Recent advances and impending challenges for the radiopharmaceutical sciences in oncology.

This paper is the first of a Series on theranostics that summarises the current landscape of the radiopharmaceutical sciences as they pertain to oncology. In this Series paper, we describe exciting developments in radiochemistry and the production of radionuclides, the development and translation of theranostics, and the application of artificial intelligence to our field. These developments are catalysing growth in the use of radiopharmaceuticals to the benefit of patients worldwide. We also highlight some of the key issues to be addressed in the coming years to realise the full potential of radiopharmaceuticals to treat cancer.

Trends in nuclear medicine and the radiopharmaceutical sciences in oncology: workforce challenges and training in the age of theranostics.

Although the promise of radionuclides for the diagnosis and treatment of disease was recognised soon after the discovery of radioactivity in the late 19th century, the systematic use of radionuclides in medicine only gradually increased over the subsequent hundred years. The past two decades, however, has seen a remarkable surge in the clinical application of diagnostic and therapeutic radiopharmaceuticals, particularly in oncology. This development is an exciting time for the use of theranostics in oncology, but the rapid growth of this area of nuclear medicine has created challenges as well. In particular, the infrastructure for the manufacturing and distribution of radiopharmaceuticals remains in development, and regulatory bodies are still optimising guidelines for this new class of drug. One issue of paramount importance for achieving equitable access to theranostics is building a sufficiently trained workforce in high-income, middle-income, and low-income countries. Here, we discuss the key challenges and opportunities that face the field as it seeks to build its workforce for the 21st century.

Cadherin-17 as a target for the immunoPET of adenocarcinoma.

PURPOSE: Cadherin-17 (CDH17) is a calcium-dependent cell adhesion protein that is overexpressed in several adenocarcinomas, including gastric, colorectal, and pancreatic adenocarcinoma. High levels of CDH17 have been linked to metastatic disease and poor prognoses in patients with these malignancies, fueling interest in the protein as a target for diagnostics and therapeutics. Herein, we report the synthesis, in vitro validation, and in vivo evaluation of a CDH17-targeted 89Zr-labeled immunoPET probe. METHODS: The CDH17-targeting mAb D2101 was modified with an isothiocyanate-bearing derivative of desferrioxamine (DFO) to produce a chelator-bearing immunoconjugate - DFO-D2101 - and flow cytometry and surface plasmon resonance (SPR) were used to interrogate its antigen-binding properties. The immunoconjugate was then radiolabeled with zirconium-89 (t1/2 ~ 3.3 days), and the serum stability and immunoreactive fraction of [89Zr]Zr-DFO-D2101 were determined. Finally, [89Zr]Zr-DFO-D2101's performance was evaluated in a trio of murine models of pancreatic ductal adenocarcinoma (PDAC): subcutaneous, orthotopic, and patient-derived xenografts (PDX). PET images were acquired over the course of 5 days, and terminal biodistribution data were collected after the final imaging time point. RESULTS: DFO-D2101 was produced with a degree of labeling of ~ 1.1 DFO/mAb. Flow cytometry with CDH17-expressing AsPC-1 cells demonstrated that the immunoconjugate binds to its target in a manner similar to its parent mAb, while SPR with recombinant CDH17 revealed that D2101 and DFO-D2101 exhibit nearly identical KD values: 8.2 × 10-9 and 6.7 × 10-9 M, respectively. [89Zr]Zr-DFO-D2101 was produced with a specific activity of 185 MBq/mg (5.0 mCi/mg), remained >80% stable in human serum over the course of 5 days, and boasted an immunoreactive fraction of >0.85. In all three murine models of PDAC, the radioimmunoconjugate yielded high contrast images, with high activity concentrations in tumor tissue and low uptake in non-target organs. Tumoral activity concentrations reached as high as >60 %ID/g in two of the cohorts bearing PDXs. CONCLUSION: Taken together, these data underscore that [89Zr]Zr-DFO-D2101 is a highly promising probe for the non-invasive visualization of CDH17 expression in PDAC. We contend that this radioimmunoconjugate could have a significant impact on the clinical management of patients with both PDAC and gastrointestinal adenocarcinoma, most likely as a theranostic imaging tool in support of CDH17-targeted therapies.

Evaluating CD133 as a Radiotheranostic Target in Small-Cell Lung Cancer.

Despite decades of work, small-cell lung cancer (SCLC) remains a frustratingly recalcitrant disease. Both diagnosis and treatment are challenges: low-dose computed tomography (the approved method used for lung cancer screening) is unable to reliably detect early SCLC, and the malignancy's 5 year survival rate stands at a paltry 7%. Clearly, the development of novel diagnostic and therapeutic tools for SCLC is an urgent, unmet need. CD133 is a transmembrane protein that is expressed at low levels in normal tissue but is overexpressed by a variety of tumors, including SCLC. We previously explored CD133 as a biomarker for a novel autoantibody-to-immunopositron emission tomography (PET) strategy for the diagnosis of SCLC, work that first suggested the promise of the antigen as a radiotheranostic target in the disease. Herein, we report the in vivo validation of a pair of CD133-targeted radioimmunoconjugates for the PET imaging and radioimmunotherapy of SCLC. To this end, [89Zr]Zr-DFO-αCD133 was first interrogated in a trio of advanced murine models of SCLC─i.e., orthotopic, metastatic, and patient-derived xenografts─with the PET probe consistently producing high activity concentrations (>%ID/g) in tumor lesions combined with low uptake in healthy tissues. Subsequently, a variant of αCD133 labeled with the ß-emitting radiometal 177Lu─[177Lu]Lu-DTPA-A″-CHX-αCD133─was synthesized and evaluated in a longitudinal therapy study in a subcutaneous xenograft model of SCLC, ultimately revealing that treatment with a dose of 9.6 MBq of the radioimmunoconjugate produced a significant increase in median survival compared to a control cohort. Taken together, these data establish CD133 as a viable target for the nuclear imaging and radiopharmaceutical therapy of SCLC.

Tuning Supramolecular Chirality in Iodinated Amphiphilic Peptides Through Tripeptide Linker Editing.

Protease-cleavable supramolecular oligopeptide nanofilaments are promising materials for targeted therapeutics and diagnostics. In these systems, single amino acid substitutions can have profound effects on the supramolecular structure and consequent proteolytic degradation, which are critical parameters for their intended applications. Herein, we describe changes to the self-assembly and proteolytic cleavage of iodine containing sequences for future translation into matrix metalloprotease (MMP-9)-activated supramolecular radio-imaging probes. We use a systematic single amino acid exchange in the tripeptide linker region of these peptide amphiphiles to provide insights into the role of each residue in the supramolecular assemblies. These modifications resulted in dramatic changes in the nature of the assembled structures formed, including an unexpected chiral inversion. By using circular dichroism, atomic force microscopy, Fourier transform infrared spectroscopy, and molecular dynamics simulations, we found that the GD loop, a common motif in ß-turn elements, induced a reversal of the chiral orientation of the assembled nanofibers. In addition to the impact on peptide packing and chirality, MMP-9-catalyzed hydrolysis was evaluated for the four peptides, with the ß-sheet content found to be a stronger determinant of enzymatic hydrolysis than supramolecular chirality. These observations provide fundamental insights into the sequence design in protease cleavable amphiphilic peptides with the potential for radio-labeling and selective biomedical applications.

Click Chemistry and Radiochemistry: An Update.

The term "click chemistry" describes a class of organic transformations that were developed to make chemical synthesis simpler and easier, in essence allowing chemists to combine molecular subunits as if they were puzzle pieces. Over the last 25 years, the click chemistry toolbox has swelled from the canonical copper-catalyzed azide-alkyne cycloaddition to encompass an array of ligations, including bioorthogonal variants, such as the strain-promoted azide-alkyne cycloaddition and the inverse electron-demand Diels-Alder reaction. Without question, the rise of click chemistry has impacted all areas of chemical and biological science. Yet the unique traits of radiopharmaceutical chemistry have made it particularly fertile ground for this technology. In this update, we seek to provide a comprehensive guide to recent developments at the intersection of click chemistry and radiopharmaceutical chemistry and to illuminate several exciting trends in the field, including the use of emergent click transformations in radiosynthesis, the clinical translation of novel probes synthesized using click chemistry, and the advent of click-based in vivo pretargeting.

Leveraging a Dual Variable Domain Immunoglobulin to Create a Site-Specifically Modified Radioimmunoconjugate.

Site-specifically modified radioimmunoconjugates exhibit superior in vitro and in vivo behavior compared to analogues synthesized via traditional stochastic methods. However, the development of approaches to site-specific bioconjugation that combine high levels of selectivity, simple reaction conditions, and clinical translatability remains a challenge. Herein, we describe a novel solution to this problem: the use of dual-variable domain immunoglobulins (DVD-IgG). More specifically, we report the synthesis, in vitro evaluation, and in vivo validation of a 177Lu-labeled radioimmunoconjugate based on HER2DVD, a DVD-IgG containing the HER2-targeting variable domains of trastuzumab and the catalytic variable domains of IgG h38C2. To this end, we first modified HER2DVD with a phenyloxadiazolyl methlysulfone-modified variant of the chelator CHX-A″-DTPA (PODS-CHX-A''-DTPA) and verified the site-specificity of the conjugation for the reactive lysines within the catalytic domains via chemical assay, MALDI-ToF mass spectrometry, and SDS-PAGE. The chelator-bearing immunoconjugate was subsequently labeled with [177Lu]Lu3+ to produce the completed radioimmunoconjugate, [177Lu]Lu-CHX-A″-DTPAPODS-HER2DVD, in >80% radiochemical conversion and a specific activity of 29.5 ± 7.1 GBq/µmol. [177Lu]Lu-CHX-A″-DTPAPODS-HER2DVD did not form aggregates upon prolonged incubation in human serum, displayed 87% stability to demetalation over a 7 days of incubation in serum, and exhibited an immunoreactive fraction of 0.95 with HER2-coated beads. Finally, we compared the pharmacokinetic profile of [177Lu]Lu-CHX-A″-DTPAPODS-HER2DVD to that of a 177Lu-labeled variant of trastuzumab in mice bearing subcutaneous HER2-expressing BT-474 human breast cancer xenografts. The in vivo performance of [177Lu]Lu-CHX-A″-DTPAPODS-HER2DVD matched that of 177Lu-labeled trastuzumab, with the former producing a tumoral activity concentration of 34.1 ± 12.1 %ID/g at 168 h and tumor-to-blood, tumor-to-liver, and tumor-to-kidney activity concentration ratios of 10.5, 9.6, and 21.8, respectively, at the same time point. Importantly, the DVD-IgG did not exhibit a substantially longer serum half-life than the traditional IgG despite its significantly larger size (202 kDa for the former vs 148 kDa for the latter). Taken together, these data suggest that DVD-IgGs represent a viable platform for the future development of highly effective site-specifically labeled radioimmunoconjugates for diagnostic imaging, theranostic imaging, and radioimmunotherapy.

Visualizing Galectin-3 Binding Protein Expression with ImmunoPET.

Galectin-3 binding protein (Gal-3BP) is a glycoprotein that is overexpressed and secreted by several cancers and has been implicated as a marker of both tumor progression and poor prognosis in melanoma, non-small cell lung cancer, head and neck squamous cell carcinoma, and breast cancer. The expression of Gal-3BP by a variety of neoplasms makes it an enticing target for both diagnostics and therapeutics, including immuno-positron emission tomography (immunoPET) probes and antibody-drug conjugates (ADCs). Herein, we report the development, in vitro characterization, and in vivo evaluation of a pair of Gal-3BP-targeting radioimmunoconjugates for 89Zr-immunoPET. A humanized anti-Gal-3BP antibody, 1959, and its corresponding ADC, 1959-sss/DM4 (DM4 = ravtansine), were modified with desferrioxamine (DFO) to yield DFO-1959 and DFO-1959-sss/DM4 immunoconjugates bearing 1-2 DFO/monoclonal antibody. Both DFO-modified immunoconjugates retained their affinity for Gal-3BP in enzyme-linked immunosorbent assay experiments. The chelator-bearing antibodies were radiolabeled with zirconium-89 (t1/2 ≈ 3.3 d) to produce radioimmunoconjugates ─ [89Zr]Zr-DFO-1959 and [89Zr]Zr-DFO-1959-sss/DM4 ─ with high specific activity (>444 MBq/mg, >12 mCi/mg) and stability (>80% intact after 168 h in human serum at 37 °C). In mice bearing subcutaneous Gal-3BP-secreting A375-MA1 xenografts, [89Zr]Zr-DFO-1959 clearly delineated tumor tissue, reaching a maximum tumoral activity concentration (54.8 ± 15.8%ID/g) and tumor-to-background contrast (tumor-to-blood = 8.0 ± 4.6) at 120 h post-injection. The administration of [89Zr]Zr-DFO-1959 to mice bearing subcutaneous Gal-3BP-expressing melanoma patient-derived xenografts produced similarly promising results. [89Zr]Zr-DFO-1959 and [89Zr]Zr-DFO-1959-sss/DM4 exhibited nearly identical pharmacokinetic profiles in the mice bearing A375-MA1 tumors, though the latter produced higher uptake in the spleen and kidneys. Both [89Zr]Zr-DFO-1959 and [89Zr]Zr-DFO-1959-sss/DM4 effectively visualized Gal-3BP-secreting tumors in murine models of melanoma. These results suggest that both probes could play a role in the clinical imaging of Gal-3BP-expressing malignancies, particularly as companion theranostics for the identification of patients likely to respond to Gal-3BP-targeted therapeutics such as 1959-sss/DM4.

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.

Lysine-Directed Site-Selective Bioconjugation for the Creation of Radioimmunoconjugates.

The synthesis of radioimmunoconjugates via the stochastic attachment of bifunctional chelators to lysines can yield heterogeneous products with suboptimal in vitro and in vivo behavior. In response to this, several site-selective approaches to bioconjugation have been developed, yet each has intrinsic drawbacks, such as the need for expensive reagents or the complexity of incorporating unnatural amino acids into IgGs. Herein, we describe the use of a simple and facile approach to lysine-directed site-selective bioconjugation for the generation of radioimmunoconjugates. This strategy relies upon on the selective modification of single lysine residues within each light chain of the monoclonal antibody (mAb) with a branched azide-bearing perfluorophenyl ester (PFP-bisN3) followed by the ligation of dibenzocyclooctyne (DBCO)-bearing payloads to these bioorthogonal handles via the strain-promoted azide-alkyne cycloaddition. This methodology was used to create [89Zr]Zr-SSKDFO-pertuzumab, a radioimmunoconjugate of the HER2-targeting mAb pertuzumab labeled with desferrioxamine (DFO) and the positron-emitting radiometal zirconium-89 (89Zr). [89Zr]Zr-SSKDFO-pertuzumab was compared to a pair of analogous probes: one synthesized via random lysine modification ([89Zr]Zr-DFO-pertuzumab) and another via thiol-maleimide chemistry ([89Zr]Zr-malDFO-pertuzumab). The bioconjugation strategy was assessed using ESI mass spectrometry, SDS-PAGE, and autoradiography. All three immunoconjugates demonstrated comparable binding to HER2 via flow cytometry and surface plasmon resonance (SPR), and 89Zr-labeled variants of each were synthesized in >99% radiochemical yield and molar activities of up to ∼55.5 GBq/µmol (10 mCi/mg). Subsequently, the in vivo behavior of this trio of 89Zr-immunoPET probes was interrogated in athymic nude mice bearing subcutaneous HER2-expressing BT-474 human breast cancer xenografts. [89Zr]Zr-SSKDFO-pertuzumab, [89Zr]Zr-malDFO-pertuzumab, and [89Zr]Zr-DFO-pertuzumab produced positron emission tomography (PET) images with high tumoral uptake and high tumor-to-healthy organ activity concentration ratios. A terminal biodistribution study complemented the PET results, revealing tumoral activity concentrations of 126.9 ± 50.3%ID/g, 86.9 ± 53.2%ID/g, and 92.5 ± 27.2%ID/g at 144 h post-injection for [89Zr]Zr-SSKDFO-pertuzumab, [89Zr]Zr-malDFO-pertuzumab, and [89Zr]Zr-DFO-pertuzumab, respectively. Taken together, the data clearly illustrate that this highly modular and facile approach to site-selective bioconjugation produces radioimmunoconjugates that are better-defined and more homogeneous than stochastically modified constructs and also exhibit excellent in vitro and in vivo performance. Furthermore, we contend that this lysine-directed strategy holds several key advantages over extant approaches to site-selective bioconjugation, especially in the context of production for the clinic.

Pretargeted PET of Osteodestructive Lesions in Dogs.

The last decade has witnessed the creation of a highly effective approach to in vivo pretargeting based on the inverse electron demand Diels-Alder (IEDDA) click ligation between tetrazine (Tz) and trans-cyclooctene (TCO). Despite the steady progression of this technology toward the clinic, concerns have persisted regarding whether this in vivo chemistry will work in humans given their larger size and blood volume. In this work, we describe the use of a 64Cu-labeled Tz radioligand ([64Cu]Cu-SarAr-Tz) and a TCO-bearing bisphosphonate (TCO-BP) for the pretargeted positron emission tomography (PET) imaging of osteodestructive lesions in a large animal model: companion dogs. First, in a small animal pilot study, healthy mice were injected with TCO-BP followed after 1 or 6 h by [64Cu]Cu-SarAr-Tz. PET images were collected 1, 6, and 24 h after the administration of [64Cu]Cu-SarAr-Tz, revealing that this approach produced high activity concentrations in the bone (>20 and >15%ID/g in the femur and humerus, respectively, at 24 h post injection) as well as high target-to-background contrast. Subsequently, companion dogs (n = 5) presenting with osteodestructive lesions were administered TCO-BP (5 or 10 mg/kg) followed 1 h later by [64Cu]Cu-SarAr-Tz (2.2-7.3 mCi; 81.4-270.1 MBq). PET scans were collected for each dog 4 h after the administration of the radioligand, and SUV values for the osteodestructive lesions, healthy bones, and kidneys were determined. In these animals, pretargeted PET clearly delineated healthy bone and produced very high activity concentrations in osteodestructive lesions. Low levels of uptake were observed in all healthy organs except for the kidneys and bladder due to the renal excretion of excess radioligand. Ultimately, this work not only illustrates that pretargeted PET with TCO-BP and [64Cu]Cu-SarAr-Tz is an effective tool for the visualization of osteodestructive lesions but also demonstrates for the first time that in vivo pretargeting based on IEDDA click chemistry is feasible in large animals.

A Theranostic Cellulose Nanocrystal-Based Drug Delivery System with Enhanced Retention in Pulmonary Metastasis of Melanoma.

Metastatic melanoma can be difficult to detect until at the advanced state that decreases the survival rate of patients. Several FDA-approved BRAF inhibitors have been used for treatment of metastatic melanoma, but overall therapeutic efficacy has been limited. Lutetium-177 (177 Lu) enables simultaneous tracking of tracer accumulation with single-photon emission computed tomography and radiotherapy. Therefore, the codelivery of 177 Lu alongside chemotherapeutic agents using nanoparticles (NPs) might improve the therapeutic outcome in metastatic melanoma. Cellulose nanocrystals (CNC NPs) can particularly deliver payloads to lung capillaries in vivo. Herein, 177 Lu-labeled CNC NPs loaded with vemurafenib ([177 Lu]Lu-CNC-V NPs) is developed and the therapeutic effect in BRAF V600E mutation-harboring YUMM1.G1 murine model of lung metastatic melanoma is investigated. The [177 Lu]Lu-CNC-V NPs demonstrate favorable radiolabel stability, drug release profile, cellular uptake, and cell growth inhibition in vitro. In vivo biodistribution reveals significant retention of the [177 Lu]Lu-CNC-V NPs in the lung, liver, and spleen. Ultimately, the median survival time of animals is doubly increased after treatment with [177 Lu]Lu-CNC-V NPs compared to control groups. The enhanced therapeutic efficacy of [177 Lu]Lu-CNC-V NPs in the lung metastatic melanoma animal model provides convincing evidence for the potential of clinical translation for theranostic CNC NP-based drug delivery systems after intravenous administration.

Synthesis and Comparative In Vivo Evaluation of Site-Specifically Labeled Radioimmunoconjugates for DLL3-Targeted ImmunoPET.

Delta-like ligand 3 (DLL3) is a therapeutic target for the treatment of small cell lung cancer, neuroendocrine prostate cancer, and isocitrate dehydrogenase mutant glioma. In the clinic, DLL3-targeted 89Zr-immunoPET has the potential to aid in the assessment of disease burden and facilitate the selection of patients suitable for therapies that target the antigen. The overwhelming majority of 89Zr-labeled radioimmunoconjugates are synthesized via the random conjugation of desferrioxamine (DFO) to lysine residues within the immunoglobulin. While this approach is admittedly facile, it can produce heterogeneous constructs with suboptimal in vitro and in vivo behavior. In an effort to circumvent these issues, we report the development and preclinical evaluation of site-specifically labeled radioimmunoconjugates for DLL3-targeted immunoPET. To this end, we modified a cysteine-engineered variant of the DLL3-targeting antibody SC16-MB1 with two thiol-reactive variants of DFO: one bearing a maleimide moiety (Mal-DFO) and the other containing a phenyloxadiazolyl methyl sulfone group (PODS-DFO). In an effort to obtain immunoconjugates with a DFO-to-antibody ratio (DAR) of 2, we explored both the reduction of the antibody with tris(2-carboxyethyl) phosphine (TCEP) as well as the use of a combination of glutathione and arginine as reducing and stabilizing agents, respectively. While exerting control over the DAR of the immunoconjugate proved cumbersome using TCEP, the use of glutathione and arginine enabled the selective reduction of the engineered cysteines and thus the formation of homogeneous immunoconjugates. A head-to-head comparison of the resulting 89Zr-radioimmunoconjugates in mice bearing DLL3-expressing H82 xenografts revealed no significant differences in tumoral uptake and showed comparable radioactivity concentrations in most healthy nontarget organs. However, 89Zr-DFOPODS-DAR2SC16-MB1 produced 30% lower uptake (3.3 ± 0.5 %ID/g) in the kidneys compared to 89Zr-DFOMal-DAR2SC16-MB1 (4.7 ± 0.5 %ID/g). In addition, H82-bearing mice injected with a 89Zr-labeled isotype-control radioimmunoconjugate synthesized using PODS exhibited ∼40% lower radioactivity in the kidneys compared to mice administered its maleimide-based counterpart. Taken together, these results demonstrate the improved in vivo performance of the PODS-based radioimmunoconjugate and suggest that a stable, well-defined DAR2 radiopharmaceutical may be suitable for the clinical immunoPET of DLL3-expressing cancers.

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.

Identification of HER2-Positive Metastases in Patients with HER2-Negative Primary Breast Cancer by Using HER2-targeted 89Zr-Pertuzumab PET/CT.

Background Human epidermal growth factor receptor 2 (HER2)-targeted therapies are successful in patients with HER2-positive malignancies; however, spatial and temporal heterogeneity of HER2 expression may prevent identification of optimal patients for these therapies. Purpose To determine whether imaging with the HER2-targeted PET tracer zirconium 89 (89Zr)-pertuzumab can depict HER2-positive metastases in women with HER2-negative primary breast cancer. Materials and Methods From January to June 2019, women with biopsy-proven HER2-negative primary breast cancer and biopsy-proven metastatic disease were enrolled in a prospective clinical trial (ClinicalTrials.gov NCT02286843) and underwent 89Zr-pertuzumab PET/CT for noninvasive whole-biopsy evaluation of potential HER2-positive metastases. 89Zr-pertuzumab-avid foci that were suspicious for HER2-positive metastases were tissue sampled and examined by pathologic analysis to document HER2 status. Results Twenty-four women (mean age, 55 years ± 11 [standard deviation]) with HER2-negative primary breast cancer were enrolled. Six women demonstrated foci at 89Zr-pertuzumab PET/CT that were suspicious for HER2-positive disease. Of these six women, three had biopsy-proven HER2-positive metastases, two had pathologic findings that demonstrated HER2-negative disease, and one had a fine-needle aspirate with inconclusive results. Conclusion Human epidermal growth factor receptor 2 (HER2)-targeted imaging with zirconium 89-pertuzumab PET/CT was successful in detecting HER2-positive metastases in women with HER2-negative primary breast cancer. This demonstrates the ability of targeted imaging to identify patients for targeted therapies that might not otherwise be considered. © RSNA, 2020 Online supplemental material is available for this article. See the editorial by Mankoff and Pantel in this issue.

DiPODS: A Reagent for Site-Specific Bioconjugation via the Irreversible Rebridging of Disulfide Linkages.

Chemoselective reactions with thiols have long held promise for the site-specific bioconjugation of antibodies and antibody fragments. Yet bifunctional probes bearing monovalent maleimides-long the "gold standard" for thiol-based ligations-are hampered by two intrinsic issues: the in vivo instability of the maleimide-thiol bond and the need to permanently disrupt disulfide linkages in order to facilitate bioconjugation. Herein, we present the synthesis, characterization, and validation of DiPODS, a novel bioconjugation reagent containing a pair of oxadiazolyl methyl sulfone moieties capable of irreversibly forming covalent bonds with two thiolate groups while simultaneously rebridging disulfide linkages. The reagent was synthesized from commercially available starting materials in 8 steps, during which rotamers were encountered and investigated both experimentally and computationally. DiPODS is designed to be modular and can thus be conjugated to any payload through a pendant terminal primary amine (DiPODS-PEG4-NH2). Subsequently, the modification of a HER2-targeting Fab with a fluorescein-conjugated variant of DiPODS (DiPODS-PEG4-FITC) reinforced the site-specificity of the reagent, illustrated its ability to rebridge disulfide linkages, and produced an immunoconjugate with in vitro properties superior to those of an analogous construct created using traditional stochastic bioconjugation techniques. Ultimately, we believe that this work has particularly important implications for the synthesis of immunoconjugates, specifically for ensuring that the attachment of cargoes to immunoglobulins is robust, irreversible, and biologically and structurally benign.

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.

Removal of Fc Glycans from [89Zr]Zr-DFO-Anti-CD8 Prevents Peripheral Depletion of CD8+ T Cells.

The N-linked biantennary glycans on the heavy chain of immunoglobulin G (IgG) antibodies (mAbs) are instrumental in the recognition of the Fc region by Fc-gamma receptors (FcγR). In the case of full-length mAb-based imaging tracers targeting immune cell populations, these Fc:FcγR interactions can potentially deplete effector cells responsible for tumor clearance. To bypass this problem, we hypothesize that the enzymatic removal of the Fc glycans will disrupt Fc:FcγR interactions and spare tracer-targeted immune cells from depletion during immunopositron emission tomography (immunoPET) imaging. Herein, we compared the in vitro and in vivo properties of 89Zr-radiolabeled CD8-specific murine mAb (anti-CD8wt, clone 2.43), a well-known depleting mAb, and its deglycosylated counterpart (anti-CD8degly). Deglycosylation was achieved via enzymatic treatment with the peptide: N-glycosidase F (PNGaseF). Both anti-CD8wt and anti-CD8degly mAbs were conjugated to p-SCN-Bn-desferrioxamine (DFO) and labeled with 89Zr. Bindings of both DFO-conjugated mAbs to FcγR and CD8+ splenocytes were compared. In vivo imaging and distribution studies were conducted to examine the specificity and pharmacokinetics of the radioimmunoconjugates in tumor-naive and CT26 colorectal tumor-bearing mice. Ex vivo analysis of CD8+ T cell population in spleens and tumors obtained postimaging were measured via flow cytometry and qRT-PCR. The removal of the Fc glycans from anti-CD8wt was confirmed via SDS-PAGE. A reduction in FcγR interaction was exhibited by DFO-anti-CD8degly, while its binding to CD8 remained unchanged. Tissue distribution showed similar pharmacokinetics of [89Zr]Zr-DFO-anti-CD8degly and the wt radioimmunoconjugate. In vivo blocking studies further demonstrated retained specificity of the deglycosylated radiotracer for CD8. From the imaging studies, no difference in accumulation in both spleens and tumors was observed between both radiotracers. Results from the flow cytometry analysis confirmed depletion of CD8+ T cells in spleens of mice administered with DFO-anti-CD8wt, whereas an increase in CD8+ T cells was shown with DFO-anti-CD8degly. No statistically significant difference in tumor infiltrating CD8+ T cells was observed in cohorts administered with the probes when compared to control unmodulated mice. CD8 mRNA levels from excised tumors showed increased transcripts of the antigen in mice administered with [89Zr]Zr-DFO-anti-CD8degly compared to mice imaged with [89Zr]Zr-DFO-anti-CD8wt. In conclusion, the removal of Fc glycans offers a straightforward approach to develop full length antibody-based imaging probes specifically for detecting CD8+ immune molecules with no consequential depletion of their target cell population in peripheral tissues.

Manipulating the In Vivo Behaviour of 68Ga with Tris(Hydroxypyridinone) Chelators: Pretargeting and Blood Clearance.

Pretargeting is widely explored in immunoPET as a strategy to reduce radiation exposure of non-target organs and allow the use of short-lived radionuclides that would not otherwise be compatible with the slow pharmacokinetic profiles of antibodies. Here we investigate a pretargeting strategy based on gallium-68 and the chelator THPMe as a high-affinity pair capable of combining in vivo. After confirming the ability of THPMe to bind 68Ga in vivo at low concentrations, the bifunctional THPMe-NCS was conjugated to a humanised huA33 antibody targeting the A33 glycoprotein. Imaging experiments performed in nude mice bearing A33-positive SW1222 colorectal cancer xenografts compared pretargeting (100 µg of THPMe-NCS-huA33, followed after 24 h by 8-10 MBq of 68Ga3+) with both a directly labelled radioimmunoconjugate (89Zr-DFO-NCS-huA33, 88 µg, 7 MBq) and a 68Ga-only negative control (8-10 MBq of 68Ga3+). Imaging was performed 25 h after antibody administration (1 h after 68Ga3+ administration for negative control). No difference between pretargeting and the negative control was observed, suggesting that pretargeting via metal chelation is not feasible using this model. However, significant accumulation of "unchelated" 68Ga3+ in the tumour was found (12.9 %ID/g) even without prior administration of THPMe-NCS-huA33, though tumour-to-background contrast was impaired by residual activity in the blood. Therefore, the 68Ga-only experiment was repeated using THPMe (20 µg, 1 h after 68Ga3+ administration) to clear circulating 68Ga3+, producing a three-fold improvement of the tumour-to-blood activity concentration ratio. Although preliminary, these results highlight the potential of THPMe as a 68Ga clearing agent in imaging applications with gallium citrate.