Targeted Radionuclide Therapy with A ¹⁷⁷Lu-labeled Anti-HER2 Nanobody

Abstract

RIT has become an attractive strategy in cancer treatment, but still faces important drawbacks due to poor tumor penetration and undesirable pharmacokinetics of the targeting vehicles. Smaller radiolabeled antibody fragments and peptides feature highly specific target accumulation, resulting in low accumulation in healthy tissue, except for the kidneys. Nanobodies are the smallest (MW < 15 kDa) functional antigen-binding fragments that are derived from heavy chain-only camelid antibodies.Here, we show that the extend of kidney retention of nanobodies is predominantly dictated by the number of polar residues in the C-terminal amino acid tag. Three nanobodies were produced with different C-terminal amino-acid tag sequences (Myc-His-tagged, His-tagged, and untagged). Dynamic planar imaging of Wistar rats with 111In-DTPA-nanobodies revealed that untagged nanobodies showed a 70 % drop in kidney accumulation compared to Myc-His-tagged nanobodies at 50 min p.i.. In addition, coinfusion of untagged nanobodies with the plasma expander Gelofusin led to a final reduction of 90 %. Similar findings were obtained with different 177Lu-DTPA-2Rs15d nanobody constructs in HER2pos tumor xenografted mice at 1 h p.i.. Kidney accumulation decreased 88 % when comparing Myc-His-tagged to untagged 2Rs15d nanobody, and 95 % with a coinfusion of Gelofusin, without affecting the tumor targeting capacity. Consequently, we identified a generic method to reduce kidney retention of radiolabeled nanobodies. Dosimetry calculations of Gelofusin-coinfused, untagged 177Lu-DTPA-2Rs15d revealed a dose of 0.90 Gy/MBq that was delivered to both tumor and kidneys and extremely low doses to healthy tissues. In a comparative study, 177Lu-DTPA-Trastuzumab supplied 6 times more radiation to the tumor than untagged 177Lu-DTPA-2Rs15d, but concomitantly also a 155, 34, 80, 26 and 4180 fold higher radioactivity burden to lung, liver, spleen, bone and blood.Most importantly, nanobody-based targeted radionuclide therapy in mice bearing small estiblashed HER2pos tumors led to an almost complete blockade of tumor growth and a significant difference in event-free survival between the treated and the control groups (P < 0.0001). Based on histology analyses, no evidence of renal inflammation, apoptosis or necrosis was obtained.In conclusion, these data highlight the importance of the amino acid composition of the nanobody's C-terminus, as it has a predominant effect on kidney retention. Moreover, we show successful nanobody-based targeted radionuclide therapy in a xenograft model and highlight the potential of radiolabeled nanobodies as a valuable adjuvant therapy candidate for treatment of minimal residual and metastatic disease.

Theranostic Radiolabeled Anti-CD20 sdAb for Targeted Radionuclide Therapy of Non-Hodgkin Lymphoma

“…The theoretical pI of his-tagged sdAb 9079 was lower (7.18) than the pI of sdAb 9077 (8.66), which could explain the lower degree of interaction of sdAb 9079 with the negatively charged lumen of the proximal tubuli in the kidneys compared with the other sdAbs. In addition, after removal of the C-tag and with a coinjection of 150 mg/kg gelofusin, its accumulation in kidneys was further decreased, in agreement with our previous work (24).…”

“…One of the most applied strategies to reduce kidney uptake is the coinjection of positively charged amino acids (34,35). In addition, the introduction of carboxylterminal (C-)tags to sdAbs dramatically increase kidney retention upon renal clearance (24). To this end, sdAbs 9077 and 9079 were produced without a C-tag for further characterization.…”

“…We also endeavored the use of sdAbs as vehicles for targeted radionuclide therapy (TRNT) in preclinical cancer models, through the therapeutic b À -particle-emitting radionuclide 177 Lu. These proof-of-concept studies showed strong signs of tumor growth inhibition with absence of toxicity in nontarget tissues (21,24).…”

“…Data obtained from the comparative ex vivo biodistribution study were time integrated for the dosimetry calculation of 177 Lu-DTPA-sdAb 9079 and 177 Lu-DTPA-rituximab (24,30). Briefly, the integrations between time 0 and 72 hours for 177 Lu-DTPA-sdAb 9079 and between 0 and 120 hours for 177 Lu-DTPA-rituximab were made using the trapezoid method.…”

Imaging Diagnostic and Therapeutic Targets: Human Epidermal Growth Factor Receptor 2

“…RIT with 90 Ytrastuzumab was even found to be applicable to BC expressing small amounts of HER2 (61). The results obtained with other candidates for RIT, such as the smaller Affibody molecules (66) and Nanobodies ( 177 Lu (67) or 90 Y (68)), have been encouraging. In addition, a first-in-human study assessing the safety of intraperitoneal a-RIT with 212 Pb-TCMC-trastuzumab was presented recently.…”