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Ganesan Vaidyanathan - One of the best experts on this subject based on the ideXlab platform.
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labeling single domain Antibody fragments with 18f using a novel residualizing prosthetic agent n succinimidyl 3 1 2 2 2 2 18f fluoroethoxy ethoxy ethoxy ethyl 1h 1 2 3 triazol 4 yl 5 guanidinomethyl benzoate
Nuclear Medicine and Biology, 2021Co-Authors: Zhengyuan Zhou, Michael R Zalutsky, Darryl Mcdougald, Rebecca Meshaw, Irina V Balyasnikova, Ganesan VaidyanathanAbstract:Abstract Introduction Labeling single domain Antibody fragments (sdAbs) with 18F 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 ([18F]RL-I), synthesized via a click reaction for labeling sdAbs with 18F, 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-[18F]fluoroethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-5-(guanidinomethyl)benzoate ([18F]SFETGMB; [18F]RL-III) designed to address these limitations. Methods [18F]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-[18F]fluoroethoxy)ethoxy)ethoxy)ethane and subsequent deprotection. The anti-HER2 sdAbs 5F7 and 2Rs15d were labeled by conjugation with [18F]RL-III and compared in a paired-label fashion to the sdAbs labeled using N-succinimidyl 4-guanidinomethyl-3-[125I]iodobenzoate ([125I]SGMIB) or N-succinimidyl 3-guanidinomethyl-5-[125I]iodobenzoate (iso-[125I]SGMIB). The 18F-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 [18F]RL-III-5F7 also was performed. Results Radiochemical yields for the synthesis of Boc2-[18F]RL-III (21.5 ± 3.4%) were significantly higher than reported for Boc2-[18F]RL-I. The overall radiochemical yields for the synthesis of [18F]RL-III-2Rs15d and [18F]RL-III-5F7 from aqueous [18F]fluoride were 1.7 ± 0.7% and 3.8 ± 2.3%, respectively. Both sdAbs, labeled using [18F]RL-III, retained affinity and immunoreactivity to HER2. Uptake and internalization of [18F]RL-III-5F7 in HER2-expressing cells was higher than that seen for [18F]RL-III-2Rs15d. Although different xenograft models were used, [18F]RL-III-2Rs15d showed relatively high uptake in a number of normal tissues, while uptake of [18F]RL-III-5F7 was mainly in tumor and kidneys with minimal background activity. Concordant with the necropsy experiments, microPET imaging with [18F]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 [18F]RL-III-5F7 in the intracranial model. Conclusions Although higher radiochemical yields than that reported for [18F]RL-I were obtained, considerable improvements are needed for this method to be of practical utility. Despite clear tumor delineation with [18F]RL-III-5F7 as early as 1 h, high activity levels in the kidneys remain a concern.
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site specific and residualizing linker for 18f labeling with enhanced renal clearance application to an anti her2 single domain Antibody fragment
The Journal of Nuclear Medicine, 2021Co-Authors: Zhengyuan Zhou, Michael R Zalutsky, Rebecca Meshaw, Ganesan VaidyanathanAbstract:Single domain Antibody fragments (sdAbs) are promising vectors for immunoPET; however, better methods for labeling sdAbs with 18F are needed. Herein, we evaluate a site-specific strategy utilizing an 18F residualizing motif and the anti-HER2 sdAb 5F7 bearing an engineered C-terminal GGC tail. Methods: 5F7GGC was site-specifically attached with a tetrazine-bearing agent via thiol-maleimide reaction. The resultant conjugate was labeled with 18F by inverse electron demand Diels-Alder cycloaddition with a trans-cyclooctene attached to 6-18F-fluoronicotinoyl moiety via a renal brush border enzyme-cleavable linker and a PEG4 chain (18F-5F7GGC). For comparisons, 5F7 sdAb was labeled using the prototypical residualizing agent, N-succinimidyl 3-(guanidinomethyl)-5-125I-iodobenzoate (iso-125I-SGMIB). The two 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. MicroPET/CT imaging after administration of 18F-5F7GGC in the above mouse model was also carried out. Results:18F-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 18F-5F7GGC was similar to that for co-incubated iso-125I-SGMIB-5F7. Likewise, the uptake of 18F-5F7GGC in BT474M1 xenografts in mice was similar to that for iso-125I-SGMIB-5F7; however, 18F-5F7GGC exhibited significantly more rapid clearance from the kidney. MicroPET/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 18F labeling strategy could facilitate clinical translation of 5F7 anti-HER2 sdAb as well as other sdAbs for immunoPET.
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site specific radioiodination of an anti her2 single domain Antibody fragment with a residualizing prosthetic agent
Nuclear Medicine and Biology, 2021Co-Authors: Yutian Feng, Ganesan Vaidyanathan, Zhengyuan Zhou, Darryl Mcdougald, Rebecca Meshaw, Michael R ZalutskyAbstract:Abstract Introduction As a consequence of their small size, high stability and high affinity, single domain Antibody fragments (sdAbs) are appealing targeting vectors for radiopharmaceutical development. With sdAbs binding to internalizing receptors like HER2, residualizing prosthetic agents can enhance tumor retention of radioiodine, which until now has been done with random labeling approaches. Herein we evaluate a site-specific strategy utilizing a radioiodinated, residualizing maleimido moiety and the anti-HER2 sdAb 5F7 bearing a GGC tail for conjugation. Methods Maleimidoethyl 3-(guanidinomethyl)-5-iodobenzoate ([131I]MEGMB) and its N-succinimidyl ester analogue, iso-[125I]SGMIB, were labeled by halodestannylation and conjugated with 5F7GGC and 5F7, respectively. Radiochemical purity, immunoreactivity and binding affinity were determined. Paired-label experiments directly compared iso-[125I]SGMIB-5F7 and [131I]MEGMIB-5F7GGC with regard to internalization/residualization and affinity on HER2-expressing SKOV-3 ovarian carcinoma cells as well as biodistribution and metabolite distribution in athymic mice with subcutaneous SKOV-3 xenografts. Results [131I]MEGMIB-5F7GGC had an immunoreactivity of 81.3% and Kd = 0.94 ± 0.27 nM. Internalization assays demonstrated high intracellular trapping for both conjugates, For example, at 1 h, intracellular retention was 50.30 ± 3.36% for [131I]MEGMIB-5F7GGC and 55.95 ± 3.27% for iso-[125I]SGMIB-5F7, while higher retention was seen for iso-[125I]SGMIB-5F7 at later time points. Peak tumor uptake was similar for both conjugates (8.35 ± 2.66%ID/g and 8.43 ± 2.84%ID/g for iso-[125I]SGMIB-5F7 and [131I]MEGMIB-5F7GGC at 1 h, respectively); however, more rapid normal tissue clearance was seen for [131I]MEGMIB-5F7GGC, with a 2-fold higher tumor-to-kidney ratio and a 3-fold higher tumor-to-liver ratio compared with co-injected iso-[125I]SGMIB-5F7. Consisted with this, generation of labeled catabolites in the kidneys was higher for [131I]MEGMIB-5F7GGC. Conclusion [131I]MEGMIB-5F7GGC offers similar tumor targeting as iso-[125I]SGMIB-5F7 but with generally lower normal tissue uptake. Advances in knowledge and implication for patient care The site specific nature of the [131I]MEGMIB reagent may facilitate clinical translation, particularly for sdAb with compromised affinity after random labeling.
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labeling a tco functionalized single domain Antibody fragment with 18f via inverse electron demand diels alder cycloaddition using a fluoronicotinyl moiety bearing tetrazine derivative
Bioorganic & Medicinal Chemistry, 2020Co-Authors: Zhengyuan Zhou, Michael R Zalutsky, Ganesan VaidyanathanAbstract:Abstract Single domain Antibody fragments (sdAbs) exhibit a rapid tumor uptake and fast blood clearance amenable for labeling with 18F (t½ = 110 min) but suffer from high kidney accumulation. Previously, we developed a method for 18F-labeling of sdAbs via trans-cyclooctene (TCO)-tetrazine (Tz) inverse electron demand Diel’s Alder cycloaddition reaction (IEDDAR) that incorporated a renal brush border enzyme (RBBE)-cleavable linker. Although >15 fold reduction in kidney activity levels was achieved, tumor uptake was compromised. Here we investigate whether replacing the [ 18 F]AlF-NOTA moiety with [ 18 F]fluoronicotinyl would rectify this problem. Anti-HER2 sdAb 5F7 was first derivatized with a TCO-containing agent that included the RBBE-cleavable linker GlyLys (GK) and a PEG chain, and then subjected to IEDDAR with 6-[ 18 F]fluoronicotinyl-PEG4-methyltetrazine to provide [ 18 F]FN-PEG4-Tz-TCO-GK-PEG4-5F7 ([ 18 F]FN-GK-5F7). For comparisons, a control lacking GK linker and 5F7 labeled using residualizing N-succinimidyl 3-guanidinomethyl-5-[125I]iodobenzoate (iso-[125I]SGMIB) also were synthesized. Radiochemical purity, affinity (KD) and immunoreactive fraction of [ 18 F]FN-GK-5F7 were 99%, 5.4 ± 0.7 nM and 72.5 ± 4.3%, respectively. Tumor uptake of [ 18 F]FN-GK-5F7 in athymic mice bearing subcutaneous SKOV3 xenografts (3.7 ± 1.2% ID/g and 3.4 ± 1.0% ID/g at 1 h and 3 h, respectively) was 2- to 3-fold lower than for co-injected iso-[125I]SGMIB-5F7 (6.9 ± 1.9 %ID/g and 8.7 ± 3.0 %ID/g). However, due to its 6-fold lower kidney activity levels, tumor-to-kidney ratios for [ 18 F]FN-GK-5F7 were 3-4 times higher than those for co-injected iso-[125I]SGMIB-5F7 as well as those observed for the 18F conjugate lacking the RBBE-cleavable linker. Micro-PET/CT imaging of [ 18 F]FN-GK-5F7 in mice with SKOV-3 subcutaneous xenografts clearly delineated tumor as early as 1 h with minimal activity in the kidneys; however, there was considerable activity in gallbladder and intestines. Although the tumor uptake of [ 18 F]FN-GK-5F7 was unexpectedly disappointing, incorporating an alternative RBBE-cleavable linker into this labeling strategy may ameliorate this problem.
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n maleimidoethyl 3 guanidinomethyl 5 131i iodobenzmide 131i megmib a residualizing prosthetic agent for site specific radioiodination of internalizing single domain Antibody fragments
The Journal of Nuclear Medicine, 2020Co-Authors: Yutian Feng, Ganesan Vaidyanathan, Zhengyuan Zhou, Darryl Mcdougald, Michael R ZalutskyAbstract:581 Objectives: Anti-HER2 single domain Antibody fragments (sdAbs; a.k.a. nanobodies, VHH molecules) are an attractive platform for delivering radionuclides for imaging and targeted radionuclide therapy (TRT). Earlier, we demonstrated excellent tumor uptake and retention as well as low normal tissue uptake when the anti-HER2 sdAb 5F7 was labeled using the residualizing agent N-succinimidyl 3-guanidinomethyl-5-[*I]iodobenzoate (iso-[*I]SGMIB). Because site-specific labeling can have advantages over random methods such as iso-[*I]SGMIB, we developed a maleimide moiety-containing analogue of iso-[*I]SGMIB, N-(maleimidoethyl)-3-(guanidinomethyl)-5-[131I]iodobenzmide ([131I]MEGMIB) and employed it for labeling a 5F7 variant having a cysteine tail (GGC) at its C-terminus (5F7GGC). Methods: The residualizing prosthetic agent MEGMIB and its protected tin precursor Boc2-MEGMTB were synthesized and characterized. Unlabeled MEGMIB was conjugated to reduced 5F7GGC and the binding affinity of the MEGMIB-5F7GGC conjugate to HER2 was measured by surface plasmon resonance. The tin precursor Boc2-MEGMTB was labeled with 131I using NCS as the oxidant and the Boc groups were removed by TFA treatment. The resultant [131I]MEGMIB was conjugated to 5F7GGC at pH of 6.3. Radiochemical purity (RCP) of the labeled conjugate was determined by SDS-PAGE and size-exclusion chromatography. Immunoreactive fraction (IRF) was determined by the Lindmo method and binding affinity (Kd) using the HER2-positive SKOV-3 ovarian cancer cell line. Paired-label internalization of [131I]MEGMIB-5F7GGC and iso-[125I]SGMIB-5F7 was determined on SKOV-3 cells. Paired-label biodistribution of [131I]MEGMIB-5F7GGC and iso-[125I]SGMIB-5F7 was assessed in nude mice bearing subcutaneous SKOV-3 xenografts. Results: [131I]MEGMIB was synthesized in 87 ± 6% (n=11) yield, comparable to yields for iso-[131I]SGMIB. Conjugation of [131I]MEGMIB to 5F7GGC was achieved in an yield of 45% ± 7% (n=10) with a RCP, Kd and IRF of [131I]MEGMIB-5F7GGC of higher than 99%, 0.94 ± 0.27 nM and 81%, respectively. Specific uptake by SKOV-3 cells after a 1 h incubation was 10.6 ± 0.6% and 8.4 ± 0.3% for 131I and 125I, respectively with the difference statistically significant. The amount of initially bound activity that was internalized at 1 h was 50.30 ± 3.36% for 131I similar to that for 125I (55.95 ± 3.27% Fig. 2). The uptake (%ID/g) of [131I]MEGMIB-5F7GGC in SKOV-3 xenografts was 8.43 ± 2.84, 6.11 ± 1.63, and 3.60 ± 1.75% ID/g at 1, 4 and 24 h, respectively; compared to 8.35 ± 2.66, 6.11 ± 1.56 and 3.13 ± 1.52% ID/g for co-injected iso-[125I]SGMIB-5F7. The kidney activity for [131I]MEGMIB-5F7GGC at 1 h (20.39 ± 5.34 %ID/g) was significantly lower (P=0.01) than that for iso-[125I]SGMIB-5F7 (41.82 ± 13.33 %ID/g). The difference in kidney levels for the two tracers also was significant at 4 h (P=0.02). Tumor-to-kidney ratios for [131I]MEGMIB-5F7GGC also were significantly higher (0.41 ± 0.09 vs 0.21 ± 0.09 at 1h; P=0.008). Conclusion: This novel residualizing prosthetic agent allows site-specific labeling of 5F7GGC, a HER2-specific sdAb with a free cysteine at the C-terminus, in good yield with excellent retention of affinity, immunoreactivity, intracellular retention and tumor localizing capacity. Importantly, tumor uptake of [131I]MEGMIB-5F7GGC was comparable to that for the previous lead agent iso-[125I]SGMIB-5F7, but with up to two fold lower uptake in the kidneys, the dose limiting tissue for radiolabeled scFv fragments. Acknowledgements: This work was supported in part by NIH Grant CA42324.
Zhengyuan Zhou - One of the best experts on this subject based on the ideXlab platform.
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labeling single domain Antibody fragments with 18f using a novel residualizing prosthetic agent n succinimidyl 3 1 2 2 2 2 18f fluoroethoxy ethoxy ethoxy ethyl 1h 1 2 3 triazol 4 yl 5 guanidinomethyl benzoate
Nuclear Medicine and Biology, 2021Co-Authors: Zhengyuan Zhou, Michael R Zalutsky, Darryl Mcdougald, Rebecca Meshaw, Irina V Balyasnikova, Ganesan VaidyanathanAbstract:Abstract Introduction Labeling single domain Antibody fragments (sdAbs) with 18F 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 ([18F]RL-I), synthesized via a click reaction for labeling sdAbs with 18F, 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-[18F]fluoroethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-5-(guanidinomethyl)benzoate ([18F]SFETGMB; [18F]RL-III) designed to address these limitations. Methods [18F]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-[18F]fluoroethoxy)ethoxy)ethoxy)ethane and subsequent deprotection. The anti-HER2 sdAbs 5F7 and 2Rs15d were labeled by conjugation with [18F]RL-III and compared in a paired-label fashion to the sdAbs labeled using N-succinimidyl 4-guanidinomethyl-3-[125I]iodobenzoate ([125I]SGMIB) or N-succinimidyl 3-guanidinomethyl-5-[125I]iodobenzoate (iso-[125I]SGMIB). The 18F-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 [18F]RL-III-5F7 also was performed. Results Radiochemical yields for the synthesis of Boc2-[18F]RL-III (21.5 ± 3.4%) were significantly higher than reported for Boc2-[18F]RL-I. The overall radiochemical yields for the synthesis of [18F]RL-III-2Rs15d and [18F]RL-III-5F7 from aqueous [18F]fluoride were 1.7 ± 0.7% and 3.8 ± 2.3%, respectively. Both sdAbs, labeled using [18F]RL-III, retained affinity and immunoreactivity to HER2. Uptake and internalization of [18F]RL-III-5F7 in HER2-expressing cells was higher than that seen for [18F]RL-III-2Rs15d. Although different xenograft models were used, [18F]RL-III-2Rs15d showed relatively high uptake in a number of normal tissues, while uptake of [18F]RL-III-5F7 was mainly in tumor and kidneys with minimal background activity. Concordant with the necropsy experiments, microPET imaging with [18F]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 [18F]RL-III-5F7 in the intracranial model. Conclusions Although higher radiochemical yields than that reported for [18F]RL-I were obtained, considerable improvements are needed for this method to be of practical utility. Despite clear tumor delineation with [18F]RL-III-5F7 as early as 1 h, high activity levels in the kidneys remain a concern.
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site specific and residualizing linker for 18f labeling with enhanced renal clearance application to an anti her2 single domain Antibody fragment
The Journal of Nuclear Medicine, 2021Co-Authors: Zhengyuan Zhou, Michael R Zalutsky, Rebecca Meshaw, Ganesan VaidyanathanAbstract:Single domain Antibody fragments (sdAbs) are promising vectors for immunoPET; however, better methods for labeling sdAbs with 18F are needed. Herein, we evaluate a site-specific strategy utilizing an 18F residualizing motif and the anti-HER2 sdAb 5F7 bearing an engineered C-terminal GGC tail. Methods: 5F7GGC was site-specifically attached with a tetrazine-bearing agent via thiol-maleimide reaction. The resultant conjugate was labeled with 18F by inverse electron demand Diels-Alder cycloaddition with a trans-cyclooctene attached to 6-18F-fluoronicotinoyl moiety via a renal brush border enzyme-cleavable linker and a PEG4 chain (18F-5F7GGC). For comparisons, 5F7 sdAb was labeled using the prototypical residualizing agent, N-succinimidyl 3-(guanidinomethyl)-5-125I-iodobenzoate (iso-125I-SGMIB). The two 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. MicroPET/CT imaging after administration of 18F-5F7GGC in the above mouse model was also carried out. Results:18F-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 18F-5F7GGC was similar to that for co-incubated iso-125I-SGMIB-5F7. Likewise, the uptake of 18F-5F7GGC in BT474M1 xenografts in mice was similar to that for iso-125I-SGMIB-5F7; however, 18F-5F7GGC exhibited significantly more rapid clearance from the kidney. MicroPET/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 18F labeling strategy could facilitate clinical translation of 5F7 anti-HER2 sdAb as well as other sdAbs for immunoPET.
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site specific radioiodination of an anti her2 single domain Antibody fragment with a residualizing prosthetic agent
Nuclear Medicine and Biology, 2021Co-Authors: Yutian Feng, Ganesan Vaidyanathan, Zhengyuan Zhou, Darryl Mcdougald, Rebecca Meshaw, Michael R ZalutskyAbstract:Abstract Introduction As a consequence of their small size, high stability and high affinity, single domain Antibody fragments (sdAbs) are appealing targeting vectors for radiopharmaceutical development. With sdAbs binding to internalizing receptors like HER2, residualizing prosthetic agents can enhance tumor retention of radioiodine, which until now has been done with random labeling approaches. Herein we evaluate a site-specific strategy utilizing a radioiodinated, residualizing maleimido moiety and the anti-HER2 sdAb 5F7 bearing a GGC tail for conjugation. Methods Maleimidoethyl 3-(guanidinomethyl)-5-iodobenzoate ([131I]MEGMB) and its N-succinimidyl ester analogue, iso-[125I]SGMIB, were labeled by halodestannylation and conjugated with 5F7GGC and 5F7, respectively. Radiochemical purity, immunoreactivity and binding affinity were determined. Paired-label experiments directly compared iso-[125I]SGMIB-5F7 and [131I]MEGMIB-5F7GGC with regard to internalization/residualization and affinity on HER2-expressing SKOV-3 ovarian carcinoma cells as well as biodistribution and metabolite distribution in athymic mice with subcutaneous SKOV-3 xenografts. Results [131I]MEGMIB-5F7GGC had an immunoreactivity of 81.3% and Kd = 0.94 ± 0.27 nM. Internalization assays demonstrated high intracellular trapping for both conjugates, For example, at 1 h, intracellular retention was 50.30 ± 3.36% for [131I]MEGMIB-5F7GGC and 55.95 ± 3.27% for iso-[125I]SGMIB-5F7, while higher retention was seen for iso-[125I]SGMIB-5F7 at later time points. Peak tumor uptake was similar for both conjugates (8.35 ± 2.66%ID/g and 8.43 ± 2.84%ID/g for iso-[125I]SGMIB-5F7 and [131I]MEGMIB-5F7GGC at 1 h, respectively); however, more rapid normal tissue clearance was seen for [131I]MEGMIB-5F7GGC, with a 2-fold higher tumor-to-kidney ratio and a 3-fold higher tumor-to-liver ratio compared with co-injected iso-[125I]SGMIB-5F7. Consisted with this, generation of labeled catabolites in the kidneys was higher for [131I]MEGMIB-5F7GGC. Conclusion [131I]MEGMIB-5F7GGC offers similar tumor targeting as iso-[125I]SGMIB-5F7 but with generally lower normal tissue uptake. Advances in knowledge and implication for patient care The site specific nature of the [131I]MEGMIB reagent may facilitate clinical translation, particularly for sdAb with compromised affinity after random labeling.
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labeling a tco functionalized single domain Antibody fragment with 18f via inverse electron demand diels alder cycloaddition using a fluoronicotinyl moiety bearing tetrazine derivative
Bioorganic & Medicinal Chemistry, 2020Co-Authors: Zhengyuan Zhou, Michael R Zalutsky, Ganesan VaidyanathanAbstract:Abstract Single domain Antibody fragments (sdAbs) exhibit a rapid tumor uptake and fast blood clearance amenable for labeling with 18F (t½ = 110 min) but suffer from high kidney accumulation. Previously, we developed a method for 18F-labeling of sdAbs via trans-cyclooctene (TCO)-tetrazine (Tz) inverse electron demand Diel’s Alder cycloaddition reaction (IEDDAR) that incorporated a renal brush border enzyme (RBBE)-cleavable linker. Although >15 fold reduction in kidney activity levels was achieved, tumor uptake was compromised. Here we investigate whether replacing the [ 18 F]AlF-NOTA moiety with [ 18 F]fluoronicotinyl would rectify this problem. Anti-HER2 sdAb 5F7 was first derivatized with a TCO-containing agent that included the RBBE-cleavable linker GlyLys (GK) and a PEG chain, and then subjected to IEDDAR with 6-[ 18 F]fluoronicotinyl-PEG4-methyltetrazine to provide [ 18 F]FN-PEG4-Tz-TCO-GK-PEG4-5F7 ([ 18 F]FN-GK-5F7). For comparisons, a control lacking GK linker and 5F7 labeled using residualizing N-succinimidyl 3-guanidinomethyl-5-[125I]iodobenzoate (iso-[125I]SGMIB) also were synthesized. Radiochemical purity, affinity (KD) and immunoreactive fraction of [ 18 F]FN-GK-5F7 were 99%, 5.4 ± 0.7 nM and 72.5 ± 4.3%, respectively. Tumor uptake of [ 18 F]FN-GK-5F7 in athymic mice bearing subcutaneous SKOV3 xenografts (3.7 ± 1.2% ID/g and 3.4 ± 1.0% ID/g at 1 h and 3 h, respectively) was 2- to 3-fold lower than for co-injected iso-[125I]SGMIB-5F7 (6.9 ± 1.9 %ID/g and 8.7 ± 3.0 %ID/g). However, due to its 6-fold lower kidney activity levels, tumor-to-kidney ratios for [ 18 F]FN-GK-5F7 were 3-4 times higher than those for co-injected iso-[125I]SGMIB-5F7 as well as those observed for the 18F conjugate lacking the RBBE-cleavable linker. Micro-PET/CT imaging of [ 18 F]FN-GK-5F7 in mice with SKOV-3 subcutaneous xenografts clearly delineated tumor as early as 1 h with minimal activity in the kidneys; however, there was considerable activity in gallbladder and intestines. Although the tumor uptake of [ 18 F]FN-GK-5F7 was unexpectedly disappointing, incorporating an alternative RBBE-cleavable linker into this labeling strategy may ameliorate this problem.
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n maleimidoethyl 3 guanidinomethyl 5 131i iodobenzmide 131i megmib a residualizing prosthetic agent for site specific radioiodination of internalizing single domain Antibody fragments
The Journal of Nuclear Medicine, 2020Co-Authors: Yutian Feng, Ganesan Vaidyanathan, Zhengyuan Zhou, Darryl Mcdougald, Michael R ZalutskyAbstract:581 Objectives: Anti-HER2 single domain Antibody fragments (sdAbs; a.k.a. nanobodies, VHH molecules) are an attractive platform for delivering radionuclides for imaging and targeted radionuclide therapy (TRT). Earlier, we demonstrated excellent tumor uptake and retention as well as low normal tissue uptake when the anti-HER2 sdAb 5F7 was labeled using the residualizing agent N-succinimidyl 3-guanidinomethyl-5-[*I]iodobenzoate (iso-[*I]SGMIB). Because site-specific labeling can have advantages over random methods such as iso-[*I]SGMIB, we developed a maleimide moiety-containing analogue of iso-[*I]SGMIB, N-(maleimidoethyl)-3-(guanidinomethyl)-5-[131I]iodobenzmide ([131I]MEGMIB) and employed it for labeling a 5F7 variant having a cysteine tail (GGC) at its C-terminus (5F7GGC). Methods: The residualizing prosthetic agent MEGMIB and its protected tin precursor Boc2-MEGMTB were synthesized and characterized. Unlabeled MEGMIB was conjugated to reduced 5F7GGC and the binding affinity of the MEGMIB-5F7GGC conjugate to HER2 was measured by surface plasmon resonance. The tin precursor Boc2-MEGMTB was labeled with 131I using NCS as the oxidant and the Boc groups were removed by TFA treatment. The resultant [131I]MEGMIB was conjugated to 5F7GGC at pH of 6.3. Radiochemical purity (RCP) of the labeled conjugate was determined by SDS-PAGE and size-exclusion chromatography. Immunoreactive fraction (IRF) was determined by the Lindmo method and binding affinity (Kd) using the HER2-positive SKOV-3 ovarian cancer cell line. Paired-label internalization of [131I]MEGMIB-5F7GGC and iso-[125I]SGMIB-5F7 was determined on SKOV-3 cells. Paired-label biodistribution of [131I]MEGMIB-5F7GGC and iso-[125I]SGMIB-5F7 was assessed in nude mice bearing subcutaneous SKOV-3 xenografts. Results: [131I]MEGMIB was synthesized in 87 ± 6% (n=11) yield, comparable to yields for iso-[131I]SGMIB. Conjugation of [131I]MEGMIB to 5F7GGC was achieved in an yield of 45% ± 7% (n=10) with a RCP, Kd and IRF of [131I]MEGMIB-5F7GGC of higher than 99%, 0.94 ± 0.27 nM and 81%, respectively. Specific uptake by SKOV-3 cells after a 1 h incubation was 10.6 ± 0.6% and 8.4 ± 0.3% for 131I and 125I, respectively with the difference statistically significant. The amount of initially bound activity that was internalized at 1 h was 50.30 ± 3.36% for 131I similar to that for 125I (55.95 ± 3.27% Fig. 2). The uptake (%ID/g) of [131I]MEGMIB-5F7GGC in SKOV-3 xenografts was 8.43 ± 2.84, 6.11 ± 1.63, and 3.60 ± 1.75% ID/g at 1, 4 and 24 h, respectively; compared to 8.35 ± 2.66, 6.11 ± 1.56 and 3.13 ± 1.52% ID/g for co-injected iso-[125I]SGMIB-5F7. The kidney activity for [131I]MEGMIB-5F7GGC at 1 h (20.39 ± 5.34 %ID/g) was significantly lower (P=0.01) than that for iso-[125I]SGMIB-5F7 (41.82 ± 13.33 %ID/g). The difference in kidney levels for the two tracers also was significant at 4 h (P=0.02). Tumor-to-kidney ratios for [131I]MEGMIB-5F7GGC also were significantly higher (0.41 ± 0.09 vs 0.21 ± 0.09 at 1h; P=0.008). Conclusion: This novel residualizing prosthetic agent allows site-specific labeling of 5F7GGC, a HER2-specific sdAb with a free cysteine at the C-terminus, in good yield with excellent retention of affinity, immunoreactivity, intracellular retention and tumor localizing capacity. Importantly, tumor uptake of [131I]MEGMIB-5F7GGC was comparable to that for the previous lead agent iso-[125I]SGMIB-5F7, but with up to two fold lower uptake in the kidneys, the dose limiting tissue for radiolabeled scFv fragments. Acknowledgements: This work was supported in part by NIH Grant CA42324.
Michael R Zalutsky - One of the best experts on this subject based on the ideXlab platform.
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labeling single domain Antibody fragments with 18f using a novel residualizing prosthetic agent n succinimidyl 3 1 2 2 2 2 18f fluoroethoxy ethoxy ethoxy ethyl 1h 1 2 3 triazol 4 yl 5 guanidinomethyl benzoate
Nuclear Medicine and Biology, 2021Co-Authors: Zhengyuan Zhou, Michael R Zalutsky, Darryl Mcdougald, Rebecca Meshaw, Irina V Balyasnikova, Ganesan VaidyanathanAbstract:Abstract Introduction Labeling single domain Antibody fragments (sdAbs) with 18F 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 ([18F]RL-I), synthesized via a click reaction for labeling sdAbs with 18F, 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-[18F]fluoroethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-5-(guanidinomethyl)benzoate ([18F]SFETGMB; [18F]RL-III) designed to address these limitations. Methods [18F]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-[18F]fluoroethoxy)ethoxy)ethoxy)ethane and subsequent deprotection. The anti-HER2 sdAbs 5F7 and 2Rs15d were labeled by conjugation with [18F]RL-III and compared in a paired-label fashion to the sdAbs labeled using N-succinimidyl 4-guanidinomethyl-3-[125I]iodobenzoate ([125I]SGMIB) or N-succinimidyl 3-guanidinomethyl-5-[125I]iodobenzoate (iso-[125I]SGMIB). The 18F-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 [18F]RL-III-5F7 also was performed. Results Radiochemical yields for the synthesis of Boc2-[18F]RL-III (21.5 ± 3.4%) were significantly higher than reported for Boc2-[18F]RL-I. The overall radiochemical yields for the synthesis of [18F]RL-III-2Rs15d and [18F]RL-III-5F7 from aqueous [18F]fluoride were 1.7 ± 0.7% and 3.8 ± 2.3%, respectively. Both sdAbs, labeled using [18F]RL-III, retained affinity and immunoreactivity to HER2. Uptake and internalization of [18F]RL-III-5F7 in HER2-expressing cells was higher than that seen for [18F]RL-III-2Rs15d. Although different xenograft models were used, [18F]RL-III-2Rs15d showed relatively high uptake in a number of normal tissues, while uptake of [18F]RL-III-5F7 was mainly in tumor and kidneys with minimal background activity. Concordant with the necropsy experiments, microPET imaging with [18F]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 [18F]RL-III-5F7 in the intracranial model. Conclusions Although higher radiochemical yields than that reported for [18F]RL-I were obtained, considerable improvements are needed for this method to be of practical utility. Despite clear tumor delineation with [18F]RL-III-5F7 as early as 1 h, high activity levels in the kidneys remain a concern.
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site specific and residualizing linker for 18f labeling with enhanced renal clearance application to an anti her2 single domain Antibody fragment
The Journal of Nuclear Medicine, 2021Co-Authors: Zhengyuan Zhou, Michael R Zalutsky, Rebecca Meshaw, Ganesan VaidyanathanAbstract:Single domain Antibody fragments (sdAbs) are promising vectors for immunoPET; however, better methods for labeling sdAbs with 18F are needed. Herein, we evaluate a site-specific strategy utilizing an 18F residualizing motif and the anti-HER2 sdAb 5F7 bearing an engineered C-terminal GGC tail. Methods: 5F7GGC was site-specifically attached with a tetrazine-bearing agent via thiol-maleimide reaction. The resultant conjugate was labeled with 18F by inverse electron demand Diels-Alder cycloaddition with a trans-cyclooctene attached to 6-18F-fluoronicotinoyl moiety via a renal brush border enzyme-cleavable linker and a PEG4 chain (18F-5F7GGC). For comparisons, 5F7 sdAb was labeled using the prototypical residualizing agent, N-succinimidyl 3-(guanidinomethyl)-5-125I-iodobenzoate (iso-125I-SGMIB). The two 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. MicroPET/CT imaging after administration of 18F-5F7GGC in the above mouse model was also carried out. Results:18F-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 18F-5F7GGC was similar to that for co-incubated iso-125I-SGMIB-5F7. Likewise, the uptake of 18F-5F7GGC in BT474M1 xenografts in mice was similar to that for iso-125I-SGMIB-5F7; however, 18F-5F7GGC exhibited significantly more rapid clearance from the kidney. MicroPET/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 18F labeling strategy could facilitate clinical translation of 5F7 anti-HER2 sdAb as well as other sdAbs for immunoPET.
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site specific radioiodination of an anti her2 single domain Antibody fragment with a residualizing prosthetic agent
Nuclear Medicine and Biology, 2021Co-Authors: Yutian Feng, Ganesan Vaidyanathan, Zhengyuan Zhou, Darryl Mcdougald, Rebecca Meshaw, Michael R ZalutskyAbstract:Abstract Introduction As a consequence of their small size, high stability and high affinity, single domain Antibody fragments (sdAbs) are appealing targeting vectors for radiopharmaceutical development. With sdAbs binding to internalizing receptors like HER2, residualizing prosthetic agents can enhance tumor retention of radioiodine, which until now has been done with random labeling approaches. Herein we evaluate a site-specific strategy utilizing a radioiodinated, residualizing maleimido moiety and the anti-HER2 sdAb 5F7 bearing a GGC tail for conjugation. Methods Maleimidoethyl 3-(guanidinomethyl)-5-iodobenzoate ([131I]MEGMB) and its N-succinimidyl ester analogue, iso-[125I]SGMIB, were labeled by halodestannylation and conjugated with 5F7GGC and 5F7, respectively. Radiochemical purity, immunoreactivity and binding affinity were determined. Paired-label experiments directly compared iso-[125I]SGMIB-5F7 and [131I]MEGMIB-5F7GGC with regard to internalization/residualization and affinity on HER2-expressing SKOV-3 ovarian carcinoma cells as well as biodistribution and metabolite distribution in athymic mice with subcutaneous SKOV-3 xenografts. Results [131I]MEGMIB-5F7GGC had an immunoreactivity of 81.3% and Kd = 0.94 ± 0.27 nM. Internalization assays demonstrated high intracellular trapping for both conjugates, For example, at 1 h, intracellular retention was 50.30 ± 3.36% for [131I]MEGMIB-5F7GGC and 55.95 ± 3.27% for iso-[125I]SGMIB-5F7, while higher retention was seen for iso-[125I]SGMIB-5F7 at later time points. Peak tumor uptake was similar for both conjugates (8.35 ± 2.66%ID/g and 8.43 ± 2.84%ID/g for iso-[125I]SGMIB-5F7 and [131I]MEGMIB-5F7GGC at 1 h, respectively); however, more rapid normal tissue clearance was seen for [131I]MEGMIB-5F7GGC, with a 2-fold higher tumor-to-kidney ratio and a 3-fold higher tumor-to-liver ratio compared with co-injected iso-[125I]SGMIB-5F7. Consisted with this, generation of labeled catabolites in the kidneys was higher for [131I]MEGMIB-5F7GGC. Conclusion [131I]MEGMIB-5F7GGC offers similar tumor targeting as iso-[125I]SGMIB-5F7 but with generally lower normal tissue uptake. Advances in knowledge and implication for patient care The site specific nature of the [131I]MEGMIB reagent may facilitate clinical translation, particularly for sdAb with compromised affinity after random labeling.
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labeling a tco functionalized single domain Antibody fragment with 18f via inverse electron demand diels alder cycloaddition using a fluoronicotinyl moiety bearing tetrazine derivative
Bioorganic & Medicinal Chemistry, 2020Co-Authors: Zhengyuan Zhou, Michael R Zalutsky, Ganesan VaidyanathanAbstract:Abstract Single domain Antibody fragments (sdAbs) exhibit a rapid tumor uptake and fast blood clearance amenable for labeling with 18F (t½ = 110 min) but suffer from high kidney accumulation. Previously, we developed a method for 18F-labeling of sdAbs via trans-cyclooctene (TCO)-tetrazine (Tz) inverse electron demand Diel’s Alder cycloaddition reaction (IEDDAR) that incorporated a renal brush border enzyme (RBBE)-cleavable linker. Although >15 fold reduction in kidney activity levels was achieved, tumor uptake was compromised. Here we investigate whether replacing the [ 18 F]AlF-NOTA moiety with [ 18 F]fluoronicotinyl would rectify this problem. Anti-HER2 sdAb 5F7 was first derivatized with a TCO-containing agent that included the RBBE-cleavable linker GlyLys (GK) and a PEG chain, and then subjected to IEDDAR with 6-[ 18 F]fluoronicotinyl-PEG4-methyltetrazine to provide [ 18 F]FN-PEG4-Tz-TCO-GK-PEG4-5F7 ([ 18 F]FN-GK-5F7). For comparisons, a control lacking GK linker and 5F7 labeled using residualizing N-succinimidyl 3-guanidinomethyl-5-[125I]iodobenzoate (iso-[125I]SGMIB) also were synthesized. Radiochemical purity, affinity (KD) and immunoreactive fraction of [ 18 F]FN-GK-5F7 were 99%, 5.4 ± 0.7 nM and 72.5 ± 4.3%, respectively. Tumor uptake of [ 18 F]FN-GK-5F7 in athymic mice bearing subcutaneous SKOV3 xenografts (3.7 ± 1.2% ID/g and 3.4 ± 1.0% ID/g at 1 h and 3 h, respectively) was 2- to 3-fold lower than for co-injected iso-[125I]SGMIB-5F7 (6.9 ± 1.9 %ID/g and 8.7 ± 3.0 %ID/g). However, due to its 6-fold lower kidney activity levels, tumor-to-kidney ratios for [ 18 F]FN-GK-5F7 were 3-4 times higher than those for co-injected iso-[125I]SGMIB-5F7 as well as those observed for the 18F conjugate lacking the RBBE-cleavable linker. Micro-PET/CT imaging of [ 18 F]FN-GK-5F7 in mice with SKOV-3 subcutaneous xenografts clearly delineated tumor as early as 1 h with minimal activity in the kidneys; however, there was considerable activity in gallbladder and intestines. Although the tumor uptake of [ 18 F]FN-GK-5F7 was unexpectedly disappointing, incorporating an alternative RBBE-cleavable linker into this labeling strategy may ameliorate this problem.
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n maleimidoethyl 3 guanidinomethyl 5 131i iodobenzmide 131i megmib a residualizing prosthetic agent for site specific radioiodination of internalizing single domain Antibody fragments
The Journal of Nuclear Medicine, 2020Co-Authors: Yutian Feng, Ganesan Vaidyanathan, Zhengyuan Zhou, Darryl Mcdougald, Michael R ZalutskyAbstract:581 Objectives: Anti-HER2 single domain Antibody fragments (sdAbs; a.k.a. nanobodies, VHH molecules) are an attractive platform for delivering radionuclides for imaging and targeted radionuclide therapy (TRT). Earlier, we demonstrated excellent tumor uptake and retention as well as low normal tissue uptake when the anti-HER2 sdAb 5F7 was labeled using the residualizing agent N-succinimidyl 3-guanidinomethyl-5-[*I]iodobenzoate (iso-[*I]SGMIB). Because site-specific labeling can have advantages over random methods such as iso-[*I]SGMIB, we developed a maleimide moiety-containing analogue of iso-[*I]SGMIB, N-(maleimidoethyl)-3-(guanidinomethyl)-5-[131I]iodobenzmide ([131I]MEGMIB) and employed it for labeling a 5F7 variant having a cysteine tail (GGC) at its C-terminus (5F7GGC). Methods: The residualizing prosthetic agent MEGMIB and its protected tin precursor Boc2-MEGMTB were synthesized and characterized. Unlabeled MEGMIB was conjugated to reduced 5F7GGC and the binding affinity of the MEGMIB-5F7GGC conjugate to HER2 was measured by surface plasmon resonance. The tin precursor Boc2-MEGMTB was labeled with 131I using NCS as the oxidant and the Boc groups were removed by TFA treatment. The resultant [131I]MEGMIB was conjugated to 5F7GGC at pH of 6.3. Radiochemical purity (RCP) of the labeled conjugate was determined by SDS-PAGE and size-exclusion chromatography. Immunoreactive fraction (IRF) was determined by the Lindmo method and binding affinity (Kd) using the HER2-positive SKOV-3 ovarian cancer cell line. Paired-label internalization of [131I]MEGMIB-5F7GGC and iso-[125I]SGMIB-5F7 was determined on SKOV-3 cells. Paired-label biodistribution of [131I]MEGMIB-5F7GGC and iso-[125I]SGMIB-5F7 was assessed in nude mice bearing subcutaneous SKOV-3 xenografts. Results: [131I]MEGMIB was synthesized in 87 ± 6% (n=11) yield, comparable to yields for iso-[131I]SGMIB. Conjugation of [131I]MEGMIB to 5F7GGC was achieved in an yield of 45% ± 7% (n=10) with a RCP, Kd and IRF of [131I]MEGMIB-5F7GGC of higher than 99%, 0.94 ± 0.27 nM and 81%, respectively. Specific uptake by SKOV-3 cells after a 1 h incubation was 10.6 ± 0.6% and 8.4 ± 0.3% for 131I and 125I, respectively with the difference statistically significant. The amount of initially bound activity that was internalized at 1 h was 50.30 ± 3.36% for 131I similar to that for 125I (55.95 ± 3.27% Fig. 2). The uptake (%ID/g) of [131I]MEGMIB-5F7GGC in SKOV-3 xenografts was 8.43 ± 2.84, 6.11 ± 1.63, and 3.60 ± 1.75% ID/g at 1, 4 and 24 h, respectively; compared to 8.35 ± 2.66, 6.11 ± 1.56 and 3.13 ± 1.52% ID/g for co-injected iso-[125I]SGMIB-5F7. The kidney activity for [131I]MEGMIB-5F7GGC at 1 h (20.39 ± 5.34 %ID/g) was significantly lower (P=0.01) than that for iso-[125I]SGMIB-5F7 (41.82 ± 13.33 %ID/g). The difference in kidney levels for the two tracers also was significant at 4 h (P=0.02). Tumor-to-kidney ratios for [131I]MEGMIB-5F7GGC also were significantly higher (0.41 ± 0.09 vs 0.21 ± 0.09 at 1h; P=0.008). Conclusion: This novel residualizing prosthetic agent allows site-specific labeling of 5F7GGC, a HER2-specific sdAb with a free cysteine at the C-terminus, in good yield with excellent retention of affinity, immunoreactivity, intracellular retention and tumor localizing capacity. Importantly, tumor uptake of [131I]MEGMIB-5F7GGC was comparable to that for the previous lead agent iso-[125I]SGMIB-5F7, but with up to two fold lower uptake in the kidneys, the dose limiting tissue for radiolabeled scFv fragments. Acknowledgements: This work was supported in part by NIH Grant CA42324.
Nick Devoogdt - One of the best experts on this subject based on the ideXlab platform.
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labeling single domain Antibody fragments with fluorine 18 using 2 3 5 6 tetrafluorophenyl 6 18f fluoronicotinate resulting in high tumor to kidney ratios
Molecular Pharmaceutics, 2019Co-Authors: Zhengyuan Zhou, Nick Devoogdt, Michael R Zalutsky, Darryl Mcdougald, Ganesan VaidyanathanAbstract:ImmunoPET agents are being investigated to assess the status of epidermal growth factor receptor 2 (HER2) in breast cancer patients with the goal of selecting those likely to benefit from HER2-targeted therapies and monitoring their progress after these treatments. We have been exploring the use of single domain Antibody fragments (sdAbs) labeled with 18F using residualizing prosthetic agents for this purpose. In this study, we have labeled two sdAbs that bind to different domains on the HER2 receptor, 2Rs15d and 5F7, using 2,3,5,6-tetrafluorophenyl 6-[18F]fluoronicotinate ([18F]TFPFN) and evaluated their HER2 targeting properties in vitro and in vivo. The overall decay-corrected radiochemical yield for the synthesis of [18F]TFPFN-2Rs15d and [18F]TFPFN-5F7 was 5.7 ± 3.6 and 4.0 ± 2.0%, respectively. The radiochemical purity of labeled sdAbs was >95%, immunoreactive fractions were about 60%, and affinity was in the low nanomolar range. Intracellularly trapped activity from [18F]TFPFN-2Rs15d and [18F]TFPFN-...
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labeling single domain Antibody fragments with fluorine 18 using 2 3 5 6 tetrafluorophenyl 6 18f fluoronicotinate resulting in high tumor to kidney ratios
Molecular Pharmaceutics, 2019Co-Authors: Zhengyuan Zhou, Nick Devoogdt, Michael R Zalutsky, Darryl Mcdougald, Ganesan VaidyanathanAbstract:ImmunoPET agents are being investigated to assess the status of epidermal growth factor receptor 2 (HER2) in breast cancer patients with the goal of selecting those likely to benefit from HER2-targeted therapies and monitoring their progress after these treatments. We have been exploring the use of single domain Antibody fragments (sdAbs) labeled with 18F using residualizing prosthetic agents for this purpose. In this study, we have labeled two sdAbs that bind to different domains on the HER2 receptor, 2Rs15d and 5F7, using 2,3,5,6-tetrafluorophenyl 6-[18F]fluoronicotinate ([18F]TFPFN) and evaluated their HER2 targeting properties in vitro and in vivo. The overall decay-corrected radiochemical yield for the synthesis of [18F]TFPFN-2Rs15d and [18F]TFPFN-5F7 was 5.7 ± 3.6 and 4.0 ± 2.0%, respectively. The radiochemical purity of labeled sdAbs was >95%, immunoreactive fractions were about 60%, and affinity was in the low nanomolar range. Intracellularly trapped activity from [18F]TFPFN-2Rs15d and [18F]TFPFN-5F7 in HER2-expressing SKOV-3 ovarian and BT474M1 breast carcinoma cells were similar to the sdAbs labeled using the previously validated radioiodination residualizing prosthetic agents N-succinimidyl 4-guanidinomethyl-3-[125I]iodobenzoate ([125I]SGMIB) and N-succinimidyl 3-guanidinomethyl-5-[125I]iodobenzoate ( iso-[125I]SGMIB). Intracellular activity was about 2-fold higher for radiolabeled 5F7 compared with 2Rs15d for both 18F and 125I. While tumor uptake of both [18F]TFPFN-2Rs15d and [18F]TFPFN-5F7 was comparable to those for the coadministered 125I-labeled sdAb, renal uptake of the 18F-labeled sdAbs was substantially lower. In microPET images, the tumor was clearly delineated in SKOV-3 and BT474 xenograft-bearing athymic mice with low levels of background activity in normal tissues, except the bladder. These results indicate that the [18F]TFPFN prosthetic group could be a valuable reagent for developing sdAb-based immunoPET imaging agents.
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an efficient method for labeling single domain Antibody fragments with 18f using tetrazine trans cyclooctene ligation and a renal brush border enzyme cleavable linker
Bioconjugate Chemistry, 2018Co-Authors: Zhengyuan Zhou, Nick Devoogdt, Michael R Zalutsky, Ganesan VaidyanathanAbstract:Single domain Antibody fragments (sdAbs) labeled with 18F have shown promise for assessing the status of oncological targets such as the human epidermal growth factor receptor 2 (HER2) by positron emission tomography (PET). Earlier, we evaluated two residualizing prosthetic agents for 18F-labeling of anti-HER2 sdAbs; however, these methods resulted in poor labeling yields and high uptake of 18F activity in the kidneys. To potentially mitigate these limitations, we have now developed an 18F labeling method that utilizes the trans-cyclooctene (TCO)-tetrazine (Tz)-based inverse-electron demand Diels–Alder reaction (IEDDAR) in tandem with a renal brush border enzyme-cleavable glycine-lysine (GK) linker in the prosthetic moiety. The HER2-targeted sdAb 2Rs15d was derivatized with TCO-GK-PEG4-NHS or TCO-PEG4-NHS, which lacks the cleavable linker. As an additional control, the non HER2-specific sdAb R3B23 was derivatized with TCO-GK-PEG4-NHS. The resultant sdAb conjugates were labeled with 18F by IEDDAR using [18...
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an efficient method for labeling single domain Antibody fragments with 18f using tetrazine trans cyclooctene ligation and a renal brush border enzyme cleavable linker
Bioconjugate Chemistry, 2018Co-Authors: Zhengyuan Zhou, Nick Devoogdt, Michael R Zalutsky, Ganesan VaidyanathanAbstract:Single domain Antibody fragments (sdAbs) labeled with 18F have shown promise for assessing the status of oncological targets such as the human epidermal growth factor receptor 2 (HER2) by positron emission tomography (PET). Earlier, we evaluated two residualizing prosthetic agents for 18F-labeling of anti-HER2 sdAbs; however, these methods resulted in poor labeling yields and high uptake of 18F activity in the kidneys. To potentially mitigate these limitations, we have now developed an 18F labeling method that utilizes the trans-cyclooctene (TCO)-tetrazine (Tz)-based inverse-electron demand Diels-Alder reaction (IEDDAR) in tandem with a renal brush border enzyme-cleavable glycine-lysine (GK) linker in the prosthetic moiety. The HER2-targeted sdAb 2Rs15d was derivatized with TCO-GK-PEG4-NHS or TCO-PEG4-NHS, which lacks the cleavable linker. As an additional control, the non HER2-specific sdAb R3B23 was derivatized with TCO-GK-PEG4-NHS. The resultant sdAb conjugates were labeled with 18F by IEDDAR using [18F]AlF-NOTA-PEG4-methyltetrazine. As a positive control, the 2Rs15d sdAb was radioiodinated using the well-characterized residualizing prosthetic agent, N-succinimidyl 4-guanidinomethyl-3-[125I]iodobenzoate ([125I]SGMIB). Synthesis of [18F]AlF-NOTA-Tz-TCO-GK-2Rs15d was achieved with an overall radiochemical yield (RCY) of 17.8 ± 1.5% ( n = 5) in 90 min, a significant improvement over prior methods (3-4% in 2-3 h). In vitro assays indicated that [18F]AlF-NOTA-Tz-TCO-GK-2Rs15d bound with high affinity and immunoreactivity to HER2. In normal mice, when normalized to coinjected [125I]SGMIB-2Rs15d, the kidney uptake of [18F]AlF-NOTA-Tz-TCO-GK-2Rs15d was 15- and 28-fold lower ( P < 0.001) than that seen for the noncleavable control ([18F]AlF-NOTA-Tz-TCO-2Rs15d) at 1 and 3 h, respectively. Uptake of [18F]AlF-NOTA-Tz-TCO-GK-2Rs15d in HER2-expressing SKOV-3 ovarian carcinoma xenografts implanted in athymic mice was about 80% of that seen for coinjected [125I]SGMIB-2Rs15d. On the other hand, kidney uptake was 5-6-fold lower, and as a result, tumor-to-kidney ratios were 4-fold higher for [18F]AlF-NOTA-Tz-TCO-GK-2Rs15d than those for [125I]SGMIB-2Rs15d. SKOV-3 xenografts were clearly delineated even at 1 h after administration of [18F]AlF-NOTA-Tz-TCO-GK-2Rs15d by Micro-PET/CT imaging with even higher contrast observed thereafter. In conclusion, this strategy warrants further evaluation for labeling small proteins such as sdAbs because it offers the benefits of good radiochemical yields and enhanced tumor-to-normal tissue ratios, particularly in the kidney.
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phase i results of cam h2 safety profile and tumor targeting in patients
Journal of Clinical Oncology, 2018Co-Authors: Marleen Keyaerts, Jens De Vos, Vicky Caveliers, Francois Duhoux, C Fontaine, Marjan Vanhoeij, Matthias Dhuyvetter, H Everaert, Pieterjan Ghykiere, Nick DevoogdtAbstract:e13017Background: CAM-H2 is a targeted radionuclide therapeutic drug (TRNT) directed at HER2 expressing cancers. The drug is a single domain Antibody fragment (sdAb) covalently linked to the toxic ...
Ellen R. Goldman - One of the best experts on this subject based on the ideXlab platform.
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importance of hypervariable region 2 for stability and affinity of a shark single domain Antibody specific for ebola virus nucleoprotein
PLOS ONE, 2016Co-Authors: George P. Anderson, Jinny L. Liu, Dan Zabetakis, Daniel D Teichler, Lisa C Shriverlake, Stephen G Lonsdale, Sarah A Goodchild, Ellen R. GoldmanAbstract:Single-Domain antibodies derived from the unique New Antigen Receptor found in sharks have numerous potential applications, ranging from diagnostic reagents to therapeutics. Shark-derived Single-Domain antibodies possess the same characteristic ability to refold after heat denaturation found in Single-Domain antibodies derived from camelid heavy-chain-only antibodies. Recently, two shark derived Single-Domain antibodies specific for the nucleoprotein of Ebola virus were described. Our evaluation confirmed their high affinity for the nucleoprotein, but found their melting temperatures to be low relative to most Single-Domain antibodies. Our first approach towards improving their stability was grafting antigen-binding regions (complementarity determining regions) of one of these Single-Domain antibodies onto a high melting temperature shark Single-Domain Antibody. This resulted in two variants: one that displayed excellent affinity with a low melting temperature, while the other had poor affinity but a higher melting temperature. These new proteins, however, differed in only 3 amino acids within the complementarity determining region 2 sequence. In shark Single-Domain antibodies, the complementarity determining region 2 is often referred to as hypervariable region 2, as this segment of the Antibody domain is truncated compared to the sequence in camelid Single-Domain antibodies and conventional heavy chain variable domains. To elucidate which of the three amino acids or combinations thereof were responsible for the affinity and stability we made the 6 double and single point mutants that covered the intermediates between these two clones. We found a single amino acid change that achieved a 10°C higher melting temperature while maintaining sub nM affinity. This research gives insights into the impact of the shark sdAb hypervariable 2 region on both stability and affinity.
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Next-generation sequencing of a single domain Antibody repertoire reveals quality of phage display selected candidates
PLoS ONE, 2016Co-Authors: Kendrick B. Turner, Jennifer Naciri, Jinny L. Liu, Ellen R. Goldman, George P. Anderson, Dan ZabetakisAbstract:Next-Generation Sequencing and bioinformatics are powerful tools for analyzing the large number of DNA sequences present in an immune library. In this work, we constructed a cDNA library of single domain antibodies from a llama immunized with staphylococcal enterotoxin B. The resulting library was sequenced, resulting in approximately 8.5 million sequences with 5.4 million representing intact, useful sequences. The sequenced library was interrogated using sequences of known SEB-binding single domain antibodies from the library obtained through phage display panning methods in a previous study. New antibodies were identified, produced, and characterized, and were shown to have affinities and melting temperatures comparable to those obtained by traditional panning methods. This demonstrates the utility of using NGS as a complementary tool to phage-displayed biopanning as a means for rapidly obtaining additional antibodies from an immune library. It also shows that phage display, using a library of high diversity, is able to select high quality antibodies even when they are low in frequency.
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enhanced stabilization of a stable single domain Antibody for seb toxin by random mutagenesis and stringent selection
Protein Engineering Design & Selection, 2014Co-Authors: Kendrick B. Turner, Dan Zabetakis, Ellen R. Goldman, George P. AndersonAbstract:Single domain antibodies, recombinant variable heavy domains derived from the unique heavy-chain only antibodies found in camelids and sharks, are exceptionally rugged due to their ability to refold following heat or chemical denaturation. In addition, a number of single domain antibodies have been found to possess high melting points which provide an even greater degree of stability; one of these, llama-derived A3, is a binder of Staphylococcal enterotoxin B and has a Tm of 83.5 °C. In this work, we utilized random mutagenesis and stringent selection in an effort to obtain variants of A3 with even higher melting points. This effort resulted in the selection of a double mutant, A3-T28I-S72I, which has a melting point of 90.0 °C and near wild-type affinity for the target antigen. We further characterized the mutations individually to determine that while both contributed to the thermal stabilization, the T28I mutation accounted for ∼ 4.1 °C of the 6.5 °C increase. This work demonstrates that by the addition of relatively subtle changes it is possible to further improve the melting temperature of single domain antibodies that are already remarkably stable.
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RESEARCH ARTICLE Open Access Isolation of a Highly Thermal Stable Lama Single Domain Antibody Specific for Staphylococcus
2013Co-Authors: Aureus Enterotoxin B, Jinny L. Liu, Dan Zabetakis, Ellen R. Goldman, George P. Anderson, Russell R Graef, Katherine A Doyle, Felicia N Sutton, Joseline Serrano-gonzález, Lynn A CooperAbstract:Background: Camelids and sharks possess a unique subclass of antibodies comprised of only heavy chains. The antigen binding fragments of these unique antibodies can be cloned and expressed as single domain antibodies (sdAbs). The ability of these small antigen-binding molecules to refold after heating to achieve their original structure, as well as their diminutive size, makes them attractive candidates for diagnostic assays. Results: Here we describe the isolation of an sdAb against Staphyloccocus aureus enterotoxin B (SEB). The clone, A3, was found to have high affinity (Kd = 75 pM) and good specificity for SEB, showing no cross reactivity to related molecules such as Staphylococcal enterotoxin A (SEA), Staphylococcal enterotoxin D (SED), and Shiga toxin. Most remarkably, this anti-SEB sdAb had an extremely high Tm of 85°C and an ability to refold after heating to 95° C. The sharp Tm determined by circular dichroism, was found to contrast with the gradual decrease observed in intrinsic fluorescence. We demonstrated the utility of this sdAb as a capture and detector molecule in Luminex based assays providing limits of detection (LODs) of at least 64 pg/mL. Conclusion: The anti-SEB sdAb A3 was found to have a high affinity and an extraordinarily high Tm and could still refold to recover activity after heat denaturation. This combination of heat resilience and strong, specific binding make this sdAb a good candidate for use in Antibody-based toxin detection technologies
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single domain Antibody quantum dot conjugates for ricin detection by both fluoroimmunoassay and surface plasmon resonance
Analytica Chimica Acta, 2013Co-Authors: George P. Anderson, Richard H Glaven, Russ W Algar, Kimihiro Susumu, Michael H Stewart, Igor L Medintz, Ellen R. GoldmanAbstract:The combination of stable biorecognition elements and robust quantum dots (QDs) has the potential to yield highly effective reporters for bioanalyses. Llama-derived single domain antibodies (sdAb) provide small thermostable recognition elements that can be easily manipulated using standard DNA methods. The sdAb was self-assembled on dihydrolipoic acid (DHLA) ligand-capped CdSe-ZnS core-shell QDs made in our laboratory through the polyhistidine tail of the protein, which coordinated to zinc ions on the QD surface. The sdAb-QD bioconjugates were then applied in both fluorometric and surface plasmon resonance (SPR) immunoassays for the detection of ricin, a potential biothreat agent. The sdAb-QD conjugates functioned in fluoroimmunoassays for the detection of ricin, providing equivalent limits of detection when compared to the same anti-ricin sdAb labeled with a conventional fluorophore. In addition, the DHLA-QD-sdAb conjugates were very effective reporter elements in SPR sandwich assays, providing more sensitive detection with a signal enhancement of ~10-fold over sdAb reporters and 2-4 fold over full sized Antibody reporters. Commercially prepared streptavidin-modified polymer-coated QDs also amplified the SPR signal for the detection of ricin when applied to locations where biotinylated anti-ricin sdAb was bound to target; however, we observed a 4-fold greater amplification when using the DHLA-QD-sdAb conjugates in this format.
