

sdAb 2Rs15d was labeled with the residualizing labels N-succinimidyl 3-((4-(4-[18F]fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(guanidinomethyl)benzoate ([18F]RL-I) and N-succinimidyl 4-guanidinomethyl-3-[125I]iodobenzoate ([125I]SGMIB), and the purity and HER2-specific binding affinity and immunoreactivity were assessed after labeling. The biodistribution of I-125- and F-18-labeled 2Rs15d was determined in SCID mice bearing subcutaneous BT474M1 xenografts. MicroPET/x-ray computed tomograph (CT) imaging of [18F]RL-I-2Rs15d was performed in this model and compared to that of nonspecific sdAb [18F]RL-I-R3B23. MicroPET/CT imaging was also done in an intracranial HER2-positive breast cancer brain metastasis model after administration of 2Rs15d-, 5F7-, and R3B23-[18F]RL-I conjugates.

Results

[18F]RL-I was conjugated to 2Rs15d in 40.8 ± 9.1 % yield and with a radiochemical purity of 97–100 %. Its immunoreactive fraction (IRF) and affinity for HER2-specific binding were 79.2 ± 5.4 % and 7.1 ± 0.4 nM, respectively. [125I]SGMIB was conjugated to 2Rs15d in 58.4 ± 8.2 % yield and with a radiochemical purity of 95–99 %; its IRF and affinity for HER2-specific binding were 79.0 ± 12.9 % and 4.5 ± 0.8 nM, respectively. Internalized radioactivity in BT474M1 cells in vitro for [18F]RL-I-2Rs15d was 43.7 ± 3.6, 36.5 ± 2.6, and 21.7 ± 1.2 % of initially bound radioactivity at 1, 2, and 4 h, respectively, and was similar to that seen for [125I]SGMIB-2Rs15d. Uptake of [18F]RL-I-2Rs15d in subcutaneous xenografts was 16–20 %ID/g over 1–3 h. Subcutaneous tumor could be clearly delineated by microPET/CT imaging with [18F]RL-I-2Rs15d but not with [18F]RL-I-R3B23. Intracranial breast cancer brain metastases could be visualized after intravenous administration of both [18F]RL-I-2Rs15d and [18F]RL-I-5F7.

Conclusions

Although radiolabeled 2Rs15d conjugates exhibited lower tumor cell retention both in vitro and in vivo than that observed previously for 5F7, given that it binds to a different epitope on HER2 from those targeted by the clinically utilized HER2-targeted therapeutic antibodies trastuzumab and pertuzumab, F-18-labeled 2Rs15d has potential for assessing HER2 status by PET imaging after trastuzumab and/or pertuzumab therapy.

Key words

Single-domain antibody Trastuzumab HER2 Fluorine-18 PET Breast cancer 

References

  1. 1.
    Schettini F, Buono G, Cardalesi C et al (2016) Hormone receptor/human epidermal growth factor receptor 2-positive breast cancer: where we are now and where we are going. Cancer Treat Rev 46:20–26CrossRefPubMedGoogle Scholar
  2. 2.
    Santa-Maria CA, Nye L, Mutonga MB et al (2016) Management of metastatic HER2-positive breast cancer: where are we and where do we go from here? Oncology (Williston Park) 30:148–155Google Scholar
  3. 3.
    Recondo G Jr, de la Vega M, Galanternik F et al (2016) Novel approaches to target HER2-positive breast cancer: trastuzumab emtansine. Cancer Manag Res 8:57–65PubMedPubMedCentralGoogle Scholar
  4. 4.
    Maximiano S, Magalhaes P, Guerreiro MP, Morgado M (2016) Trastuzumab in the treatment of breast cancer. BioDrugs 30:75–86CrossRefPubMedGoogle Scholar
  5. 5.
    Gebhart G, Flamen P, De Vries EG et al (2016) Imaging diagnostic and therapeutic targets: human epidermal growth factor receptor 2. J Nucl Med 57(Suppl 1):81S–88SCrossRefPubMedGoogle Scholar
  6. 6.
    Nitta H, Kelly BD, Allred C et al (2016) The assessment of HER2 status in breast cancer: the past, the present, and the future. Pathol Int 66:313–324CrossRefPubMedGoogle Scholar
  7. 7.
    Karagoz Ozen DS, Ozturk MA, Aydin O et al (2014) Receptor expression discrepancy between primary and metastatic breast cancer lesions. Oncol Res Treat 37:622–626CrossRefPubMedGoogle Scholar
  8. 8.
    Rack B, Zombirt E, Trapp E et al (2016) Comparison of HER2 expression in primary tumor and disseminated tumor cells in the bone marrow of breast cancer patients. Oncology 90:232–238CrossRefPubMedGoogle Scholar
  9. 9.
    Kramer-Marek G, Oyen WJ (2016) Targeting the human epidermal growth factor receptors: imaging biomarkers from bench to bedside. J Nucl Med 57:996–1001CrossRefPubMedGoogle Scholar
  10. 10.
    Sorensen J, Velikyan I, Sandberg D et al (2016) Measuring HER2-receptor expression in metastatic breast cancer using [68Ga]ABY-025 affibody PET/CT. Theranostics 6:262–271CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Mendler CT, Gehring T, Wester HJ et al (2015) 89Zr-labeled versus 124I-labeled αHER2 Fab with optimized plasma half-life for high-contrast tumor imaging in vivo. J Nucl Med 56:1112–1118CrossRefPubMedGoogle Scholar
  12. 12.
    Ma T, Sun X, Cui L et al (2014) Molecular imaging reveals trastuzumab-induced epidermal growth factor receptor downregulation in vivo. J Nucl Med 55:1002–1007CrossRefPubMedGoogle Scholar
  13. 13.
    Olafsen T, Sirk SJ, Olma S et al (2012) ImmunoPET using engineered antibody fragments: fluorine-18 labeled diabodies for same-day imaging. Tumour Biol 33:669–677CrossRefPubMedGoogle Scholar
  14. 14.
    Olafsen T, Kenanova VE, Sundaresan G et al (2005) Optimizing radiolabeled engineered anti-p185HER2 antibody fragments for in vivo imaging. Cancer Res 65:5907–5916CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Kijanka M, Dorresteijn B, Oliveira S, van Bergen en Henegouwen PM (2015) Nanobody-based cancer therapy of solid tumors. Nanomedicine 10:161–174CrossRefPubMedGoogle Scholar
  16. 16.
    De Meyer T, Muyldermans S, Depicker A (2014) Nanobody-based products as research and diagnostic tools. Trends Biotechnol 32:263–270CrossRefPubMedGoogle Scholar
  17. 17.
    Keyaerts M, Xavier C, Heemskerk J et al (2016) Phase I study of 68Ga-HER2-nanobody for PET/CT assessment of HER2 expression in breast carcinoma. J Nucl Med 57:27–33CrossRefPubMedGoogle Scholar
  18. 18.
    Xavier C, Blykers A, Vaneycken I et al (2016) 18F-nanobody for PET imaging of HER2 overexpressing tumors. Nucl Med Biol 43:247–252CrossRefPubMedGoogle Scholar
  19. 19.
    Vaidyanathan G, McDougald D, Choi J et al (2016) Preclinical evaluation of 18F-labeled anti-HER2 nanobody conjugates for imaging HER2 receptor expression by immuno-PET. J Nucl Med 57:967–973CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Revets HM BC, Hoogenboom HM. (2011) Amino acid sequences directed against HER2 and polypeptides comprising the same for the treatment of cancers and/or tumors. US Patent 2011/0059090 AlGoogle Scholar
  21. 21.
    Rockberg J, Schwenk JM, Uhlen M (2009) Discovery of epitopes for targeting the human epidermal growth factor receptor 2 (HER2) with antibodies. Mol Oncol 3:238–247CrossRefPubMedGoogle Scholar
  22. 22.
    Kramer-Marek G, Gijsen M, Kiesewetter DO et al (2012) Potential of PET to predict the response to trastuzumab treatment in an ErbB2-positive human xenograft tumor model. J Nucl Med 53:629–637CrossRefPubMedGoogle Scholar
  23. 23.
    Vaneycken I, Devoogdt N, Van Gassen N et al (2011) Preclinical screening of anti-HER2 nanobodies for molecular imaging of breast cancer. FASEB J 25:2433–2446CrossRefPubMedGoogle Scholar
  24. 24.
    Xavier C, Vaneycken I, D’Huyvetter M et al (2013) Synthesis, preclinical validation, dosimetry, and toxicity of 68Ga-NOTA-anti-HER2 nanobodies for iPET imaging of HER2 receptor expression in cancer. J Nucl Med 54:776–784CrossRefPubMedGoogle Scholar
  25. 25.
    Vaidyanathan G, McDougald D, Choi J et al (2016) N-Succinimidyl 3-((4-(4-[18F]fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(guanidinomethyl)ben zoate ([18F]SFBTMGMB): a residualizing label for 18F-labeling of internalizing biomolecules. Org Biomol Chem 14:1261–1271CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Vaidyanathan G, Zalutsky MR (2007) Synthesis of N-succinimidyl 4-guanidinomethyl-3-[*I]iodobenzoate: a radio-iodination agent for labeling internalizing proteins and peptides. Nat Protoc 2:282–286CrossRefPubMedGoogle Scholar
  27. 27.
    Pruszynski M, Koumarianou E, Vaidyanathan G et al (2013) Targeting breast carcinoma with radioiodinated anti-HER2 nanobody. Nucl Med Biol 40:52–59CrossRefPubMedGoogle Scholar
  28. 28.
    Gray MA, Tao RN, DePorter SM et al (2016) A nanobody activation immunotherapeutic that selectively destroys HER2-positive breast cancer cells. Chembiochem 17:155–158CrossRefPubMedGoogle Scholar
  29. 29.
    Lemaire M, D’Huyvetter M, Lahoutte T et al (2014) Imaging and radioimmunotherapy of multiple myeloma with anti-idiotypic nanobodies. Leukemia 28:444–447CrossRefPubMedGoogle Scholar
  30. 30.
    Yu Z, Xia W, Wang HY et al (2006) Antitumor activity of an Ets protein, PEA3, in breast cancer cell lines MDA-MB-361DYT2 and BT474M1. Mol Carcinog 45:667–675CrossRefPubMedGoogle Scholar
  31. 31.
    Choi J, Vaidyanathan G, Koumarianou E et al (2014) N-Succinimidyl guanidinomethyl iodobenzoate protein radiohalogenation agents: influence of isomeric substitution on radiolabeling and target cell residualization. Nucl Med Biol 41:802–812CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Lindmo T, Boven E, Cuttitta F et al (1984) Determination of the immunoreactive fraction of radiolabeled monoclonal antibodies by linear extrapolation to binding at infinite antigen excess. J Immunol Meth 72:77–89CrossRefGoogle Scholar
  33. 33.
    Kanojia D, Balyasnikova IV, Morshed RA et al (2015) Neural stem cells secreting anti-HER2 antibody improve survival in a preclinical model of HER2 overexpressing breast cancer brain metastases. Stem Cells 33:2985–2994CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Velikyan I, Wennborg A, Feldwisch J et al (2016) Good manufacturing practice production of [68Ga]Ga-ABY-025 for HER2 specific breast cancer imaging. Am J Nucl Med Mol Imaging 6:135–153PubMedPubMedCentralGoogle Scholar
  35. 35.
    Trousil S, Hoppmann S, Nguyen QD et al (2014) Positron emission tomography imaging with 18F-labeled ZHER2:2891 affibody for detection of HER2 expression and pharmacodynamic response to HER2-modulating therapies. Clin Cancer Res 20:1632–1643CrossRefPubMedGoogle Scholar
  36. 36.
    Pruszynski M, Koumarianou E, Vaidyanathan G et al (2014) Improved tumor targeting of anti-HER2 nanobody through N-succinimidyl 4-guanidinomethyl-3-iodobenzoate radiolabeling. J Nucl Med 55:650–656CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Sanchez-Crespo A (2013) Comparison of gallium-68 and fluorine-18 imaging characteristics in positron emission tomography. Appl Radiat Isot 76:55–62CrossRefPubMedGoogle Scholar
  38. 38.
    D’Huyvetter M, Aerts A, Xavier C et al (2012) Development of 177Lu-nanobodies for radioimmunotherapy of HER2-positive breast cancer: evaluation of different bifunctional chelators. Contrast Media Mol Imaging 7:254–264CrossRefPubMedGoogle Scholar
  39. 39.
    D’Huyvetter M, Vincke C, Xavier C et al (2014) Targeted radionuclide therapy with a 177Lu-labeled anti-HER2 nanobody. Theranostics 4:708–720CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Banappagari S, McCall A, Fontenot K et al (2013) Design, synthesis and characterization of peptidomimetic conjugate of BODIPY targeting HER2 protein extracellular domain. Eur J Med Chem 65:60–69CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Fuentes G, Scaltriti M, Baselga J, Verma CS (2011) Synergy between trastuzumab and pertuzumab for human epidermal growth factor 2 (Her2) from colocalization: an in silico based mechanism. Breast Cancer Res 13:R54CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Xu FJ, Yu YH, Bae DS et al (1997) Radioiodinated antibody targeting of the HER-2/neu oncoprotein. Nucl Med Biol 24:451–459CrossRefPubMedGoogle Scholar
  43. 43.
    Langmuir VK, Mendonca HL, Woo DV (1992) Comparisons between two monoclonal antibodies that bind to the same antigen but have differing affinities: uptake kinetics and 125I-antibody therapy efficacy in multicell spheroids. Cancer Res 52:4728–4734PubMedGoogle Scholar
  44. 44.
    Wargalla UC, Reisfeld RA (1989) Rate of internalization of an immunotoxin correlates with cytotoxic activity against human tumor cells. Proc Natl Acad Sci U S A 86:5146–5150CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Ward DM, Kaplan J (1990) The rate of internalization of different receptor-ligand complexes in alveolar macrophages is receptor-specific. Biochem J 270:369–374CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Stemmler HJ, Schmitt M, Harbeck N et al (2006) Application of intrathecal trastuzumab (Herceptin trademark) for treatment of meningeal carcinomatosis in HER2-overexpressing metastatic breast cancer. Oncol Rep 15:1373–1377PubMedGoogle Scholar
  47. 47.
    Stemmler HJ, Schmitt M, Willems A et al (2007) Ratio of trastuzumab levels in serum and cerebrospinal fluid is altered in HER2-positive breast cancer patients with brain metastases and impairment of blood-brain barrier. Anti-Cancer Drugs 18:23–28CrossRefPubMedGoogle Scholar
  48. 48.
    Dijkers EC, Oude Munnink TH, Kosterink JG et al (2010) Biodistribution of 89Zr-trastuzumab and PET imaging of HER2-positive lesions in patients with metastatic breast cancer. Clin Pharmacol Ther 87:586–592CrossRefPubMedGoogle Scholar

Copyright information

© World Molecular Imaging Society 2017

Authors and Affiliations

  • Zhengyuan Zhou
    • 1
  • Ganesan Vaidyanathan
    • 1
  • Darryl McDougald
    • 1
  • Choong Mo Kang
    • 1
  • Irina Balyasnikova
    • 2
  • Nick Devoogdt
    • 3
  • Angeline N. Ta
    • 4
  • Brian R. McNaughton
    • 4
  • Michael R. Zalutsky
    • 1
  1. 1.Department of RadiologyDuke University Medical CenterDurhamUSA
  2. 2.The Feinberg School of MedicineNorthwestern UniversityChicagoUSA
  3. 3.In Vivo Cellular and Molecular Imaging LaboratoryVrije Universiteit BrusselBrusselsBelgium
  4. 4.Department of ChemistryColorado State UniversityFort CollinsUSA

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