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Introduction: Single domain antibody fragments (sdAbs) are an appealing scaffold for radiopharmaceutical development due to their small size (~15 kDa), high solubility, high stability, and excellent tumor penetration. Previously, we developed NB7 sdAb, which has very high affinity for an epitope on PSMA that is different from those targeted by small molecule PSMA inhibitors. Herein, we evaluated NB7 after radioiodination using [*I]SGMIB (1,3,4-isomer) and iso-[*I]SGMIB (1,3,5-isomer), as well as their 211 At-labeled analogues., Methods: [*I]SGMIB, iso-[*I]SGMIB, [ 211 At]SAGMB, and iso-[ 211 At]SAGMB conjugates of NB7 sdAb were synthesized and their binding affinity, cell uptake and internalization were assessed in PSMA + PC3 PIP and PSMA - PC3 flu cells. Biodistribution studies were performed in mice bearing PSMA + PC3 PIP xenografts. First, a single-label experiment evaluated the tissue distribution of a NB7 bearing a His 6 -tag (NB7H6) and labeled with iso-[ 125 I]SGMIB. Three paired-label experiments then were performed to compare: a) NB7 labeled using [*I]SGMIB and iso-[*I]SGMIB, b) 131 I- vs 211 At-labeled NB7 conjugates and c) [ 125 I]SGMIB-NB7H6 to the small molecule PSMA inhibitor [ 131 I]YF2., Results: All NB7 radioconjugates bound specifically to PSMA with dissociation constants, K d , in the low nM range (1.4-6.4 nM). An initial biodistribution study demonstrated good tumor uptake for iso-[ 125 I]SGMIB-NB7H6 (7.2 ± 1.5 % ID/g at 1 h) and no deleterious effect of the His 6 -tag on renal activity levels, which declined to 3.1 ± 1.1 % ID/g by 4 h. Paired-label biodistribution found no distinction between the two SGMIB isomer NB7 conjugates with the [ 131 I]SGMIB-NB7-to-iso-[ 125 I]SGMIB-NB7 tumor uptake ratios not significantly different from unity: 1.06 ± 0.08 at 1 h, 1.04 ± 0.12 at 4 h, and 1.07 ± 0.09 at 24 h. Both isomer conjugates cleared rapidly from normal tissues and exhibited very low uptake in thyroid, lacrimal and salivary glands. Paired-label biodistribution of [ 131 I]SGMIB-NB7H6 and [ 211 At]SAGMB-NB7H6 demonstrated similar tumor uptake and kidney clearance for the two radioconjugates. However, levels of 211 At in thyroid, stomach, salivary and lacrimal glands were significantly higher (P < 0.05) that those for 131 I suggesting greater dehalogenation for [ 211 At]SAGMB-NB7H6. Finally, co-administration of [ 125 I]SGMIB-NB7H6 and [ 131 I]YF2 demonstrated good tumor uptake for both with considerably more rapid renal clearance for the NB7 radioconjugate., Conclusion: NB7 radioconjugates exhibited good accumulation in PSMA-positive xenografts with rapid clearance from kidney and other normal tissues. We conclude that NB7 is a potentially useful scaffold for developing PSMA-targeted theranostics with different characteristics than current small molecule and antibody-based approaches., Competing Interests: Declaration of competing interest LR and NP have filed a patent application that includes the NB7 single domain antibody fragment. The authors declare that they have no other known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Single-domain antibody fragments (sdAbs) are attractive for targeted α-particle therapy, particularly with 211 At, because of their rapid accumulation in tumor and clearance from normal tissues. Here, we evaluate the therapeutic potential of this strategy with 5F7 and VHH&#95;1028-2 sdAbs that bind with high affinity to domain IV of human epidermal growth factor receptor type 2 (HER2). Methods: The HER2-specific sdAbs and HER2-irrelevant VHH&#95;2001 were labeled using N -succinimidyl-3- 211 At-astato-5-guanidinomethyl benzoate ( iso - 211 At-SAGMB). The cytotoxicity of iso- 211 At-SAGMB-5F7 and iso- 211 At-SAGMB-VHH&#95;2001 were compared on HER2-expressing BT474 breast carcinoma cells. Three experiments in mice with subcutaneous BT474 xenografts were performed to evaluate the therapeutic effectiveness of single doses of iso- 211 At-SAGMB-5F7 (0.7-3.0 MBq), iso- 211 At-SAGMB-VHH&#95;1028 (1.0-3.0 MBq), and iso- 211 At-SAGMB-VHH&#95;1028 and iso- 211 At-SAGMB-VHH&#95;2001 (∼1.0 MBq). Results: Clonogenic survival of BT474 cells was reduced after exposure to iso- 211 At-SAGMB-5F7 (D 0  = 1.313 kBq/mL) whereas iso- 211 At-SAGMB-VHH&#95;2001 was ineffective. Dose-dependent tumor growth inhibition was observed with 211 At-labeled HER2-specific 5F7 and VHH&#95;1028 but not with HER2-irrelevant VHH&#95;2001. At the 3.0-MBq dose, complete tumor regression was seen in 3 of 4 mice treated with iso- 211 At-SAGMB-5F7 and 8 of 11 mice treated with iso- 211 At-SAGMB-VHH&#95;1028; prolongation in median survival was 495% and 414%, respectively. Conclusion: Combining rapidly internalizing, high-affinity HER2-targeted sdAbs with the iso- 211 At-SAGMB residualizing prosthetic agent is a promising strategy for targeted α-particle therapy of HER2-expressing cancers., (© 2023 by the Society of Nuclear Medicine and Molecular Imaging.)

Radiopharmaceutical therapy (RPT) is an attractive strategy for treatment of disseminated cancers including those overexpressing the HER2 receptor including breast, ovarian and gastroesophageal carcinomas. Single-domain antibody fragments (sdAbs) exemplified by the HER2-targeted VHH&#95;1028 evaluated herein are attractive for RPT because they rapidly accumulate in tumor and clear faster from normal tissues than intact antibodies. In this study, VHH&#95;1028 was labeled using the residualizing prosthetic agent N-succinimidyl 3-guanidinomethyl 5-[ 131 I]iodobenzoate (iso-[ 131 I]SGMIB) and its tissue distribution evaluated in the HER2-expressing SKOV-3 ovarian and BT474 breast carcinoma xenograft models. In head-to-head comparisons to [ 131 I]SGMIB-2Rs15d, a HER2-targeted radiopharmaceutical currently under clinical investigation, iso-[ 131 I]SGMIB-VHH&#95;1028 exhibited significantly higher tumor uptake and significantly lower kidney accumulation. The results demonstrated 2.9 and 6.3 times more favorable tumor-to-kidney radiation dose ratios in the SKOV-3 and BT474 xenograft models, respectively. Iso-[ 131 I]SGMIB-VHH&#95;1028 was prepared using a solid-phase extraction method for purification of the prosthetic agent intermediate Boc 2 -iso-[ 131 I]SGMIB that reproducibly scaled to therapeutic-level doses and obviated the need for its HPLC purification. Single-dose (SKOV-3) and multiple-dose (BT474) treatment regimens demonstrated that iso-[ 131 I]SGMIB-VHH&#95;1028 was well tolerated and provided significant tumor growth delay and survival prolongation. This study suggests that iso-[ 131 I]SGMIB-VHH&#95;1028 is a promising candidate for RPT of HER2-expressing cancers and further development is warranted., (© 2022. The Author(s).)

Single-domain antibody fragments (sdAbs) are promising vectors for immuno-PET; however, better methods for labeling sdAbs with 18 F are needed. Herein, we evaluate a site-specific strategy using an 18 F residualizing motif and the anti-epidermal growth factor receptor 2 (HER2) sdAb 5F7 bearing an engineered C-terminal GGC tail (5F7GGC). Methods: 5F7GGC was site-specifically attached with a tetrazine-bearing agent via thiol-maleimide reaction. The resultant conjugate was labeled with 18 F by inverse electron demand Diels-Alder cycloaddition with a trans -cyclooctene attached to 6- 18 F-fluoronicotinoyl moiety via a renal brush border enzyme-cleavable linker and a PEG 4 chain ( 18 F-5F7GGC). For comparisons, 5F7 sdAb was labeled using the prototypical residualizing agent, N -succinimidyl 3-(guanidinomethyl)-5- 125 I-iodobenzoate ( iso - 125 I-SGMIB). The 2 labeled sdAbs were compared in paired-label studies performed in the HER2-expressing BT474M1 breast carcinoma cell line and athymic mice bearing BT474M1 subcutaneous xenografts. Small-animal PET/CT imaging after administration of 18 F-5F7GGC in the above mouse model was also performed. Results: 18 F-5F7GGC was synthesized in an overall radiochemical yield of 8.9% ± 3.2% with retention of HER2 binding affinity and immunoreactivity. The total cell-associated and intracellular activity for 18 F-5F7GGC was similar to that for coincubated iso - 125 I-SGMIB-5F7. Likewise, the uptake of 18 F-5F7GGC in BT474M1 xenografts in mice was similar to that for iso - 125 I-SGMIB-5F7; however, 18 F-5F7GGC exhibited significantly more rapid clearance from the kidney. Small-animal PET/CT imaging confirmed high uptake and retention in the tumor with very little background activity at 3 h except in the bladder. Conclusion: This site-specific and residualizing 18 F-labeling strategy could facilitate clinical translation of 5F7 anti-HER2 sdAb as well as other sdAbs for immuno-PET., (© 2021 by the Society of Nuclear Medicine and Molecular Imaging.)

Introduction: Labeling single domain antibody fragments (sdAbs) with 18 F is an attractive strategy for immunoPET. Earlier, we developed a residualizing label, N-succinimidyl 3-((4-(4-fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(guanidinomethyl)benzoate ([ 18 F]RL-I), synthesized via a click reaction for labeling sdAbs with 18 F, that has attractive features but suffered from modest radiochemical yields and suboptimal hydrophobicity. Herein, we have evaluated the potential utility of an analogous agent, N-succinimidyl 3-(1-(2-(2-(2-(2-[ 18 F]fluoroethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-5-(guanidinomethyl)benzoate ([ 18 F]SFETGMB; [ 18 F]RL-III) designed to address these limitations., Methods: [ 18 F]RL-III was synthesized by the click reaction between 3-((2,3-bis(tert-butoxycarbonyl)guanidino)methyl)-5-ethynylbenzoate and 1-azido-2-(2-(2-(2-[ 18 F]fluoroethoxy)ethoxy)ethoxy)ethane and subsequent deprotection. The anti-HER2 sdAbs 5F7 and 2Rs15d were labeled by conjugation with [ 18 F]RL-III and compared in a paired-label fashion to the sdAbs labeled using N-succinimidyl 4-guanidinomethyl-3-[ 125 I]iodobenzoate ([ 125 I]SGMIB) or N-succinimidyl 3-guanidinomethyl-5-[ 125 I]iodobenzoate (iso-[ 125 I]SGMIB). The 18 F-labeled sdAbs were evaluated in vitro using HER2-expressing breast and ovarian carcinoma cells (BT474/BT474M1 and SKOV-3) and in vivo in athymic mice bearing subcutaneous SKOV-3 or BT474 xenografts. PET imaging of athymic mice bearing either subcutaneous BT474 or intracranial BT474M1Br-Fluc xenografts after administration of [ 18 F]RL-III-5F7 also was performed., Results: Radiochemical yields for the synthesis of Boc 2 -[ 18 F]RL-III (21.5 ± 3.4%) were significantly higher than reported for Boc 2 -[ 18 F]RL-I. The overall radiochemical yields for the synthesis of [ 18 F]RL-III-2Rs15d and [ 18 F]RL-III-5F7 from aqueous [ 18 F]fluoride were 1.7 ± 0.7% and 3.8 ± 2.3%, respectively. Both sdAbs, labeled using [ 18 F]RL-III, retained affinity and immunoreactivity to HER2. Uptake and internalization of [ 18 F]RL-III-5F7 in HER2-expressing cells was higher than that seen for [ 18 F]RL-III-2Rs15d. Although different xenograft models were used, [ 18 F]RL-III-2Rs15d showed relatively high uptake in a number of normal tissues, while uptake of [ 18 F]RL-III-5F7 was mainly in tumor and kidneys with minimal background activity. Concordant with the necropsy experiments, microPET imaging with [ 18 F]RL-III-5F7 in the BT474 subcutaneous model demonstrated clear delineation of the tumor (12.2 ± 5.1% ID/g) with minimal background activity except in kidneys. A tumor uptake (max) of 0.98%ID/g and a tumor-to-normal brain ratio of 9.8:1 were observed for [ 18 F]RL-III-5F7 in the intracranial model., Conclusions: Although higher radiochemical yields than that reported for [ 18 F]RL-I were obtained, considerable improvements are needed for this method to be of practical utility. Despite clear tumor delineation with [ 18 F]RL-III-5F7 as early as 1 h, high activity levels in the kidneys remain a concern., Competing Interests: Declaration of competing interest Michael R. Zalutsky holds ownership interest and is a board member for Cereius, Inc. Ganesan Vaidyanathan is a consultant and shareholder for Cereius, Inc. There are no other potential conflicts of interest relevant to this article., (Copyright © 2021 Elsevier Inc. All rights reserved.)