Harnessing 64Cu/67Cu for a theranostic approach to pretargeted radioimmunotherapy.

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, t1/2 = 12.7 h) and beta particle-emitting copper-67 (67Cu, t1/2 = 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.

Targeting Triple Negative Breast Cancer with a Nucleus-Directed p53 Tetramerization Domain Peptide.

Triple negative breast cancer (TNBC) has no targeted detection or treatment method. Mutant p53 (mtp53) is overexpressed in >80% of TNBCs, and the stability of mtp53 compared to the instability of wild-type p53 (wtp53) in normal cells makes mtp53 a promising TNBC target for diagnostic and theranostic imaging. We generated Cy5p53Tet, a novel nucleus-penetrating mtp53-oligomerization-domain peptide (mtp53ODP) to the tetramerization domain (TD) of mtp53. This mtp53ODP contains the p53 TD sequence conjugated to a Cy5 fluorophore for near-infrared fluorescence imaging (NIRF). In vitro co-immunoprecipitation and glutaraldehyde cross-linking showed a direct interaction between mtp53 and Cy5p53Tet. Confocal microscopy and flow cytometry demonstrated higher uptake of Cy5p53Tet in the nuclei of TNBC MDA-MB-468 cells with mtp53 R273H than in ER-positive MCF7 cells with wtp53. Furthermore, depletion of mtp53 R273H caused a decrease in the uptake of Cy5p53Tet in nuclei. In vivo analysis of the peptide in mice bearing MDA-MB-468 xenografts showed that Cy5p53Tet could be detected in tumor tissue 12 min after injection. In these in vivo experiments, significantly higher uptake of Cy5p53Tet was observed in mtp53-expressing MDA-MB-468 xenografts compared with the wtp53-expressing MCF7 tumors. Cy5p53Tet has clinical potential as an intraoperative imaging agent for fluorescence-guided surgery, and the mtp53ODP scaffold shows promise for modification in the future to enable the delivery of a wide variety of payloads including radionuclides and toxins to mtp53-expressing TNBC tumors.

A Molecularly Targeted Intraoperative Near-Infrared Fluorescence Imaging Agent for High-Grade Serous Ovarian Cancer.

Ovarian cancer is the fifth leading cause of cancer deaths among women, accounting for more deaths than any other cancer of the female reproductive system. The foundation of its management consists of cytoreductive surgery (CRS) followed by systemic chemotherapy, with the completeness of surgical resection consistently identified as one of the most important prognostic factors for the disease. The goal of our investigation is the development of a near-infrared fluorescence (NIRF) imaging agent for the intraoperative imaging of high-grade serous ovarian cancer (HGSOC). As surgeons are currently limited to the visual and manual assessment of tumor tissue during CRS, this technology could facilitate more complete resections as well as serve important functions at other points in the surgical management of the disease. Elevated levels of cancer antigen 125 (CA125) have proven a useful biomarker of HGSOC, and the CA125-targeting antibody B43.13 has shown potential as a platform for immunoPET imaging in murine models of ovarian cancer. Herein, we report the development of a NIRF imaging agent based on B43.13: ssB43.13-IR800. We site-specifically modified the heavy chain glycans of B43.13 with the near-infrared dye IRDye 800CW using a chemoenzymatic approach developed in our laboratories. SDS-PAGE analysis confirmed the specificity of the conjugation reaction, and flow cytometry, immunostaining, and fluorescence microscopy verified the specific binding of ssB43.13-IR800 to CA125-expressing OVCAR3 human ovarian cancer cells. NIRF imaging studies demonstrated that ssB43.13-IR800 can be used to image CA125-expressing HGSOC tumors in subcutaneous, orthotopic, and patient-derived xenograft mouse models. Finally, ex vivo analyses confirmed that ssB43.13-IR800 can bind and identify CA125-expressing cells in primary tumor and metastatic lymph node samples from human patients with HGSOC.

89Zr-Labeled AR20.5: A MUC1-Targeting ImmunoPET Probe.

High expression levels of the tumor-associated antigen MUC1 have been correlated with tumor aggressiveness, poor response to therapy, and poor survival in several tumor types, including breast, pancreatic, and epithelial ovarian cancer. Herein, we report the synthesis, characterization, and in vivo evaluation of a novel radioimmunoconjugate for the immuno-positron emission tomography (immunoPET) imaging of MUC1 expression based on the AR20.5 antibody. To this end, we modified AR20.5 with the chelator desferrioxamine (DFO) and labeled it with the positron-emitting radiometal zirconium-89 (t1/2 ~3.3 d) to produce [89Zr]Zr-DFO-AR20.5. In subsequent in vivo experiments in athymic nude mice bearing subcutaneous MUC1-expressing ovarian cancer xenografts, [89Zr]Zr-DFO-AR20.5 clearly delineated tumor tissue, producing a tumoral activity concentration of 19.1 ± 6.4 percent injected dose per gram (%ID/g) at 120 h post-injection and a tumor-to-muscle activity concentration ratio of 42.4 ± 10.6 at the same time point. Additional PET imaging experiments in mice bearing orthotopic MUC1-expressing ovarian cancer xenografts likewise demonstrated that [89Zr]Zr-DFO-AR20.5 enables the visualization of tumor tissue-including metastatic lesions-with promising tumor-to-background contrast.

Dual Radionuclide Theranostic Pretargeting.

Recent years have played witness to the advent of nuclear theranostics: the synergistic use of "matched pair" radiopharmaceuticals for diagnostic imaging and targeted radiotherapy. In this investigation, we report the extension of this concept to in vivo pretargeting based on the rapid and bioorthogonal inverse electron demand Diels-Alder reaction between tetrazine (Tz) and trans-cyclooctene (TCO). We demonstrate that a single injection of a TCO-modified immunoconjugate can be used as a platform for pretargeted PET imaging and radiotherapy via the sequential administration of a pair of Tz-bearing radioligands labeled with the positron-emitting radiometal copper-64 (t1/2 ≈ 12.7 h) and the beta-emitting radiometal lutetium-177 (t1/2 ≈ 6.7 days). More specifically, a mouse model of human colorectal carcinoma received a dose of the A33 antigen-targeting immunoconjugate huA33-TCO, followed 24 and 48 h later by injections of [64Cu]Cu-SarAr-Tz and [177Lu]Lu-DOTA-PEG7-Tz, respectively. This approach produces high activity concentrations of both radioligands in tumor tissue (16.4 ± 2.7 %ID/g for [64Cu]Cu-SarAr-Tz at 48 h post-injection and 18.1 ± 2.1 %ID/g for [177Lu]Lu-DOTA-PEG7-Tz at 120 h post-injection) as well as promising tumor-to-healthy organ activity concentration ratios. Ultimately, we believe that this work could not only have important implications in nuclear theranostics-most excitingly with isotopologue-based radioligand pairs such as [64Cu]Cu-SarAr-Tz and [67Cu]Cu-SarAr-Tz-but also in the delivery of fractionated doses during pretargeted radioimmunotherapy.

Toward the Optimization of Click-Mediated Pretargeted Radioimmunotherapy.

Pretargeted radioimmunotherapy (PRIT) based on the inverse electron demand Diels-Alder reaction has shown promise in murine models of disease, yet the radiation dosimetry of this approach must be optimized to make it a viable clinical option. To this end, we have leveraged two recent developments in pretargeted imaging-dendritic scaffolds and masking agents-to improve the dosimetric profile of a proof-of-concept PRIT system that is based on the huA33 antibody, a 177Lu-labeled tetrazine radioligand ([177Lu]Lu-DOTA-PEG7-Tz), and a mouse model of A33 antigen-expressing colorectal carcinoma. Pretargeting using an huA33 immunoconjugate bearing a trans-cyclooctene-decorated dendritic scaffold (sshuA33-DEN-TCO) produced significantly higher tumoral activity concentrations at 120 h post-injection (23.0 ± 2.2 %ID/g) than those achieved with an analogous, dendrimer-lacking immunoconjugate (12.7 ± 2.6 %ID/g). However, pretargeting using sshuA33-DEN-TCO also resulted in increased activity concentrations in the blood at the same time point (1.9 ± 0.4 %ID/g) compared to the dendrimer-lacking construct (0.7 ± 0.2 %ID/g), thereby curtailing improvements to the tumor-to-blood therapeutic ratio of the system. In order to circumvent this issue, a tetrazine-labeled, dextran-based masking agent (Tz-DP) was injected prior to the radioligand to prevent the ligation between [177Lu]Lu-DOTA-PEG7-Tz and circulating immunoconjugate. This approach dramatically decreased the absorbed dose to the blood but also attenuated the absorbed dose to the tumor and increased the absorbed dose to the lungs. Ultimately, these data suggest that dendritic scaffolds and masking agents could be used to improve the dosimetry of PRIT, but the combination of these technologies will require extensive optimization.

Click-Mediated Pretargeted Radioimmunotherapy of Colorectal Carcinoma.

Pretargeted radioimmunotherapy (PRIT) based on the inverse electron demand Diels-Alder (IEDDA) reaction between tetrazine (Tz) and trans-cyclooctene (TCO) represents a promising strategy for leveraging the affinity and specificity of antibodies without their pharmacokinetic drawbacks. Herein, we present an investigation of the in vivo efficacy and dosimetry of a PRIT strategy for colorectal carcinoma based on the ligation between a 177Lu-labeled Tz radioligand (177Lu-DOTA-PEG7-Tz) and a TCO-bearing immunoconjugate of the huA33 antibody (huA33-TCO). Biodistribution studies in tumor-bearing mice using intervals of 24, 48, and 72 h between the administration of huA33-TCO and 177Lu-DOTA-PEG7-Tz revealed that a 24 h lag time produced the most promising in vivo results: high activity concentrations in the tumor (21.2 %ID/g ± 2.9 at 24 h postinjection), low uptake in nontarget tissues, and favorable dosimetry (an effective dose of 0.054 mSv/MBq). A subsequent longitudinal therapy study revealed striking differences between both the survival and tumor growth of the treatment and control cohorts, clearly underscoring the promise of this approach for the radiotherapy of colorectal carcinoma.

The Influence of Glycans-Specific Bioconjugation on the FcγRI Binding and In vivo Performance of 89Zr-DFO-Pertuzumab.

Rationale: The overwhelming majority of radioimmunoconjugates are produced via random conjugation methods predicated on attaching bifunctional chelators to the lysines of antibodies. However, this approach inevitably produces poorly defined and heterogeneous immunoconjugates because antibodies have several lysines distributed throughout their structure. To circumvent this issue, we have previously developed a chemoenzymatic bioconjugation strategy that site-specifically appends cargoes to the biantennary heavy chain glycans attached to CH2 domains of the immunoglobulin's Fc region. In the study at hand, we explore the effects of this approach to site-specific bioconjugation on the Fc receptor binding and in vivo behavior of radioimmunoconjugates. Methods: We synthesized three desferrioxamine (DFO)-labeled immunoconjugates based on the HER2-targeting antibody pertuzumab: one using random bioconjugation methods (DFO-nsspertuzumab) and two using variants of our chemoenzymatic protocol (DFO-sspertuzumab-EndoS and DFO-sspertuzumab-ßGal). Subsequently, we characterized these constructs and evaluated their ability to bind HER2, human FcγRI (huFcγRI), and mouse FcγRI (muFcγRI). After radiolabeling the immunoconjugates with zirconium-89, we conducted PET imaging and biodistribution studies in two different mouse models of HER2-expressing breast cancer. Results: MALDI-ToF and SDS-PAGE analysis confirmed the site-specific nature of the bioconjugation, and flow cytometry and surface plasmon resonance (SPR) revealed that all three immunoconjugates bind HER2 as effectively as native pertuzumab. Critically, however, SPR experiments also illuminated that DFO-sspertuzumab-EndoS possesses an attenuated binding affinity for huFcγRI (17.4 ± 0.3 nM) compared to native pertuzumab (4.7 ± 0.2 nM), DFO-nsspertuzumab (4.1 ± 0.1 nM), and DFO-sspertuzumab-ßGal (4.7 ± 0.2 nM). ImmunoPET and biodistribution experiments in athymic nude mice bearing HER2-expressing BT474 human breast cancer xenografts yielded no significant differences in the in vivo behavior of the radioimmunoconjugates. Yet experiments in tumor-bearing humanized NSG mice revealed that 89Zr-DFO-sspertuzumab-EndoS produces higher activity concentrations in the tumor (111.8 ± 39.9 %ID/g) and lower activity concentrations in the liver and spleen (4.7 ± 0.8 %ID/g and 13.1 ± 4.0 %ID/g, respectively) than its non-site-specifically labeled cousin, a phenomenon we believe stems from the altered binding of the former to huFcγRI. Conclusion: These data underscore that this approach to site-specific bioconjugation not only produces more homogeneous and well-defined radioimmunoconjugates than traditional methods but may also improve their in vivo performance in mouse models by reducing binding to FcγRI.

A brief overview of metal complexes as nuclear imaging agents.

Metallic radionuclides have been instrumental in the field of nuclear imaging for over half a century. While recent years have played witness to a dramatic rise in the use of radiometals as labels for chelator-bearing biomolecules, imaging agents based solely on coordination compounds of radiometals have long played a critical role in the discipline as well. In this work, we seek to provide a brief overview of metal complex-based radiopharmaceuticals for positron emission tomography (PET) and single photon emission computed tomography (SPECT). More specifically, we have focused on imaging agents in which the metal complex itself rather than a pendant biomolecule or targeting moiety is responsible for the in vivo behavior of the tracer. This family of compounds contains metal complexes based on an array of different nuclides as well as probes that have been used for the imaging of a variety of pathologies, including infection, inflammation, cancer, and heart disease. Indeed, two of the defining traits of transition metal complexes-modularity and redox chemistry-have both been creatively leveraged in the development of imaging agents. In light of our audience, particular attention is paid to structure and mechanism, though clinical data is addressed as well. Ultimately, it is our hope that this review will not only educate readers about some of the seminal work performed in this space over the last 30 years but also spur renewed interest in the creation of radiopharmaceuticals based on small metal complexes.

The Impact of FcγRI Binding on Immuno-PET.

Antibodies are promising vectors for PET imaging. However, the high uptake of radioimmunoconjugates in nontarget tissues such as the liver and spleen hampers their performance as radiotracers. This off-target uptake can lead to suboptimal tumor-to-background activity concentration ratios, decreasing the contrast of images and reducing their diagnostic utility. A possible cause of this uptake is the sequestration of radioimmunoconjugates by immune cells bearing Fc-γ-receptors (FcγR) that bind to the Fc regions of antibodies. Methods: Since the heavy chain glycans influence the affinity of FcγR for the Fc domain, we set out to investigate whether radioimmunoconjugates with truncated glycans would exhibit altered binding to FcγRI and, in turn, improved in vivo performance. Using the HER2-targeting antibody trastuzumab, we synthesized a series of desferrioxamine-bearing immunoconjugates with differing glycosylation states and interrogated their FcγRI binding via surface plasmon resonance, enzyme-linked immunosorbent assay, and flow cytometry. Furthermore, we labeled these immunoconjugates with 89Zr and explored their biodistribution in athymic nude, NSG, and humanized NSG mice bearing human epidermal growth factor receptor 2-expressing human breast cancer xenografts. Results: We observed a strong correlation between the impaired in vitro FcγRI binding of deglycosylated immunoconjugates and significant decreases in the in vivo off-target uptake of the corresponding 89Zr-labeled radioimmunoconjugates (i.e., liver activity concentrations are reduced by ∼3.5-fold in humanized NSG mice). These reductions in off-target uptake were accompanied by concomitant increases in the tumoral activity concentrations of the glycoengineered radioimmunoconjugates, ultimately yielding improved tumor-to-healthy organ contrast and higher quality PET images. Conclusion: Our findings suggest that the deglycosylation of antibodies represents a facile strategy for improving the quality of immuno-PET in animal models as well as in certain patient populations.

Pretargeting of internalizing trastuzumab and cetuximab with a 18F-tetrazine tracer in xenograft models.

BACKGROUND: Pretargeting-based approaches are being investigated for radioimmunoimaging and therapy applications to reduce the effective radiation burden to the patient. To date, only a few studies have used short-lived radioisotopes for pretargeting of antibodies, and such examples with internalizing antibodies are even rarer. Herein, we have investigated pretargeting methodology using inverse electron-demand Diels-Alder (IEDDA) for tracing two clinically relevant, internalizing monoclonal antibodies, cetuximab and trastuzumab. RESULTS: Bioorthogonal reaction between tetrazine and trans-cyclooctene (TCO) was used for tracing cetuximab and trastuzumab in vivo with a fluorine-18 (t ½ = 109.8 min) labelled tracer. TCO-cetuximab or TCO-trastuzumab was administered 24, 48, or 72 h prior to the injection of tracer to A431 or BT-474 tumour-bearing mice, respectively. With cetuximab, the highest tumour-to-blood ratios were achieved when the lag time between antibody and tracer injections was 72 h. With trastuzumab, no difference was observed between different lag times. For both antibodies, the tumour could be clearly visualized in the PET images with the highest tumour uptake of 3.7 ± 0.1%ID/g for cetuximab and 1.5 ± 0.1%ID/g for trastuzumab as quantified by ex vivo biodistribution. In vivo IEDDA reaction was observed in the blood for both antibodies, but with trastuzumab, this was to a much lower degree than with cetuximab. CONCLUSIONS: We could successfully visualize the tumours by using cetuximab and trastuzumab in pretargeted PET imaging despite the challenging circumstances where the antibody is internalized and there is still some unbound antibody circulating in the blood flow. This clearly demonstrates the potential of a pretargeted approach for targeting internalizing antigens and warrants development of pharmacokinetic optimization of the biorthogonal reactants to this end.