Use of Fc-Engineered Antibodies as Clearing Agents to Increase Contrast During PET (original) (raw)
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Selective depletion of radiolabeled HER2-specific antibody for contrast improvement during PET
mAbs
The prolonged in vivo persistence of antibodies results in high background and poor contrast during their use as molecular imaging agents for positron emission tomography (PET). We have recently described a class of engineered Fc fusion proteins that selectively deplete antigen-specific antibodies without affecting the levels of antibodies of other specificities. Here, we demonstrate that these Fc fusions (called Seldegs, for selective degradation) can be used to clear circulating, radiolabeled HER2-specific antibody during diagnostic imaging of HER2-positive tumors in mice. The analyses show that Seldegs have considerable promise for the reduction of whole-body exposure to radiolabel and improvement of contrast during PET.
ImmunoPET using engineered antibody fragments: fluorine-18 labeled diabodies for same-day imaging
Tumor Biology, 2012
Combining the specificity of tumor-targeting antibodies with the sensitivity and quantification offered by positron emission tomography (PET) provides tremendous opportunities for molecular characterization of tumors in vivo. Until recently, significant challenges have been faced when attempting to combine antibodies which show long biological half-lives and positron-emitting radionuclides with comparably short physical half-lives, in particular 18 F (half-life, 109 min). A fast and simple microwave-assisted method of generating N-succinimidyl-4-[ 18 F]fluorobenzoate has been developed and employed for radiolabeling a small, rapidly targeting HER2-specific engineered antibody fragment, the cysdiabody. Using this tracer, HER2-positive tumor xenografts in mice were detected at 1-4 h post-injection by microPET. This confirms the rapid kinetics of [ 18 F]fluorobenzoyl cys-diabody localization, and demonstrates the feasibility of same-day immunoPET imaging. This approach can be broadly applied to antibodies targeting cell surface biomarkers for molecular imaging of tumors and should be highly translatable for clinical use.
Journal of Nuclear Medicine
Prolonged clearance kinetics have hampered the development of intact antibodies as imaging agents, despite their ability to effectively deliver radionuclides to tumor targets in vivo. Genetically engineered antibody fragments display rapid, high-level tumor uptake coupled with rapid clearance from the circulation in the athymic mouse/LS174T xenograft model. The anticarcinoembryonic antigen (CEA) T84.66 minibody (single-chain Fv fragment [scFv]-C H 3 dimer, 80 kDa) and T84.66 diabody (noncovalent dimer of scFv, 55 kDa) exhibit pharmacokinetics favorable for radioimmunoimaging. The present work evaluated the minibody or diabody labeled with 124 I, for imaging tumor-bearing mice using a high-resolution small-animal PET system. Methods: Labeling was conducted with 0.2-0.3 mg of protein and 65-98 MBq (1.7-2.6 mCi) of 124 I using an iodination reagent. Radiolabeling efficiencies ranged from 33% to 88%, and immunoreactivity was 42% (diabody) or Ͼ90% (minibody). In vivo distribution was evaluated in athymic mice bearing paired LS174T human colon carcinoma (CEA-positive) and C6 rat glioma (CEA-negative) xenografts. Mice were injected via the tail vein with 1.9 -3.1 MBq (53-85 Ci) of 124 I-minibody or with 3.1 MBq (85 Ci) of 124 I-diabody and imaged at 4 and 18 h by PET. Some mice were also imaged using 18 F-FDG 2 d before imaging with 124 I-minibody. Results: PET images using 124 I-labeled minibody or diabody showed specific localization to the CEA-positive xenografts and relatively low activity elsewhere in the mice, particularly by 18 h. Target-to-background ratios for the LS174T tumors versus soft tissues using 124 I-minibody were 3.05 at 4 h and 11.03 at 18 h. Similar values were obtained for the 124 Idiabody (3.95 at 4 h and 10.93 at 18 h). These results were confirmed by direct counting of tissues after the final imaging. Marked reduction of normal tissue activity, especially in the abdominal region, resulted in high-contrast images at 18 h for the 124 I-anti-CEA diabody. CEA-positive tumors as small as 11 mg (Ͻ3 mm in diameter) could be imaged, and 124 I-anti-CEA minibodies, compared with 18 F-FDG, demonstrated highly specific localization. Conclusion: 124 I labeling of engineered antibody fragments provides a promising new class of tumor-specific probes for PET imaging of tumors and metastases.
Theranostics, 2019
Antibody-based PET tracers are exceptionally well-suited for determination of the in vivo biodistribution and quantification of therapeutic antibodies. The continued expansion in antibody-based therapeutics has accordingly driven the development towards more robust conjugation strategies in order to reliably predict the performance of such agents. We therefore aimed to evaluate the effect of site-specific labeling by enzymatic remodeling on the stability, immuno-reactivity and tumor-targeting properties of the monoclonal antibody (mAb) trastuzumab and compare it to conventional, random labeling in a HER2-positive xenograft mouse model. Methods: Trastuzumab was conjugated with the p-SCN-Bn-Desferrioxamine (SCN-Bn-DFO) chelator randomly on lysine residues or site-specifically on enzymatically modified glycans using either β-galactosidase or endoglycosidase S2 prior to 89 Zr radiolabeling. 89 Zr-DFO-trastuzumab was injected into SK-OV-3 tumor-bearing NMRI nude mice. The antibody dose was titrated with either 100 µg or 500 µg of unlabeled trastuzumab. Mice underwent small animal PET/CT imaging 24, 70 and 120 hours post-injection for longitudinal assessment. Parallel experiments were conducted with an isotype control matched antibody. In vivo imaging was supported by conventional ex vivo biodistribution and HER2 immuno-histochemistry. Furthermore, site-specifically labeled 89 Zr-DFO-trastuzumab was evaluated in a panel of subcutaneous patient-derived xenograft (PDX) models. Additionally, the affinity, in vitro stability and immuno-reactivity were assessed for all tracers. Results: Site-specific labeling significantly increased PET tumor uptake (One-way ANOVA, p<0.0001) at all time-points when compared to random labeling. Mean tumor uptakes were 6.7 ± 1.7, 13.9 ± 3.3 and 15.3 ± 3.8 % injected dose per gram tissue (%ID/g) at 70 hours post-injection, for random, β-galactosidase or endoglycosidase S2 labeled probes, respectively. Co-injection with unlabeled trastuzumab increased the circulation time of tracers but did not alter tumor uptake notably. Site-specific probes presented with a superior in vitro stability and immuno-reactivity compared to the randomly labeled probe. Ex vivo biodistribution confirmed the data obtained by in vivo PET imaging, and site-specific 89 Zr-DFO-trastuzumab successfully detected HER2-positive tumors in PDX mouse models. Conclusion: 89 Zr-DFO-trastuzumab is well-matched for specific immuno-PET imaging of HER2-positive tumors and site-specific labeling of trastuzumab by the SiteClick TM technology minimizes the impact of the Ivyspring International Publisher
Journal of Nuclear Medicine, 2009
To optimize in vivo tissue uptake kinetics and clearance of engineered monoclonal antibody (mAb) fragments for radiotherapeutic and radiodiagnostic applications, we compared the biodistribution and tumor localization of four 111 In-and 86 Y-labeled antibody formats, derived from a single antimindin/RG-1 mAb, in a prostate tumor model. The IgG, diabody, single-chain variable domain (scFv), and novel miniantibody formats, composed of the human IgE-C H 4 and a modified IgG1 hinge linked to scFv domains, were compared. Methods: Antibodies were first derivatized with the bifunctional chelator CHX-A$-diethylenetriamine pentaacetic acid and then bound to the radiometal to create radiolabeled immunoconjugates. Human LNCaP xenografts were grown in nude mice, and 111 In-or 86 Y-labeled antibodies were administered intravenously. Tissues were harvested at different times, and the level of antibody deposition was determined by measuring radioactivity. Whole-body small-animal PET of mice receiving 86 Y-labeled antibodies was performed at 6 time points and colocalized with simultaneous micro-CT imaging. Results: The biodistributions of 111 In and 86 Y antibodies were quite similar. The blood, tumor, kidney, and liver tissues contained varying levels of radioactivity. The antibody accumulation in the tumor correlated with molecular size. The IgG steadily increased with time to 24.1 percentage injected dose per gram (%ID/g) at 48 h. The miniantibody accumulated at a similar rate to reach a lower level (14.2 %ID/g) at 48 h but with a higher tumor-to-blood ratio than the IgG. Tumor accumulation of the diabody peaked at 3 h, reaching a much lower level (3.7 %ID/g). A combination of rapid clearance and lower relative affinity of the scFv precluded deposition in the tumor. Small-animal PET results correlated well with the biodistribution results, with similar tumor localization patterns. Conclusion: The larger antibody formats (IgG and miniantibody) gave higher tumor uptake levels than did the smaller formats (diabody and scFv). These larger formats may be more suitable for radioimmunotherapy applications, evidenced by the preclinical efficacy previously shown by a report on the IgG format. The smaller formats were rapidly cleared from circulation, and the diabody, which accumulated in the tumor, may be more suitable for radiodiagnostic applications.
Prolonged clearance kinetics have hampered the development of intact antibodies as imaging agents, despite their ability to effectively deliver radionuclides to tumor targets in vivo. Genetically engineered antibody fragments display rapid, high-level tumor uptake coupled with rapid clearance from the circulation in the athymic mouse/LS174T xenograft model. The anticarcinoembryonic antigen (CEA) T84.66 minibody (single-chain Fv fragment [scFv]-C H 3 dimer, 80 kDa) and T84.66 diabody (noncovalent dimer of scFv, 55 kDa) exhibit pharmacokinetics favorable for radioimmunoimaging. The present work evaluated the minibody or diabody labeled with 124 I, for imaging tumor-bearing mice using a high-resolution small-animal PET system. Methods: Labeling was conducted with 0.2-0.3 mg of protein and 65-98 MBq (1.7-2.6 mCi) of 124 I using an iodination reagent. Radiolabeling efficiencies ranged from 33% to 88%, and immunoreactivity was 42% (diabody) or Ͼ90% (minibody). In vivo distribution was evaluated in athymic mice bearing paired LS174T human colon carcinoma (CEA-positive) and C6 rat glioma (CEA-negative) xenografts. Mice were injected via the tail vein with 1.9 -3.1 MBq (53-85 Ci) of 124 I-minibody or with 3.1 MBq (85 Ci) of 124 I-diabody and imaged at 4 and 18 h by PET. Some mice were also imaged using 18 F-FDG 2 d before imaging with 124 I-minibody. Results: PET images using 124 I-labeled minibody or diabody showed specific localization to the CEA-positive xenografts and relatively low activity elsewhere in the mice, particularly by 18 h. Target-to-background ratios for the LS174T tumors versus soft tissues using 124 I-minibody were 3.05 at 4 h and 11.03 at 18 h. Similar values were obtained for the 124 Idiabody (3.95 at 4 h and 10.93 at 18 h). These results were confirmed by direct counting of tissues after the final imaging. Marked reduction of normal tissue activity, especially in the abdominal region, resulted in high-contrast images at 18 h for the 124 I-anti-CEA diabody. CEA-positive tumors as small as 11 mg (Ͻ3 mm in diameter) could be imaged, and 124 I-anti-CEA minibodies, compared with 18 F-FDG, demonstrated highly specific localization. Conclusion: 124 I labeling of engineered antibody fragments provides a promising new class of tumor-specific probes for PET imaging of tumors and metastases.
Journal of Nuclear Medicine, 2009
Human epidermal growth factor receptor type 2 (HER2) is a tyrosine kinase, which is often overexpressed in many carcinomas. Imaging HER2 expression in malignant tumors can provide important prognostic and predictive diagnostic information. The use of anti-HER2 tracers labeled with positron-emitting radionuclides may increase the sensitivity of HER2 imaging. The goal of this study was to compare directly 2 approaches for developing anti-HER2 PET tracers: a 124 I-labeled monoclonal antibody and a small (7-kDa) scaffold protein, the Affibody molecule. Methods: The anti-HER2 Affibody Z HER2:342 and humanized monoclonal antibody trastuzumab were labeled with 124/125 I using p-iodobenzoate (PIB) as a linker. Cellular processing of both tracers by HER2-expressing cells was investigated. The biodistributions of 124 I-PIB-Z HER2:342 and 125 I-PIB-trastuzumab were compared in BALB/C nu/nu mice bearing HER2-expressing NCI-N87 xenografts using paired labels. Small-animal PET of 124 I-PIB-Z HER2:342 and 124 I-PIB-trastuzumab in tumor-bearing mice was performed at 6, 24, and 72 h after injection. Results: Both radioiodinated Z HER2:342 and trastuzumab bound specifically to HER2-expressing cells in vitro and specifically targeted HER2-expressing xenografts in vivo. Radioiodinated trastuzumab was more rapidly internalized and degraded, which resulted in better retention of radioactivity delivered by Z HER2:342 . Total uptake of trastuzumab in tumors was higher than that of 124 I-PIB-Z HER2:342 . However, tumor-to-organ ratios were appreciably higher for 124 I-PIB-Z HER2:342 due to the more rapid clearance of radioactivity from blood and normal organs. The ex vivo results were confirmed by small-animal PET. Conclusion: The use of the small scaffold targeting Affibody provides better contrast in HER2 imaging than does the monoclonal antibody.
Journal of Nuclear Medicine Official Publication Society of Nuclear Medicine, 2003
Prolonged clearance kinetics have hampered the development of intact antibodies as imaging agents, despite their ability to effectively deliver radionuclides to tumor targets in vivo. Genetically engineered antibody fragments display rapid, high-level tumor uptake coupled with rapid clearance from the circulation in the athymic mouse/LS174T xenograft model. The anticarcinoembryonic antigen (CEA) T84.66 minibody (single-chain Fv fragment [scFv]-C H 3 dimer, 80 kDa) and T84.66 diabody (noncovalent dimer of scFv, 55 kDa) exhibit pharmacokinetics favorable for radioimmunoimaging. The present work evaluated the minibody or diabody labeled with 124 I, for imaging tumor-bearing mice using a high-resolution small-animal PET system. Methods: Labeling was conducted with 0.2-0.3 mg of protein and 65-98 MBq (1.7-2.6 mCi) of 124 I using an iodination reagent. Radiolabeling efficiencies ranged from 33% to 88%, and immunoreactivity was 42% (diabody) or Ͼ90% (minibody). In vivo distribution was evaluated in athymic mice bearing paired LS174T human colon carcinoma (CEA-positive) and C6 rat glioma (CEA-negative) xenografts. Mice were injected via the tail vein with 1.9 -3.1 MBq (53-85 Ci) of 124 I-minibody or with 3.1 MBq (85 Ci) of 124 I-diabody and imaged at 4 and 18 h by PET. Some mice were also imaged using 18 F-FDG 2 d before imaging with 124 I-minibody. Results: PET images using 124 I-labeled minibody or diabody showed specific localization to the CEA-positive xenografts and relatively low activity elsewhere in the mice, particularly by 18 h. Target-to-background ratios for the LS174T tumors versus soft tissues using 124 I-minibody were 3.05 at 4 h and 11.03 at 18 h. Similar values were obtained for the 124 Idiabody (3.95 at 4 h and 10.93 at 18 h). These results were confirmed by direct counting of tissues after the final imaging. Marked reduction of normal tissue activity, especially in the abdominal region, resulted in high-contrast images at 18 h for the 124 I-anti-CEA diabody. CEA-positive tumors as small as 11 mg (Ͻ3 mm in diameter) could be imaged, and 124 I-anti-CEA minibodies, compared with 18 F-FDG, demonstrated highly specific localization. Conclusion: 124 I labeling of engineered antibody fragments provides a promising new class of tumor-specific probes for PET imaging of tumors and metastases.
Engineered Antibody Fragments with Infinite Affinity as Reporter Genes for PET Imaging
2008
Reporter gene imaging has great potential for many clinical applica- tions including the tracking of transplanted cells and monitoring of gene therapy. However, currently available reporter gene- reporter probe combinations have significant limitations with the biodistribution of the reporter probe and the specificity and immu- nogenicity of the reporter gene. The objective of the present study was to evaluate a
Improved Characteristics of RANKL Immuno-PET Imaging Using Radiolabeled Antibody Fab Fragments
Pharmaceutics
Purpose: RANKL expression in the tumor microenvironment has been identified as a biomarker of immune suppression, negating the effect of some cancer immunotherapies. Previously we had developed a radiotracer based on the FDA-approved RANKL-specific antibody denosumab, [89Zr]Zr-DFO-denosumab, enabling successful immuno-PET imaging. Radiolabeled denosumab, however, showed long blood circulation and delayed tumor uptake, potentially limiting its applications. Here we aimed to develop a smaller radiolabeled denosumab fragment, [64Cu]Cu-NOTA-denos-Fab, that would ideally show faster tumor accumulation and better diffusion into the tumor for the visualization of RANKL. Experimental design: Fab fragments were prepared from denosumab using papain and conjugated to a NOTA chelator for radiolabeling with 64Cu. The bioconjugates were characterized in vitro using SDS-PAGE analysis, and the binding affinity was assessed using a radiotracer cell binding assay. Small animal PET imaging evaluated t...