Dataset Information

Pretargeted PET Imaging Using a Site-Specifically Labeled Immunoconjugate.

ABSTRACT: In recent years, both site-specific bioconjugation techniques and bioorthogonal pretargeting strategies have emerged as exciting technologies with the potential to improve the safety and efficacy of antibody-based nuclear imaging. In the work at hand, we have combined these two approaches to create a pretargeted PET imaging strategy based on the rapid and bioorthogonal inverse electron demand Diels-Alder reaction between a (64)Cu-labeled tetrazine radioligand ((64)Cu-Tz-SarAr) and a site-specifically modified huA33-trans-cyclooctene immunoconjugate ((ss)huA33-PEG12-TCO). A bioconjugation strategy that harnesses enzymatic transformations and strain-promoted azide-alkyne click chemistry was used to site-specifically append PEGylated TCO moieties to the heavy chain glycans of the colorectal cancer-targeting huA33 antibody. Preclinical in vivo validation studies were performed in athymic nude mice bearing A33 antigen-expressing SW1222 human colorectal carcinoma xenografts. To this end, mice were administered (ss)huA33-PEG12-TCO via tail vein injection and-following accumulation intervals of 24 or 48 h-(64)Cu-Tz-SarAr. PET imaging and biodistribution studies reveal that this strategy clearly delineates tumor tissue as early as 1 h post-injection (6.7 ± 1.7%ID/g at 1 h p.i.), producing images with excellent contrast and high tumor-to-background activity concentration ratios (tumor:muscle = 21.5 ± 5.6 at 24 h p.i.). Furthermore, dosimetric calculations illustrate that this pretargeting approach produces only a fraction of the overall effective dose (0.0214 mSv/MBq; 0.079 rem/mCi) of directly labeled radioimmunoconjugates. Ultimately, this method effectively facilitates the high contrast pretargeted PET imaging of colorectal carcinoma using a site-specifically modified immunoconjugate.

SUBMITTER: Cook BE

PROVIDER: S-EPMC5102008 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Pretargeted PET Imaging Using a Site-Specifically Labeled Immunoconjugate.

Cook Brendon E BE Adumeau Pierre P Membreno Rosemery R Carnazza Kathryn E KE Brand Christian C Reiner Thomas T Agnew Brian J BJ Lewis Jason S JS Zeglis Brian M BM

Bioconjugate chemistry 20160714 8

In recent years, both site-specific bioconjugation techniques and bioorthogonal pretargeting strategies have emerged as exciting technologies with the potential to improve the safety and efficacy of antibody-based nuclear imaging. In the work at hand, we have combined these two approaches to create a pretargeted PET imaging strategy based on the rapid and bioorthogonal inverse electron demand Diels-Alder reaction between a (64)Cu-labeled tetrazine radioligand ((64)Cu-Tz-SarAr) and a site-specifi ...[more]

PMID: 27356886

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Project description:The epidermal growth factor receptor (EGFR) serves as an attractive target for cancer molecular imaging and therapy. Our previous positron emission tomography (PET) studies showed that the EGFR-targeting affibody molecules (64)Cu-DOTA-ZEGFR:1907 and (18)F-FBEM-ZEGFR:1907 can discriminate between high and low EGFR-expression tumors and have the potential for patient selection for EGFR-targeted therapy. Compared with (64)Cu, (18)F may improve imaging of EGFR-expression and is more suitable for clinical application, but the labeling reaction of (18)F-FBEM-ZEGFR:1907 requires a long synthesis time. The aim of the present study is to develop a new generation of (18)F labeled affibody probes (Al(18)F-NOTA-ZEGFR:1907 and (18)F-CBT-ZEGFR:1907) and to determine whether they are suitable agents for imaging of EGFR expression. The first approach consisted of conjugating ZEGFR:1907 with NOTA and radiolabeling with Al(18)F to produce Al(18)F-NOTA-ZEGFR:1907. In a second approach the prosthetic group (18)F-labeled-2-cyanobenzothiazole ((18)F-CBT) was conjugated to Cys-ZEGFR:1907 to produce (18)F-CBT-ZEGFR:1907. Binding affinity and specificity of Al(18)F-NOTA-ZEGFR:1907 and (18)F-CBT-ZEGFR:1907 to EGFR were evaluated using A431 cells. Biodistribution and PET studies were conducted on mice bearing A431 xenografts after injection of Al(18)F-NOTA-ZEGFR:1907 or (18)F-CBT-ZEGFR:1907 with or without coinjection of unlabeled affibody proteins. The radiosyntheses of Al(18)F-NOTA-ZEGFR:1907 and (18)F-CBT-ZEGFR:1907 were completed successfully within 40 and 120 min with a decay-corrected yield of 15% and 41% using a 2-step, 1-pot reaction and 2-step, 2-pot reaction, respectively. Both probes bound to EGFR with low nanomolar affinity in A431 cells. Although (18)F-CBT-ZEGFR:1907 showed instability in vivo, biodistribution studies revealed rapid and high tumor accumulation and quick clearance from normal tissues except the bones. In contrast, Al(18)F-NOTA-ZEGFR:1907 demonstrated high in vitro and in vivo stability, high tumor uptake, and relative low uptake in most of the normal organs except the liver and kidneys at 3 h after injection. The specificity of both probes for A431 tumors was confirmed by their lower uptake on coinjection of unlabeled affibody. PET studies showed that Al(18)F-NOTA-ZEGFR:1907 and (18)F-CBT-ZEGFR:1907 could clearly identify EGFR positive tumors with good contrast. Two strategies for (18)F-labeling of affibody molecules were successfully developed as two model platforms using NOTA or CBT coupling to affibody molecules that contain an N-terminal cysteine. Al(18)F-NOTA-ZEGFR:1907 and (18)F-CBT-ZEGFR:1907 can be reliably obtained in a relatively short time. Biodistribution and PET studies demonstrated that Al(18)F-NOTA-ZEGFR:1907 is a promising PET probe for imaging EGFR expression in living mice.

Project description:To develop imaging and therapeutic agents, antibodies are often conjugated randomly to a chelator/radioisotope or drug using a primary amine (NH2) of lysine or sulfhydryl (SH) of cysteine. Random conjugation to NH2 or SH groups can require extreme conditions and may affect target recognition/binding and must therefore be tested. In the present study, nimotuzumab was site-specifically labeled using ∆N-SpyCatcher/SpyTag with different chelators and radiometals. Nimotuzumab is a well-tolerated anti-EGFR antibody with low skin toxicities. First, ΔN-SpyCatcher was reduced using tris(2-carboxyethyl)phosphine (TCEP), which was followed by desferoxamine-maleimide (DFO-mal) conjugation to yield a reactive ΔN-SpyCatcher-DFO. The ΔN-SpyCatcher-DFO was reacted with nimotuzumab-SpyTag to obtain stable nimotuzumab-SpyTag-∆N-SpyCatcher-DFO. Radiolabeling was performed with 89Zr, and the conjugate was used for the in vivo microPET imaging of EGFR-positive MDA-MB-468 xenografts. Similarly, ∆N-SpyCatcher was conjugated to an eighteen-membered macrocyclic chelator macropa-maleimide and used to radiolabel nimotuzumab-SpyTag with actinium-225 (225Ac) for in vivo radiotherapy studies. All constructs were characterized using biolayer interferometry, flow cytometry, radioligand binding assays, HPLC, and bioanalyzer. MicroPET/CT imaging showed a good tumor uptake of 89Zr-nimotuzumab-SpyTag-∆N-SpyCatcher with 6.0 ± 0.6%IA/cc (n = 3) at 48 h post injection. The EC50 of 225Ac-nimotuzumab-SpyTag-∆N-SpyCatcher and 225Ac-control-IgG-SpyTag-∆N-SpyCatcher against an EGFR-positive cell-line (MDA-MB-468) was 3.7 ± 3.3 Bq/mL (0.04 ± 0.03 nM) and 18.5 ± 4.4 Bq/mL (0.2 ± 0.04 nM), respectively. In mice bearing MDA-MB-468 EGFR-positive xenografts, 225Ac-nimotuzumab-SpyTag-∆N-SpyCatcher significantly (p = 0.0017) prolonged the survival of mice (64 days) compared to 225Ac-control IgG (28.5 days), nimotuzumab (28.5 days), or PBS-treated mice (30 days). The results showed that the conjugation and labeling using SpyTag/∆N-SpyCatcher to nimotuzumab did not significantly (p > 0.05) alter the receptor binding of nimotuzumab compared with a non-specific conjugation approach. 225Ac-nimotuzumab-SpyTag-∆N-SpyCatcher was effective in vitro and in an EGFR-positive triple negative breast cancer xenograft model.

Project description:Background: The pretargeted imaging strategy using inverse electron demand Diels-Alder (IEDDA) cycloaddition between a trans-cyclooctene (TCO) and tetrazine (Tz) has emerged and rapidly grown as a promising concept to improve radionuclide imaging and therapy in oncology. This strategy has mostly relied on the use of radiolabeled Tz together with TCO-modified targeting vectors leading to a rapid growth of the number of available radiolabeled tetrazines, while only a few radiolabeled TCOs are currently reported. Here, we aim to develop novel and structurally diverse 18F-labeled cis-dioxolane-fused TCO (d-TCO) derivatives to further expand the bioorthogonal toolbox for in vivo ligation and evaluate their potential for positron emission tomography (PET) pretargeted imaging. Results: A small series of d-TCO derivatives were synthesized and tested for their reactivity against tetrazines, with all compounds showing fast reaction kinetics with tetrazines. A fluorescence-based pretargeted blocking study was developed to investigate the in vivo ligation of these compounds without labor-intensive prior radiochemical development. Two compounds showed excellent in vivo ligation results with blocking efficiencies of 95 and 97%. Two novel 18F-labeled d-TCO radiotracers were developed, from which [18F]MICA-214 showed good in vitro stability, favorable pharmacokinetics, and moderate in vivo stability. Micro-PET pretargeted imaging with [18F]MICA-214 in mice bearing LS174T tumors treated with tetrazine-modified CC49 monoclonal antibody (mAb) (CC49-Tz) showed significantly higher uptake in tumor tissue in the pretargeted group (CC49-Tz 2.16 ± 0.08% ID/mL) when compared to the control group with nonmodified mAb (CC49 1.34 ± 0.07% ID/mL). Conclusions: A diverse series of fast-reacting fluorinated d-TCOs were synthesized. A pretargeted blocking approach in tumor-bearing mice allowed the choice of a lead compound with fast reaction kinetics with Tz. A novel 18F-labeled d-TCO tracer was developed and used in a pretargeted PET imaging approach, allowing specific tumor visualization in a mouse model of colorectal cancer. Although further optimization of the radiotracer is needed to enhance the tumor-to-background ratios for pretargeted imaging, we anticipate that the 18F-labeled d-TCO will find use in studies where increased hydrophilicity and fast bioconjugation are required.

Dataset Information

Pretargeted PET Imaging Using a Site-Specifically Labeled Immunoconjugate.

Publications

Pretargeted PET Imaging Using a Site-Specifically Labeled Immunoconjugate.

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