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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.)

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.)