Targeted radionuclide therapy with A 177Lu-labeled anti-HER2 nanobody

Matthias D'Huyvetter, Cécile Vincke, Catarina Xavier, An Aerts, Nathalie Impens, Sarah Baatout, Hendrik De Raeve, Serge Muyldermans, Vicky Caveliers, Nick Devoogdt, Tony Lahoutte, Matthias D'Huyvetter, Cécile Vincke, Catarina Xavier, An Aerts, Nathalie Impens, Sarah Baatout, Hendrik De Raeve, Serge Muyldermans, Vicky Caveliers, Nick Devoogdt, Tony Lahoutte

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.

Keywords: 177-Lutetium.; HER2; nanobody; targeted radionuclide therapy.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
Zimmerman - polarity score plot for the various 2Rs15d nanobody formats. (A) untagged 2Rs15d; (B) His-tagged 2Rs15d and (C) Myc-His-tagged 2Rs15d. Introduction of charged amino acids at the nanobody's C-terminus has a major impact on the polarity score.
Figure 2
Figure 2
(Radio-)chromatographic analyses of various untagged 2Rs15d conjugates. (A) unconjugated nanobody, (B) CHX-A”-DTPA-2Rs15d, (C) 1B4M-DTPA-2Rs15d, (D)177Lu-DTPA-2Rs15d, (E)111In-DTPA-2Rs15d; (A-C) SEC on Superdex 10/30, (D) radio-SEC on Superdex 75 5/150GL; (E) radio-HPLC on PLRP-S. The R-times of the major peaks are shown in each graph.
Figure 3
Figure 3
Accumulation of radioactivity in kidneys in function of time after injection of various 111In-labeled 2Rs15d formats in healthy Wistar rats (n=3); (A-D) A selection of images are shown obtained by gamma camera dynamic scintigraphy; (A) 2Rs15d-Myc-His-tag, (B) 2Rs15d-His-tag, (C) untagged 2Rs15d, (D) untagged 2Rs15d coinfused with 80 mg/kg Gelofusin. (E) Time-activity curves of renal retention in rats (n=3 per condition). Lowest accumulation in kidneys was observed for the 111In-labeled untagged 2Rs15d that was coinfused with 150 mg/kg Gelofusin.
Figure 4
Figure 4
Ex vivo biodistribution analyses of 177Lu-labeled 2Rs15d constructs in HER2pos tumor xenografted mice, at 1 h p.i. Animals were injected with 21.5 MBq (4 µg) radioconjugates. Columns, mean (n=3); bars, SD. Kidney accumulation decreased significantly by removing the C-terminal amino acid tag, and by a coinfusion with Gelofusin. Tumor targeting was not affected.
Figure 5
Figure 5
Tumor growth monitoring during targeted radionuclide therapy. Tumor volumes were quantified using (A) bioluminescence imaging (ph/s/cm2/sr) or (B) caliper measurements (mm3), in function of time (days). Animals (n=8 per group) were treated with a weekly injection of untagged 177Lu-labeled 2Rs15d (20.7 ± 0.4 MBq) and in the control groups with PBS or 177Lu-labeled BCII10 (19.3 ± 0.8 MBq). All treatments occurred with a 150 mg/kg Gelofusin coinjection. In terms of tumor growth, important differences were observed between both control groups and the treated group, for both caliper and bioluminescence measurements.
Figure 6
Figure 6
(A) Event-free survival during targeted radionuclide therapy. Events are defined as 1. Mortality; 2. > 20 % weight loss; 3. Ulcerating tumor tissue; 4. Tumor volume > 250 mm3. Animals (n=8 per group) were treated with a weekly injection of untagged 177Lu-labeled 2Rs15d (20.7 ± 0.4 MBq) and in the control groups with PBS or 177Lu-labeled BCII10 (19.3 ± 0.8 MBq). All treatments occurred with a 150 mg/kg Gelofusin coinjection. (B) Renal histopathology. Kidneys of 177Lu-dosed animal groups were compared to the PBS-treated animal group. Sections were H&E stained and examined for evidence of renal toxicity. No differences in renal histology were observed between the animal groups that received (B.1) PBS, (B.2) untagged 177Lu-labeled BCII10 or (B.3) untagged 177Lu-labeled 2Rs15d.

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