BACKGROUND: Targeted alpha therapy represents an advanced and rapidly evolving form of precision cancer treatment with increasing importance in recent years. The alpha-emitter 225Ac plays a key role in this clinical development due to its attractive physical and chemical properties. In this context, the macropa chelator has favorable characteristics in terms of labeling conditions and complex stability, making its derivatives exceptionally appealing for 225Ac-labeling of heat-sensitive biomolecules. However, preclinical evaluation of such 225Ac-containing molecules and comprehensive assessment of their pharmacokinetics, dosimetry and radiobiology necessitate a suitable diagnostic counterpart. Due to its attractive radiation properties, 133La represents an adequate positron-emitting radionuclide to form a matched pair with 225Ac for macropa-based radiopharmaceuticals. Herein, we describe the preparation and radiopharmacological characterization of macropa-functionalized, 133La/225Ac-labeled single-domain antibodies (sdAbs) targeting the epidermal growth factor receptor (EGFR) to demonstrate the general suitability of this theranostic pair of radionuclides. RESULTS: The synthesis of a clickable, bicyclononyne-modified macropa chelator and its site-specific conjugation to azide-modified, monovalent and biparatopic sdAbs is presented. Subsequent labeling at room temperature (rt) for 15 min resulted in molar activities of 30 MBq/nmol for 133La and 0.5 MBq/nmol for 225Ac, respectively. In vitro studies using the 133La-labeled sdAbs revealed comparable binding characteristics, but an enhanced cellular internalization of the biparatopic variant compared to its monovalent counterparts. This increased uptake consequently resulted in higher cytotoxicity of the 225Ac-labeled biparatopic conjugate. In vivo PET imaging of the 133La-labeled conjugates indicated comparable uptake and retention of the mono- and biparatopic variants in liver and kidneys, with the former showing slightly higher tumor accumulation. Ex vivo biodistribution studies conducted with 225Ac-labeled conjugates largely confirmed the findings obtained by PET imaging, albeit with a marginally higher tumor accumulation of the biparatopic 225Ac-radioimmunoconjugate. Final histological examinations of tumor and kidney tissues showed DNA damage in the renal cortex of the 225Ac-radioimmunoconjugate-treated mice, but no differences in the number of γ-H2AX-positive cells in the corresponding tumor tissues could be detected. CONCLUSIONS: We present a comprehensive study on the theranostic application of 133La and 225Ac for antibody-based biomolecules and lay the foundation for the future application of this matched pair of radionuclides towards labeling of heat-sensitive, macropa-functionalized radiopharmaceuticals in general. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s41181-025-00354-7.

Czech Advanced Technology and Research Institute Palacký University Olomouc Czech Republic

German Cancer Consortium Partner Site Dresden Fetscherstraße 74 01307 Dresden Germany

Helmholtz Zentrum Dresden Rossendorf Institute of Radiopharmaceutical Cancer Research Dresden Germany

Institute of Clinical and Molecular Pathology Faculty of Medicine and Dentistry Palacký University Olomouc Olomouc Czech Republic

Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacký University Olomouc Czech Republic

Laboratory of Experimental Medicine University Hospital Olomouc Czech Republic

National Center for Tumor Diseases Dresden University Hospital Carl Gustav Carus Fetscherstraße 74 01307 Dresden Germany

School of Science Faculty of Chemistry and Food Chemistry Technische Universität Dresden Dresden Germany

Zobrazit více v PubMed

Akiba H, Tsumoto K. Development and activities, including Immunocomplex formation, of biparatopic antibodies and alternative scaffold proteins. Translational Regul Sci. 2020;2(1):1–6.

Beltran Hernandez I, Rompen R, Rossin R, Xenaki KT, Katrukha EA, Nicolay K, van Bergen En Henegouwen P, Grull H, Oliveira S. Imaging of tumor spheroids, Dual-Isotope SPECT, and autoradiographic analysis to assess the tumor uptake and distribution of different nanobodies. Mol Imaging Biol. 2019;21(6):1079–88. PubMed

Bidkar AP, Zerefa L, Yadav S, VanBrocklin HF, Flavell RR. Actinium-225 targeted alpha particle therapy for prostate cancer. Theranostics. 2024;14(7):2969–92. PubMed PMC

Blei MK, Waurick L, Reissig F, Kopka K, Stumpf T, Drobot B, Kretzschmar J, Mamat C. Equilibrium thermodynamics of Macropa complexes with selected metal isotopes of radiopharmaceutical interest. Inorg Chem. 2023;62(50):20699–709. PubMed PMC

Bodei L, Herrmann K, Schoder H, Scott AM, Lewis JS. Radiotheranostics in oncology: current challenges and emerging opportunities. Nat Rev Clin Oncol. 2022;19(8):534–50. PubMed PMC

Brandt F, Ullrich M, Laube M, Kopka K, Bachmann M, Löser R, Pietzsch J, Pietzsch HJ, van den Hoff J, Wodtke R. Clickable albumin binders for modulating the tumor uptake of targeted radiopharmaceuticals. J Med Chem. 2022;65(1):710–33. PubMed

Brühlmann SA, Kreller M, Pietzsch HJ, Kopka K, Mamat C, Walther M, Reissig F. Efficient production of the PET radionuclide PubMed PMC

Brühlmann SA, Walther M, Blei MK, Mamat C, Kopka K, Freudenberg R, Kreller M. Scalability study on [ PubMed PMC

Burkett BJ, Bartlett DJ, McGarrah PW, Lewis AR, Johnson DR, Berberoglu K, Pandey MK, Packard AT, Halfdanarson TR, Hruska CB. Johnson GB and Kendi AT. A review of theranostics: perspectives on emerging approaches and clinical advancements. Radiol Imaging Cancer. 2023;5(4):e220157. PubMed PMC

Castillo Seoane D, de Saint-Hubert M, Crabbe M, Struelens L, Koole M. Targeted alpha therapy: a critical review of translational dosimetry research with emphasis on actinium-225. Q J Nucl Med Mol Imaging. 2020;64(3):265–77. PubMed

Cieslik P, Kubeil M, Zarschler K, Ullrich M, Brandt F, Anger K, Wadepohl H, Kopka K, Bachmann M, Pietzsch J, Stephan H. Comba P. Toward personalized medicine: one Chelator for imaging and therapy with Lutetium-177 and Actinium-225. J Am Chem Soc. 2022;144(47):21555–67. PubMed

Coll RP, Bright SJ, Martinus DKJ, Georgiou DK, Sawakuchi GO, Manning HC. Alpha Particle-Emitting radiopharmaceuticals as Cancer therapy: biological basis, current status, and future outlook for therapeutics discovery. Mol Imaging Biol. 2023;25(6):991–1019. PubMed PMC

Currie GM, Rohren EM. Sharpening the blade of precision theranostics. Semin Nucl Med. 2025. PubMed

Debie P, Lafont C, Defrise M, Hansen I, van Willigen DM, van Leeuwen FWB, Gijsbers R, D’Huyvetter M, Devoogdt N, Lahoutte T, Mollard P, Hernot S. Size and affinity kinetics of nanobodies influence targeting and penetration of solid tumours. J Control Release. 2020;317:34–42. PubMed

Dommerholt J, Schmidt S, Temming R, Hendriks LJ, Rutjes FP, van Hest JC, Lefeber DJ, Friedl P, van Delft FL. Readily accessible bicyclononynes for bioorthogonal labeling and three-dimensional imaging of living cells. Angew Chem Int Ed Engl. 2010;49(49):9422–5. PubMed PMC

Fan J, Zhuang X, Yang X, Xu Y, Zhou Z, Pan L, Chen S. A multivalent biparatopic EGFR-targeting nanobody drug conjugate displays potent anticancer activity in solid tumor models. Signal Transduct Target Ther. 2021;6(1):320. PubMed PMC

Fujimori K, Covell DG, Fletcher JE, Weinstein JN. A modeling analysis of monoclonal antibody percolation through tumors: a binding-site barrier. J Nucl Med. 1990;31(7):1191–8. PubMed

Gainkam LO, Huang L, Caveliers V, Keyaerts M, Hernot S, Vaneycken I, Vanhove C, Revets H, De Baetselier P, Lahoutte T. Comparison of the biodistribution and tumor targeting of two PubMed

Gainkam LO, Caveliers V, Devoogdt N, Vanhove C, Xavier C, Boerman O, Muyldermans S, Bossuyt A, Lahoutte T. Localization, mechanism and reduction of renal retention of technetium-99m labeled epidermal growth factor receptor-specific nanobody in mice. Contrast Media Mol Imaging. 2011a;6(2):85–92. PubMed

Gainkam LO, Keyaerts M, Caveliers V, Devoogdt N, Vanhove C, Van Grunsven L, Muyldermans S, Lahoutte T. Correlation between epidermal growth factor receptor-specific nanobody uptake and tumor burden: a tool for noninvasive monitoring of tumor response to therapy. Mol Imaging Biol. 2011b;13(5):940–8. PubMed

Heukers R, Vermeulen JF, Fereidouni F, Bader AN, Voortman J, Roovers RC, Gerritsen HC, van Bergen En Henegouwen PM. Endocytosis of EGFR requires its kinase activity and N-terminal transmembrane dimerization motif. J Cell Sci. 2013;126(Pt 21):4900–12. PubMed

Hooijman EL, Radchenko V, Ling SW, Konijnenberg M, Brabander T, Koolen SLW, de Blois E. Implementing Ac-225 labelled radiopharmaceuticals: practical considerations and (pre-)clinical perspectives. EJNMMI Radiopharm Chem. 2024;9(1):9. PubMed PMC

Huang L, Gainkam LO, Caveliers V, Vanhove C, Keyaerts M, De Baetselier P, Bossuyt A, Revets H, Lahoutte T. SPECT imaging with PubMed

Hurley K, Cao M, Huang H, Wang Y. Targeted alpha therapy (TAT) with Single-Domain antibodies (Nanobodies). Cancers (Basel). 2023;15(13):3493. PubMed PMC

Jang A, Kendi AT, Johnson GB, Halfdanarson TR, Sartor O. Targeted Alpha-Particle therapy: A review of current trials. Int J Mol Sci. 2023;24(14):11626. PubMed PMC

Jovcevska I, Muyldermans S. Therapeutic Potential Nanobodies BioDrugs. 2020;34(1):11–26. PubMed PMC

Jumapili NA, Zivalj M, Barthelmess RM, Raes G, De Groof TWM, Devoogdt N, Stijlemans B, Vincke C, Van Ginderachter JA. A few good reasons to use nanobodies for cancer treatment. Eur J Immunol. 2023;53(9):e2250024. PubMed

Jurcic JG. Targeted Alpha-Particle therapy for hematologic malignancies. J Med Imaging Radiat Sci. 2019;50(4 Suppl 1):S53–7. PubMed

Juweid M, Neumann R, Paik C, Perez-Bacete MJ, Sato J, van Osdol W, Weinstein JN. Micropharmacology of monoclonal antibodies in solid tumors: direct experimental evidence for a binding site barrier. Cancer Res. 1992;52(19):5144–53. PubMed

Kadassery KJ, King AP, Fayn S, Baidoo KE, MacMillan SN, Escorcia FE, Wilson JJ. H PubMed PMC

Karges J, Jakubaszek M, Mari C, Zarschler K, Goud B, Stephan H, Gasser G. Synthesis and characterization of an epidermal growth factor Receptor-Selective Ru PubMed PMC

Keenan MA, Stabin MG, Segars WP, Fernald MJ. RADAR realistic animal model series for dose assessment. J Nucl Med. 2010;51(3):471–6. PubMed PMC

Kelkar SS, Reineke TM. Theranostics: combining imaging and therapy. Bioconjug Chem. 2011;22(10):1879–903. PubMed

King AP, Lin FI, Escorcia FE. Why bother with alpha particles? Eur J Nucl Med Mol Imaging. 2021;49(1):7–17. PubMed

Kozempel J, Mokhodoeva O, Vlk M. Progress in targeted Alpha-Particle therapy. What we learned about recoils release from in vivo generators. Molecules. 2018;23(3):581. PubMed PMC

Krasniqi A, Bialkowska M, Xavier C, Van der Jeught K, Muyldermans S, Devoogdt N, D’Huyvetter M. Pharmacokinetics of radiolabeled dimeric SdAbs constructs targeting human CD20. N Biotechnol. 2018;45:69–79. PubMed

Kurfurstova D, Bartkova J, Vrtel R, Mickova A, Burdova A, Majera D, Mistrik M, Kral M, Santer FR, Bouchal J, Bartek J. DNA damage signalling barrier, oxidative stress and treatment-relevant DNA repair factor alterations during progression of human prostate cancer. Mol Oncol. 2016;10(6):879–94. PubMed PMC

Lepareur N, Ramee B, Mougin-Degraef M, Bourgeois M. Clinical advances and perspectives in targeted radionuclide therapy. Pharmaceutics. 2023;15(6):1733. PubMed PMC

Levantini E, Maroni G, Del Re M, Tenen DG. EGFR signaling pathway as therapeutic target in human cancers. Semin Cancer Biol. 2022;85:253–75. PubMed

Li C, Wen B, Wang L, Feng H, Xia X, Ding Z, Gao B, Zhang Y, Lan. X. PubMed

Makvandi M, Dupis E, Engle JW, Nortier FM, Fassbender ME, Simon S, Birnbaum ER, Atcher RW, John KD, Rixe O, Norenberg JP. Alpha-Emitters and targeted alpha therapy in oncology: from basic science to clinical investigations. Target Oncol. 2018;13(2):189–203. PubMed

McDevitt MR, Ma D, Lai LT, Simon J, Borchardt P, Frank RK, Wu K, Pellegrini V, Curcio MJ, Miederer M, Bander NH, Scheinberg DA. Tumor therapy with targeted atomic nanogenerators. Science. 2001;294(5546):1537–40. PubMed

Miederer M, Benesova-Schafer M, Mamat C, Kastner D, Pretze M, Michler E, Brogsitter C, Kotzerke J, Kopka K. Scheinberg DA and McDevitt MR. Alpha-Emitting radionuclides: current status and future perspectives. Pharmaceuticals (Basel). 2024;17(1):76. PubMed PMC

Morgenstern A, Apostolidis C, Kratochwil C, Sathekge M, Krolicki L, Bruchertseifer F. An overview of targeted alpha therapy with PubMed PMC

Morgenstern A, Lilley LM, Stein BW, Kozimor SA, Batista ER, Yang P. Computer-Assisted design of macrocyclic chelators for Actinium-225 radiotherapeutics. Inorg Chem. 2021;60(2):623–32. PubMed

Mourtada F, Tomiyoshi K, Sims-Mourtada J, Mukai-Sasaki Y, Yagihashi T, Namiki Y, Murai T, Yang DJ, Inoue T. Actinium-225 targeted agents: where are we now?? Brachytherapy. 2023;22(6):697–708. PubMed PMC

Movahedi K, Schoonooghe S, Laoui D, Houbracken I, Waelput W, Breckpot K, Bouwens L, Lahoutte T, De Baetselier P, Raes G, Devoogdt N, Van Ginderachter JA. Nanobody-based targeting of the macrophage mannose receptor for effective in vivo imaging of tumor-associated macrophages. Cancer Res. 2012;72(16):4165–77. PubMed

Muyldermans S. Nanobodies: natural single-domain antibodies. Annu Rev Biochem. 2013;82:775–97. PubMed

Nelson BJB, Wilson J, Andersson JD, Wuest F. High yield cyclotron production of a novel PubMed PMC

Nelson BJB, Andersson JD, Wuest F, Radiolanthanum. Promising theranostic radionuclides for PET, alpha, and Auger-Meitner therapy. Nucl Med Biol. 2022a;110–111:59–66. PubMed

Nelson BJB, Ferguson S, Wuest M, Wilson J, Duke MJM, Richter S, Soenke-Jans H, Andersson JD, Juengling F, Wuest F. First in vivo and Phantom imaging of Cyclotron-Produced PubMed PMC

Nelson BJB, Wilson J, Andersson JD, Wuest F. Theranostic imaging surrogates for targeted alpha therapy: progress in production, purification, and applications. Pharmaceuticals (Basel). 2023;16(11):1622. PubMed PMC

Niquille DL, Fitzgerald KM, Gera N. Biparatopic antibodies: therapeutic applications and prospects. MAbs. 2024;16(1):2310890. PubMed PMC

Price EW, Orvig C. Matching chelators to radiometals for radiopharmaceuticals. Chem Soc Rev. 2014;43(1):260–90. PubMed

Reissig F, Bauer D, Zarschler K, Novy Z, Bendova K, Ludik MC, Kopka K, Pietzsch HJ, Petrik M, Mamat C. Towards targeted alpha therapy with Actinium-225: chelators for mild condition radiolabeling and targeting PSMA-A proof of concept study. Cancers (Basel). 2021;13(8):1974. PubMed PMC

Reissig F, Zarschler K, Novy Z, Petrik M, Bendova K, Kurfurstova D, Bouchal J, Ludik MC, Brandt F, Kopka K, Khoylou M, Pietzsch HJ, Hajduch M, Mamat C. Modulating the Pharmacokinetic profile of Actinium-225-labeled macropa-derived radioconjugates by dual targeting of PSMA and albumin. Theranostics. 2022;12(17):7203–15. PubMed PMC

Robertson AKH, Ramogida CF, Schaffer P, Radchenko V. Development of PubMed PMC

Roovers RC, Vosjan MJ, Laeremans T, el Khoulati R, de Bruin RC, Ferguson KM, van Verkleij AJ. Dongen GA and Van Bergen En Henegouwen PM. A biparatopic anti-EGFR nanobody efficiently inhibits solid tumour growth. Int J Cancer. 2011;129(8):2013–24. PubMed PMC

Roscher M, Bakos G, Benesova M. Atomic Nanogenerators in Targeted Alpha Therapies: Curie’s Legacy in Modern Cancer Management. Pharmaceuticals (Basel). 2020;13(4):76. PubMed PMC

Rudnick SI, Adams GP. Affinity and avidity in antibody-based tumor targeting. Cancer Biother Radiopharm. 2009;24(2):155–61. PubMed PMC

Rudnick SI, Lou J, Shaller CC, Tang Y, Klein-Szanto AJ, Weiner LM, Marks JD, Adams GP. Influence of affinity and antigen internalization on the uptake and penetration of Anti-HER2 antibodies in solid tumors. Cancer Res. 2011;71(6):2250–9. PubMed PMC

Schmidt MM, Wittrup KD. A modeling analysis of the effects of molecular size and binding affinity on tumor targeting. Mol Cancer Ther. 2009;8(10):2861–71. PubMed PMC

Schmitz KR, Bagchi A, van Roovers RC. Bergen En Henegouwen PM and Ferguson KM. Structural evaluation of EGFR Inhibition mechanisms for nanobodies/vhh domains. Structure. 2013;21(7):1214–24. PubMed PMC

Sgouros G, Hobbs R, Josefsson A. Dosimetry and radiobiology of Alpha-Particle emitting radionuclides. Curr Radiopharm. 2018;11(3):209–14. PubMed

Sharifi J, Khirehgesh MR, Akbari B, Soleymani B, Mansouri K. Hu7CG2: A novel humanized Anti-Epidermal growth factor receptor (EGFR) biparatopic nanobody. Mol Biotechnol. 2021;63(6):525–33. PubMed

Singh G, Zarschler K, Hunoldt S, Martinez IIS, Ruehl CL, Matterna M, Bergmann R, Mathe D, Hegedus N, Bachmann M, Comba P. Versatile Bispidine-Based bifunctional chelators for PubMed PMC

Thiele NA, Brown V, Kelly JM, Amor-Coarasa A, Jermilova U, MacMillan SN, Nikolopoulou A, Ponnala S, Ramogida CF, Robertson AKH, Rodriguez-Rodriguez C, Schaffer P, Williams C Jr., Babich JW, Radchenko V, Wilson JJ. An Eighteen-Membered macrocyclic ligand for Actinium-225 targeted alpha therapy. Angew Chem Int Ed Engl. 2017;56(46):14712–7. PubMed

Thurber GM, Schmidt MM, Wittrup KD. Factors determining antibody distribution in tumors. Trends Pharmacol Sci. 2008;29(2):57–61. PubMed PMC

Tripathy RK, Pande AH. Molecular and functional insight into anti-EGFR nanobody: theranostic implications for malignancies. Life Sci. 2024;345:122593. PubMed

Tronchin S, Forster JC, Hickson K, Bezak E. Dosimetry in targeted alpha therapy. A systematic review: current findings and what is needed. Phys Med Biol. 2022;67(9). PubMed

Ullrich M, Bergmann R, Peitzsch M, Zenker EF, Cartellieri M, Bachmann M, Ehrhart-Bornstein M, Block NL, Schally AV, Eisenhofer G, Bornstein SR, Pietzsch J, Ziegler CG. Multimodal somatostatin receptor theranostics using [(64)Cu]Cu-/[(177)Lu]Lu-DOTA-(Tyr(3))octreotate and AN-238 in a mouse pheochromocytoma model. Theranostics. 2016;6(5):650–65. PubMed PMC

Vahidfar N, Eppard E, Farzanehfar S, Yordanova A, Fallahpoor M, Ahmadzadehfar H. An impressive approach in nuclear medicine: theranostics. PET Clin. 2021;16(3):327–40. PubMed

Wang Z. ErbB receptors and Cancer. Methods Mol Biol. 2017;1652:3–35. PubMed

Weinstein JN, van Osdol W. Early intervention in cancer using monoclonal antibodies and other biological ligands: micropharmacology and the binding site barrier. Cancer Res. 1992;52(9 Suppl):s2747–51. PubMed

White JM, Escorcia FE, Viola NT. Perspectives on metals-based radioimmunotherapy (RIT): moving forward. Theranostics. 2021;11(13):6293–314. PubMed PMC

Xenaki KT, Oliveira S, van Bergen En Henegouwen PMP. Antibody or antibody fragments: implications for molecular imaging and targeted therapy of solid tumors. Front Immunol. 2017;8:1287. PubMed PMC

Yang H, Zhang C, Yuan Z, Rodriguez-Rodriguez C, Robertson A, Radchenko V, Perron R, Gendron D, Causey P, Gao F, Benard F, Schaffer P. Synthesis and evaluation of a macrocyclic Actinium-225 chelator, quality control and in vivo evaluation of PubMed

Zarschler K, Witecy S, Kapplusch F, Foerster C, Stephan H. High-yield production of functional soluble single-domain antibodies in the cytoplasm of Escherichia coli. Microb Cell Fact. 2013;12:97. PubMed PMC

Zarschler K, Prapainop K, Mahon E, Rocks L, Bramini M, Kelly PM, Stephan H, Dawson KA. Diagnostic nanoparticle targeting of the EGF-receptor in complex biological conditions using single-domain antibodies. Nanoscale. 2014;6(11):6046–56. PubMed