A Pretargeted Approach for the Multimodal PET/NIRF Imaging of Colorectal Cancer.
ABSTRACT: The complementary nature of positron emission tomography (PET) and near-infrared fluorescence (NIRF) imaging makes the development of strategies for the multimodal PET/NIRF imaging of cancer a very enticing prospect. Indeed, in the context of colorectal cancer, a single multimodal PET/NIRF imaging agent could be used to stage the disease, identify candidates for surgical intervention, and facilitate the image-guided resection of the disease. While antibodies have proven to be highly effective vectors for the delivery of radioisotopes and fluorophores to malignant tissues, the use of radioimmunoconjugates labeled with long-lived nuclides such as 89Zr poses two important clinical complications: high radiation doses to the patient and the need for significant lag time between imaging and surgery. In vivo pretargeting strategies that decouple the targeting vector from the radioactivity at the time of injection have the potential to circumvent these issues by facilitating the use of positron-emitting radioisotopes with far shorter half-lives. Here, we report the synthesis, characterization, and in vivo validation of a pretargeted strategy for the multimodal PET and NIRF imaging of colorectal carcinoma. This approach is based on the rapid and bioorthogonal ligation between a trans-cyclooctene- and fluorophore-bearing immunoconjugate of the huA33 antibody (huA33-Dye800-TCO) and a 64Cu-labeled tetrazine radioligand (64Cu-Tz-SarAr). In vivo imaging experiments in mice bearing A33 antigen-expressing SW1222 colorectal cancer xenografts clearly demonstrate that this approach enables the non-invasive visualization of tumors and the image-guided resection of malignant tissue, all at only a fraction of the radiation dose created by a directly labeled radioimmunoconjugate. Additional in vivo experiments in peritoneal and patient-derived xenograft models of colorectal carcinoma reinforce the efficacy of this methodology and underscore its potential as an innovative and useful clinical tool.
Project description:The specificity of antibodies have made immunoconjugates promising vectors for the delivery of radioisotopes to cancer cells; however, their long pharmacologic half-lives necessitate the use of radioisotopes with long physical half-lives, a combination that leads to high radiation doses to patients. Therefore, the development of targeting modalities that harness the advantages of antibodies without their pharmacokinetic limitations is desirable. To this end, we report the development of a methodology for pretargeted PET imaging based on the bioorthogonal Diels-Alder click reaction between tetrazine and transcyclooctene.A proof-of-concept system based on the A33 antibody, SW1222 colorectal cancer cells, and (64)Cu was used. The huA33 antibody was covalently modified with transcyclooctene, and a NOTA-modified tetrazine was synthesized and radiolabeled with (64)Cu. Pretargeted in vivo biodistribution and PET imaging experiments were performed with athymic nude mice bearing A33 antigen-expressing, SW1222 colorectal cancer xenografts.The huA33 antibody was modified with transcyclooctene to produce a conjugate with high immunoreactivity, and the (64)Cu-NOTA-labeled tetrazine ligand was synthesized with greater than 99% purity and a specific activity of 9-10 MBq/?g. For in vivo experiments, mice bearing SW1222 xenografts were injected with transcyclooctene-modified A33; after allowing 24 h for accumulation of the antibody in the tumor, the mice were injected with (64)Cu-NOTA-labeled tetrazine for PET imaging and biodistribution experiments. At 12 h after injection, the retention of uptake in the tumor (4.1 ± 0.3 percent injected dose per gram), coupled with the fecal excretion of excess radioligand, produced images with high tumor-to-background ratios. PET imaging and biodistribution experiments performed using A33 directly labeled with either (64)Cu or (89)Zr revealed that although absolute tumor uptake was higher with the directly radiolabeled antibodies, the pretargeted system yielded comparable images and tumor-to-muscle ratios at 12 and 24 h after injection. Further, dosimetry calculations revealed that the (64)Cu pretargeting system resulted in only a fraction of the absorbed background dose of A33 directly labeled with (89)Zr (0.0124 mSv/MBq vs. 0.4162 mSv/MBq, respectively).The high quality of the images produced by this pretargeting approach, combined with the ability of the methodology to dramatically reduce nontarget radiation doses to patients, marks this system as a strong candidate for clinical translation.
Project description:The complementary nature of positron emission tomography (PET) and optical imaging (OI) has fueled increasing interest in the development of multimodal PET/OI probes that can be employed during the diagnosis, staging, and surgical treatment of cancer. Due to their high selectivity and affinity, antibodies have emerged as promising platforms for the development of hybrid PET/OI agents. However, the lack of specificity of many bioconjugation reactions can threaten immunoreactivity and lead to poorly defined constructs. To circumvent this issue, we have developed a chemoenzymatic strategy for the construction of multimodal PET/OI immunoconjugates that have been site-specifically labeled on the heavy chain glycans. The methodology consists of four steps: (1) the enzymatic removal of the terminal galactose residues on the heavy chain glycans; (2) the enzymatic incorporation of azide-bearing galactose (GalNAz) residues into the heavy chain glycans; (3) the strain-promoted click conjugation of chelator- and fluorophore-modified dibenzocyclooctynes to the azide-modified sugars; and (4) the radiolabeling of the immunoconjugate. For proof-of-concept, a model system was created using the colorectal cancer-targeting antibody huA33, the chelator desferrioxamine (DFO), the positron-emitting radiometal (89)Zr, and the near-infrared fluorescent dye Alexa Fluor 680. The bioconjugation strategy is robust and reproducible, reliably producing well-defined and immunoreactive conjugates labeled with (89)Zr, Alexa Fluor 680, or an easily and precisely tuned mixture of the two reporters. In in vivo PET and fluorescence imaging experiments, a hybrid (89)Zr- and Alexa Fluor 680-labeled huA33 conjugate displayed high levels of specific uptake (>45% ID/g) in athymic nude mice bearing A33 antigen-expressing SW1222 colorectal cancer xenografts.
Project description:Pertuzumab is clinically employed in the treatment of cancers over-expressing human epidermal growth factor receptor 2 (HER2). Herein, we developed dual-labeled pertuzumab with a radionuclide (<sup>89</sup>Zr) and a near-infrared fluorophore (IRDye 800CW) to investigate the feasibility of utilizing dual-labeled monoclonal antibodies (mAbs) with numerous imaging modalities for preoperative imaging and image-guided surgery in ovarian cancer models. MAbs were dually-labeled with <sup>89</sup>Zr and IRDye 800CW to generate <sup>89</sup>Zr-Df-pertuzumab-800CW or <sup>89</sup>Zr-Df-IgG-800CW. Serial positron emission tomography (PET) and near-infrared fluorescence (NIRF) images were acquired up to 72 hours after injection of dual-labeled mAbs to map the tracers' biodistributions. After the last time point, image-guided tumor resection was executed using different modalities (NIRF, Cerenkov luminescence [CL], and ? particle imaging) and <i>ex vivo</i> studies including biodistribution assays and histology analysis were performed to confirm the <i>in vivo</i> imaging data. SKOV3 ovarian cancer cells showed high expression of HER2 and pertuzumab conjugated with Df and IRDye 800CW maintained its binding affinity for these cells. For PET imaging in subcutaneous xenograft ovarian cancer models, <sup>89</sup>Zr-Df-pertuzumab-800CW showed a significantly higher tumor-to-muscle ratio compared to the nonspecific <sup>89</sup>Zr-Df-IgG-800CW from 24 hours after injection through the last time point (72 h: 30.7 ± 7.4 vs. 7.5 ± 1.8, <i>P</i> < 0.01, <i>n</i> = 3-4). During image-guided surgery, three imaging modalities including NIRF, CL, and ? particle imaging could detect ovarian cancer in both subcutaneous and orthotopic models and each exhibited its own imaging characteristics. In addition, <i>ex vivo</i> imaging and biodistribution studies as well as histology analysis corroborated the <i>in vivo</i> imaging results. Therefore, we concluded that this single radiolabeled tracer can provide all-in-one contrast for multiple imaging modalities. The dual-labeled mAbs may hold promise to be employed for image-guided tumor surgery as well as diagnosis and staging through balancing out the strengths and weaknesses of various modalities such as PET/CT, NIRF, CL, and ? particle imaging.
Project description: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.
Project description:Pretargeted PET imaging has emerged as an effective strategy for merging the exquisite selectivity of antibody-based targeting vectors with the rapid pharmacokinetics of radiolabeled small molecules. We previously reported the development of a strategy for the pretargeted PET imaging of colorectal cancer based on the bioorthogonal inverse electron demand Diels-Alder reaction between a tetrazine-bearing radioligand and a transcyclooctene-modified huA33 immunoconjugate. Although this method effectively delineated tumor tissue, its clinical potential was limited by the somewhat sluggish clearance of the radioligand through the gastrointestinal tract. Herein, we report the development and in vivo validation of a pretargeted strategy for the PET imaging of colorectal carcinoma with dramatically improved pharmacokinetics. Two novel tetrazine constructs, Tz-PEG7-NOTA and Tz-SarAr, were synthesized, characterized, and radiolabeled with (64)Cu in high yield (>90%) and radiochemical purity (>99%). PET imaging and biodistribution experiments in healthy mice revealed that although (64)Cu-Tz-PEG7-NOTA is cleared via both the gastrointestinal and urinary tracts, (64)Cu-Tz-SarAr is rapidly excreted by the renal system alone. On this basis, (64)Cu-Tz-SarAr was selected for further in vivo evaluation. To this end, mice bearing A33 antigen-expressing SW1222 human colorectal carcinoma xenografts were administered huA33-TCO, and the immunoconjugate was given 24 h to accumulate at the tumor and clear from the blood, after which (64)Cu-Tz-SarAr was administered via intravenous tail vein injection. PET imaging and biodistribution experiments revealed specific uptake of the radiotracer in the tumor at early time points (5.6 ± 0.7 %ID/g at 1 h p.i.), high tumor-to-background activity ratios, and rapid elimination of unclicked radioligand. Importantly, experiments with longer antibody accumulation intervals (48 and 120 h) yielded slight decreases in tumoral uptake but also concomitant increases in tumor-to-blood activity concentration ratios. This new strategy offers dosimetric benefits as well, yielding a total effective dose of 0.041 rem/mCi, far below the doses produced by directly labeled (64)Cu-NOTA-huA33 (0.133 rem/mCi) and (89)Zr-DFO-huA33 (1.54 rem/mCi). Ultimately, this pretargeted PET imaging strategy boasts a dramatically improved pharmacokinetic profile compared to our first generation system and is capable of clearly delineating tumor tissue with high image contrast at only a fraction of the radiation dose created by directly labeled radioimmunoconjugates.
Project description:<h4>Purpose</h4>There is a clinical need for agents that target glioma cells for non-invasive and intraoperative imaging to guide therapeutic intervention and improve the prognosis of glioma. Matrix metalloproteinase (MMP)-14 is overexpressed in glioma with negligible expression in normal brain, presenting MMP-14 as an attractive biomarker for imaging glioma. In this study, we designed a peptide probe containing a near-infrared fluorescence (NIRF) dye/quencher pair, a positron emission tomography (PET) radionuclide, and a moiety with high affinity to MMP-14. This novel substrate-binding peptide allows dual modality imaging of glioma only after cleavage by MMP-14 to activate the quenched NIRF signal, enhancing probe specificity and imaging contrast.<h4>Methods</h4>MMP-14 expression and activity in human glioma tissues and cells were measured in vitro by immunofluorescence and gel zymography. Cleavage of the novel substrate and substrate-binding peptides by glioma cells in vitro and glioma xenograft tumors in vivo was determined by NIRF imaging. Biodistribution of the radiolabeled MMP-14-binding peptide or substrate-binding peptide was determined in mice bearing orthotopic patient-derived xenograft (PDX) glioma tumors by PET imaging.<h4>Results</h4>Glioma cells with MMP-14 activity showed activation and retention of NIRF signal from the cleaved peptides. Resected mouse brains with PDX glioma tumors showed tumor-to-background NIRF ratios of 7.6-11.1 at 4 h after i.v. injection of the peptides. PET/CT images showed localization of activity in orthotopic PDX tumors after i.v. injection of <sup>68</sup>Ga-binding peptide or <sup>64</sup>Cu-substrate-binding peptide; uptake of the radiolabeled peptides in tumors was significantly reduced (p < 0.05) by blocking with the non-labeled-binding peptide. PET and NIRF signals correlated linearly in the orthotopic PDX tumors. Immunohistochemistry showed co-localization of MMP-14 expression and NIRF signal in the resected tumors.<h4>Conclusions</h4>The novel MMP-14 substrate-binding peptide enabled PET/NIRF imaging of glioma models in mice, warranting future image-guided resection studies with the probe in preclinical glioma models.
Project description:The novel synthesis of a dual-modality, pentamethine cyanine (Cy5) fluorescent, 18F positron emission tomography (PET) imaging probe is reported. The probe shows a large extinction coefficient and large quantum yield in the biologically transparent, near-infrared window (650-900 nm) for in vivo fluorescent imaging. This fluorophore bears the isotope, 18F, giving a 18F-PET/near-infrared fluorescent (NIRF), bi-modal imaging probe, that combines the long-term stability of NIRF and the unlimited penetration depth of PET imaging. The bi-modal probe is labeled with 18F in a quick, one-step reaction, which is important in working with the rapid decay of 18F. The bi-modal probe bears a free carboxyl group, highlighting a PET/NIRF synthon that can be conjugated onto many advanced biomolecules for biomarker-specific in vivo dual-modal PET/NIR tumor imaging, confocal histology, and utility in multi-fluorophore, fluorescence-guided surgery. Its potential in vivo biocompatibility is explored in a quick proof-of-principal in vivo study. The dye is delivered to A549 xenograft flank-tumors to generate PET and NIRF signals at the tumor site. The tumor distribution is confirmed in ex vivo gamma counting and imaging. Pentamethine cyanine (Cy5) has the ability to preferentially accumulate in tumor xenografts. We substitute the PET/NIRF probe for Cy5, and explore this phenomenon.
Project description:Over the past decade, theranostic imaging has emerged as a powerful clinical tool in oncology for identifying patients likely to respond to targeted therapies and for monitoring the response of patients to treatment. Herein, we report a theranostic approach to pretargeted radioimmunotherapy (PRIT) based on a pair of radioisotopes of copper: positron-emitting copper-64 (64Cu, t 1/2 = 12.7 h) and beta particle-emitting copper-67 (67Cu, t 1/2 = 61.8 h). This strategy is predicated on the in vivo ligation between a trans-cyclooctene (TCO)-bearing antibody and a tetrazine (Tz)-based radioligand via the rapid and bioorthogonal inverse electron-demand Diels-Alder reaction. Longitudinal therapy studies were conducted in a murine model of human colorectal carcinoma using an immunoconjugate of the huA33 antibody modified with TCO (huA33-TCO) and a 67Cu-labeled Tz radioligand ([67Cu]Cu-MeCOSar-Tz). The injection of huA33-TCO followed 72 h later by the administration of 18.5, 37.0, or 55.5 MBq of [67Cu]Cu-MeCOSar-Tz produced a dose-dependent therapeutic response, with the median survival time increasing from 68 d for the lowest dose to >200 d for the highest. Furthermore, we observed that mice that received the highest dose of [67Cu]Cu-MeCOSar-Tz in a fractionated manner exhibited improved hematological values without sacrificing therapeutic efficacy. Dual radionuclide experiments in which a single administration of huA33-TCO was followed by separate injections of [64Cu]Cu-MeCOSar-Tz and [67Cu]Cu-MeCOSar-Tz revealed that the positron emission tomography images produced by the former accurately predicted the efficacy of the latter. In these experiments, a correlation was observed between the tumoral uptake of [64Cu]Cu-MeCOSar-Tz and the subsequent therapeutic response to [67Cu]Cu-MeCOSar-Tz.
Project description:Overexpression of CD146 has been correlated with aggressiveness, recurrence rate, and poor overall survival in hepatocellular carcinoma (HCC) patients. In this study, we set out to develop a CD146-targeting probe for high-contrast noninvasive in vivo positron emission tomography (PET) and near-infrared fluorescence (NIRF) imaging of HCCs. YY146, an anti-CD146 monoclonal antibody, was employed as a targeting molecule to which we conjugated the zwitterionic near-infrared fluorescence (NIRF) dye ZW800-1 and the chelator deferoxamine (Df). This enabled labeling of Df-YY146-ZW800 with (89)Zr and its subsequent detection using PET and NIRF imaging, all without compromising antibody binding properties. Two HCC cell lines expressing high (HepG2) and low (Huh7) levels of CD146 were employed to generate subcutaneous (s.c.) and orthotopic xenografts in athymic nude mice. Sequential PET and NIRF imaging performed after intravenous injection of (89)Zr-Df-YY146-ZW800 into tumor-bearing mice unveiled prominent and persistent uptake of the tracer in HepG2 tumors that peaked at 31.65 ± 7.15 percentage of injected dose per gram (%ID/g; n=4) 72 h post-injection. Owing to such marked accumulation, tumor delineation was successful by both PET and NIRF, which facilitated the fluorescence image-guided resection of orthotopic HepG2 tumors, despite the relatively high liver background. CD146-negative Huh7 and CD146-blocked HepG2 tumors exhibited significantly lower (89)Zr-Df-YY146-ZW800 accretion (6.1 ± 0.5 and 8.1 ± 1.0 %ID/g at 72 h p.i., respectively; n=4), demonstrating the CD146-specificity of the tracer in vivo. Ex vivo biodistribution and immunofluorescent staining corroborated the accuracy of the imaging data and correlated tracer uptake with in situ CD146 expression. Overall, (89)Zr-Df-YY146-ZW800 showed excellent properties as a PET/NIRF imaging agent, including high in vivo affinity and specificity for CD146-expressing HCC. CD146-targeted molecular imaging using dual-labeled YY146 has great potential for early detection, prognostication, and image-guided surgical resection of liver malignancies.
Project description:Multifunctional mesoporous silica nanoparticles (MSN) with well-integrated multimodality imaging properties have generated increasing research interest in the past decade. However, limited progress has been made in developing MSN-based multimodality imaging agents to image tumors. We describe the successful conjugation of, copper-64 ((64)Cu, t1/2 = 12.7 h), 800CW (a near-infrared fluorescence [NIRF] dye), and TRC105 (a human/murine chimeric IgG1 monoclonal antibody) to the surface of MSN via well-developed surface engineering procedures, resulting in a dual-labeled MSN for in vivo targeted positron emission tomography (PET) imaging/NIRF imaging of the tumor vasculature. Pharmacokinetics and tumor targeting efficacy/specificity in 4T1 murine breast tumor-bearing mice were thoroughly investigated through various in vitro, in vivo, and ex vivo experiments. Dual-labeled MSN is an attractive candidate for future cancer theranostics.