Introduction With a molecular weight an order of magnitude lower than antibodies but possessing comparable affinities, Nanobodies (Nbs) are attractive as targeting agents for cancer diagnosis and therapy. An anti-HER2 Nb could be utilized to determine HER2 status in breast cancer patients prior to trastuzumab treatment. This provided motivation for the generation of HER2-specific 5F7GGC Nb, its radioiodination and evaluation for targeting HER2 expressing tumors. Methods 5F7GGCNb was radioiodinated with 125I using Iodogen and with 131I using the residualizing agent Nε-(3-[131I]iodobenzoyl)-Lys5-Nα-maleimido-Gly1-GEEEK ([131I]IB-Mal-D-GEEEK) used previously successfully with intact antibodies. Paired-label internalization assays using BT474M1 cells and tissue distribution experiments in athymic mice bearing BT474M1 xenografts were performed to compare the two labeled Nb preparations. Results The radiochemical yields for Iodogen and [131I]IB-Mal-D-GEEEK labeling were 83.6±5.0%(n= 10) and 59.6±9.4% (n = 15), respectively. The immunoreactivity of labeled proteins was preserved as confirmed by in vitro and in vivo binding to tumor cells. Biodistribution studies showed that Nb radiolabeled using [131I]IB-Mal-D-GEEEK, compared with the directly labeled Nb, had a higher tumor uptake (4.65 ± 0.61% ID/g vs. 2.92 ± 0.24% ID/g at 8 h), faster blood clearance, lower accumulation in non-target organs except kidneys, and as a result, higher concomitant tumor-to-blood and tumor-to-tissue ratios. Conclusions Taken together, these results demonstrate that 5F7GGC anti-HER2 Nb labeled with residualizing [131I]IB-Mal-D-GEEEK had better tumor targeting properties compared to the directly labeled Nb suggesting the potential utility of this Nb conjugate for SPECT (123I)and PET imaging (124I) of patients with HER2-expressing tumors.

Nanobodies are approximately 15-kDa proteins based on the smallest functional fragments of naturally occurring heavy chain–only antibodies and represent an attractive platform for the development of molecularly targeted agents for cancer diagnosis and therapy. Because the human epidermal growth factor receptor type 2 (HER2) is overexpressed in breast and ovarian carcinoma, as well as in other malignancies, HER2-specific Nanobodies may be valuable radiodiagnostics and therapeutics for these diseases. The aim of the present study was to evaluate the tumor-targeting potential of anti-HER2 5F7GGC Nanobody after radioiodination with the residualizing agent N-succinimidyl 4-guanidinomethyl 3-125/131I-iodobenzoate (*I-SGMIB). Methods The 5F7GGC Nanobody was radiolabeled using *I-SGMIBand, for comparison, withNε-(3-*I-iodobenzoyl)-Lys5-Nα-maleimido-Gly1-GEEEK (*I-IB-Mal-D-GEEEK), another residualizing agent, and by direct radioiodination using IODO-GEN (125I-Nanobody). The 3 labeled Nanobodies were evaluated in affinity measurements, and paired-label internalization assays were performed on HER2-expressing BT474M1 breast carcinoma cells and in paired-label tissue distribution measurements in mice bearing subcutaneous BT474M1 xenografts. Results *I-SGMIB-Nanobody was produced in 50.4% ± 3.6% radiochemical yield and exhibited a dissociation constant of 1.5 ± 0.5 nM. Internalization assays demonstrated that intracellular retention of radioactivity was up to 1.5-fold higher for *I-SGMIB-Nanobody than for coincubated 125I-Nanobody or *I-IB-Mal-D-GEEEK-Nanobody. Peak tumor uptake for *I-SGMIB-Nanobody was 24.50% ± 9.89% injected dose/g at 2 h, 2- to 4-fold higher than observed with other labeling methods, and was reduced by 90% with trastuzumab blocking, confirming the HER2 specificity of localization. Moreover, normal-organ clearance was fastest for *I-SGMIB-Nanobody, such that tumor–to–normal-organ ratios greater than 50:1 were reached by 24 h in all tissues except lungs and kidneys, for which the values were 10.4 ± 4.5 and 5.2 ± 1.5, respectively. Conclusion Labeling anti-HER2 Nanobody 5F7GGC with *I-SGMIB yields a promising new conjugate for targeting HER2-expressing malignancies. Further research is needed to determine the potential utility of *I-SGMIB-5F7GGC labeled with 124I, 123I, and 131I for PET and SPECT imaging and for targeted radiotherapy, respectively.

Alpha-particle emitters have a high linear energy transfer and short range, offering the potential for treating micrometastases while sparing normal tissues. We developed a urea-based, 211 At-labeled small molecule targeting prostate-specific membrane antigen (PSMA) for the treatment of micrometastases due to prostate cancer (PC). Methods: PSMA-targeted (2S)-2-(3-(1-carboxy-5-(4-211 At-astatobenzamido) pentyl)ureido)-pentanedioic acid ( 211 At-6) was synthesized. Cellular uptake and clonogenic survival were tested in PSMA-positive (PSMA1) PC3 PIP and PSMA-negative (PSMA−) PC3 flu human PC cells after 211 At-6 treatment. The antitumor efficacy of 211 At-6 was evaluated in mice bearing PSMA1 PC3 PIP and PSMA-PC3 flu flank xenografts at a 740-kBq dose and in mice bearing PSMA1, luciferase-expressing PC3-ML micrometastases. Biodistribution was determined in mice bearing PSMA1 PC3 PIP and PSMA-PC3 flu flank xenografts. Suborgan distribution was evaluated using α-camera images, and microscale dosimetry was modeled. Longterm toxicity was assessed in mice for 12 mo. Results: 211 At-6 treatment resulted in PSMA-specific cellular uptake and decreased clonogenic survival in PSMA1 PC3 PIP cells and caused significant tumor growth delay in PSMA1 PC3 PIP flank tumors. Significantly improved survival was achieved in the newly developed PSMA1 micrometastatic PC model. Biodistribution showed uptake of 211 At-6 in PSMA1 PC3 PIP tumors and in kidneys. Microscale kidney dosimetry based on α-camera images and a nephron model revealed hot spots in the proximal renal tubules. Long-term toxicity studies confirmed that the dose-limiting toxicity was late radiation nephropathy. Conclusion: PSMA-targeted 211 At-6 α-particle radiotherapy yielded significantly improved survival in mice bearing PC micrometastases after systemic administration. 211 At-6 also showed uptake in renal proximal tubules resulting in late nephrotoxicity, highlighting the importance of long-term toxicity studies and microscale dosimetry.