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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 (177)Lu-labeled radioimmunoconjugate based on (HER2)DVD, 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 (HER2)DVD 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 [(177)Lu]Lu(3+) to produce the completed radioimmunoconjugate — [(177)Lu]Lu-CHX-A″-DTPA(PODS)-(HER2)DVD — in >80% radiochemical conversion and a specific activity of 29.5 ± 7.1 GBq/μmol. [(177)Lu]Lu-CHX-A″-DTPA(PODS)-(HER2)DVD did not form aggregates upon prolonged incubation in human serum, displayed 87% stability to demetallation over a 7 d incubation in serum, and exhibited an immunoreactive fraction of 0.95 with HER2-coated beads. Finally, we compared the pharmacokinetic profile of [(177)Lu]Lu-CHX-A″-DTPA(PODS)-(HER2)DVD to that of a (177)Lu-labeled variant of trastuzumab in mice bearing subcutaneous HER2-expressing BT-474 human breast cancer xenografts. The in vivo performance of [(177)Lu]Lu-CHX-A″-DTPA(PODS)-(HER2)DVD matched that of (177)Lu-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 timepoint. 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.

This document contains all supporting materials and methods as well as supplemental figures and tables.

Supplemental data detailing the methods and results for the synthesis and in vitro characterization of the radioimmunoconjugates, longitudinal PET imaging and ex vivo analyses in various models described within the main text.

Supplementary Data

Purpose:Glioblastoma multiforme is a highly aggressive form of brain cancer whose location, tendency to infiltrate healthy surrounding tissue, and heterogeneity significantly limit survival, with scant progress having been made in recent decades.Experimental Design:123I-MAPi (Iodine-123 Meitner-Auger PARP1 inhibitor) is a precise therapeutic tool composed of a PARP1 inhibitor radiolabeled with an Auger- and gamma-emitting iodine isotope. Here, the PARP inhibitor, which binds to the DNA repair enzyme PARP1, specifically targets cancer cells, sparing healthy tissue, and carries a radioactive payload within reach of the cancer cells' DNA.Results:The high relative biological efficacy of Auger electrons within their short range of action is leveraged to inflict DNA damage and cell death with high precision. The gamma ray emission of 123I-MAPi allows for the imaging of tumor progression and therapy response, and for patient dosimetry calculation. Here we demonstrated the efficacy and specificity of this small-molecule radiotheranostic in a complex preclinical model. In vitro and in vivo studies demonstrate high tumor uptake and a prolonged survival in mice treated with 123I-MAPi when compared with vehicle controls. Different methods of drug delivery were investigated to develop this technology for clinical applications, including convection enhanced delivery and intrathecal injection.Conclusions:Taken together, these results represent the first full characterization of an Auger-emitting PARP inhibitor which demonstrate a survival benefit in mouse models of GBM and confirm the high potential of 123I-MAPi for clinical translation.

Purpose:Interest in targeted alpha-therapy has surged due to α-particles' high cytotoxicity. However, the widespread clinical use of this approach could be limited by on-/off-target toxicities. Here, we investigated the inverse electron-demand Diels–Alder ligation between an 225Ac-labeled tetrazine radioligand and a trans-cyclooctene–bearing anti-CA19.9 antibody (5B1) for pretargeted α-radioimmunotherapy (PRIT) of pancreatic ductal adenocarcinoma (PDAC). This alternative strategy is expected to reduce nonspecific toxicities as compared with conventional radioimmunotherapy (RIT).Experimental Design: A side-by-side comparison of 225Ac-PRIT and conventional RIT using a directly 225Ac-radiolabeled immunoconjugate evaluates the therapeutic efficacy and toxicity of both methodologies in PDAC murine models.Results:A comparative biodistribution study of the PRIT versus RIT methodology underscored the improved pharmacokinetic properties (e.g., prolonged tumor uptake and increased tumor-to-tissue ratios) of the PRIT approach. Cerenkov imaging coupled to PRIT confirmed the in vivo biodistribution of 225Ac-radioimmunoconjugate but—importantly—further allowed for the ex vivo monitoring of 225Ac's radioactive daughters' redistribution. Human dosimetry was extrapolated from the mouse biodistribution and confirms the clinical translatability of 225Ac-PRIT. Furthermore, longitudinal therapy studies performed in subcutaneous and orthotopic PDAC models confirm the therapeutic efficacy of 225Ac-PRIT with the observation of prolonged median survival compared with control cohorts. Finally, a comparison with conventional RIT highlighted the potential of 225Ac-PRIT to reduce hematotoxicity while maintaining therapeutic effectiveness.Conclusions:The ability of 225Ac-PRIT to deliver a radiotherapeutic payload while simultaneously reducing the off-target toxicity normally associated with RIT suggests that the clinical translation of this approach will have a profound impact on PDAC therapy.

Supplemental data

The Notch ligand DLL3 has emerged as a novel therapeutic target expressed in small cell lung cancer (SCLC) and high-grade neuroendocrine carcinomas. Rovalpituzumab teserine (Rova-T; SC16LD6.5) is a first-in-class DLL3-targeted antibody–drug conjugate with encouraging initial safety and efficacy profiles in SCLC in the clinic. Here we demonstrate that tumor expression of DLL3, although orders of magnitude lower in surface protein expression than typical oncology targets of immunoPET, can serve as an imaging biomarker for SCLC. We developed 89Zr-labeled SC16 antibody as a companion diagnostic agent to facilitate selection of patients for treatment with Rova-T based on a noninvasive interrogation of the in vivo status of DLL3 expression using PET imaging. Despite low cell-surface abundance of DLL3, immunoPET imaging with 89Zr-labeled SC16 antibody enabled delineation of subcutaneous and orthotopic SCLC tumor xenografts as well as distant organ metastases with high sensitivity. Uptake of the radiotracer in tumors was concordant with levels of DLL3 expression and, most notably, DLL3 immunoPET yielded rank-order correlation for response to SC16LD6.5 therapy in SCLC patient–derived xenograft models. Cancer Res; 77(14); 3931–41. ©2017 AACR.

Supplementary Figure from ImmunoPET of Ovarian and Pancreatic Cancer with AR9.6, a Novel MUC16-Targeted Therapeutic Antibody

Supplementary Date from ImmunoPET of Ovarian and Pancreatic Cancer with AR9.6, a Novel MUC16-Targeted Therapeutic Antibody

Supplementary Table from ImmunoPET of Ovarian and Pancreatic Cancer with AR9.6, a Novel MUC16-Targeted Therapeutic Antibody

Purpose:Advances in our understanding of the contribution of aberrant glycosylation to the pro-oncogenic signaling and metastasis of tumor cells have reinvigorated the development of mucin-targeted therapies. Here, we validate the tumor-targeting ability of a novel monoclonal antibody (mAb), AR9.6, that binds MUC16 and abrogates downstream oncogenic signaling to confer a therapeutic response.Experimental Design:The in vitro and ex vivo validation of the binding of AR9.6 to MUC16 was achieved via flow cytometry, radioligand binding assay (RBA), and immunohistochemistry (IHC). The in vivo MUC16 targeting of AR9.6 was validated by creating a 89Zr-labeled radioimmunoconjugate of the mAb and utilizing immunoPET and ex vivo biodistribution studies in xenograft models of human ovarian and pancreatic cancer.Results:Flow cytometry, RBA, and IHC revealed that AR9.6 binds to ovarian and pancreatic cancer cells in an MUC16-dependent manner. The in vivo radiopharmacologic profile of 89Zr-labeled AR9.6 in mice bearing ovarian and pancreatic cancer xenografts confirmed the MUC16-dependent tumor targeting by the radioimmunoconjugate. Radioactivity uptake was also observed in the distant lymph nodes (LNs) of mice bearing xenografts with high levels of MUC16 expression (i.e., OVCAR3 and Capan-2). IHC analyses of these PET-positive LNs highlighted the presence of shed antigen as well as necrotic, phagocytized, and actively infiltrating neoplastic cells. The humanization of AR9.6 did not compromise its ability to target MUC16-expressing tumors.Conclusions:The unique therapeutic mechanism of AR9.6 combined with its excellent in vivo tumor targeting makes it a highly promising theranostic agent. huAR9.6 is poised for clinical translation to impact the management of metastatic ovarian and pancreatic cancers.

A critical benchmark in the development of antibody-based therapeutics is demonstration of efficacy in preclinical mouse models of human disease, many of which rely on immunodeficient mice. However, relatively little is known about how the biology of various immunodeficient strains impacts the in vivo fate of these drugs. Here we used immunoPET radiotracers prepared from humanized, chimeric, and murine mAbs against four therapeutic oncologic targets to interrogate their biodistribution in four different strains of immunodeficient mice bearing lung, prostate, and ovarian cancer xenografts. The immunodeficiency status of the mouse host as well as both the biological origin and glycosylation of the antibody contributed significantly to the anomalous biodistribution of therapeutic monoclonal antibodies in an Fc receptor-dependent manner. These findings may have important implications for the preclinical evaluation of Fc-containing therapeutics and highlight a clear need for biodistribution studies in the early stages of antibody drug development.Significance: Fc/FcγR-mediated immunobiology of the experimental host is a key determinant to preclinical in vivo tumor targeting and efficacy of therapeutic antibodies. Cancer Res; 78(7); 1820–32. ©2018 AACR.

The last decade has played witness to 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 (64)Cu-labeled Tz radioligand ([(64)Cu]Cu-SarAr-Tz) and a TCO-bearing bisphosphonate (TCO-BP) for the pretargeted 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 [(64)Cu]Cu-SarAr-Tz. PET images were collected 1, 6, and 24 h after the administration of [(64)Cu]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 [(64)Cu]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 bone, 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 [(64)Cu]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.

Matrix metalloproteinase (MMP) enzymes are over-expressed by some metastatic cancers, in which they are responsible for the degradation and remodeling of the extracellular matrix. In recent years, MMPs have emerged as promising targets for enzyme-responsive diagnostic probes because oligopeptides can be designed to be selectively hydrolyzed by exposure to these enzymes. With the ultimate goal of developing radio-iodinated peptides as supramolecular building blocks for MMP-sensitive tools for nuclear imaging and therapy, we designed three MMP-9-responsive peptides containing either tyrosine or iodotyrosine, in order to assess the impact of iodotyrosine introduction to peptide structure and cleavage kinetics. We found that the peptides containing iodotyrosine underwent more rapid and more complete hydrolysis by MMP-9. While the peptides under investigation were predominantly disordered, it was found that iodination increased the degree of aromatic residue-driven aggregation of the peptides. We determined that these iodination-related trends stem from improved overall intramolecular order through H- and halogen bonding, in addition to intermolecular organization of the self-assembled peptides due to steric and electrostatic effects introduced by the halogenated tyrosine. These fundamental observations provide insights for the development of enzyme-triggered peptide aggregation tools for localized radioactive iodine- based tumor imaging.

Because of their rapid tumor accumulation and normal tissue clearance, ~15-kDa single domain antibody fragments (sdAbs, also known as nanobodies) are an attractive vehicle for developing targeted radiotherapeutics labeled with the 7.2-h α-particle emitting radiohalogen (211)At. We have developed a site-specific (211)At-labeled prosthetic agent — iso-[(211)At]AGMB-PODS — that combines a functionality for residualizing radioactivity in tumor cells with a phenyloxadiazolyl methylsulfone (PODS) moiety for linking it to an sdAb. This reagent was evaluated for thiol-selective conjugation to anti-HER2 5F7 sdAb modified to have a C-terminus GGC tail for this purpose. The iso-[(211)At]AGMB-PODS-5F7GGC conjugate and its (131)I-labeled analogue were synthesized in good radiochemical yields with retention of high binding affinity and immunoreactivity. The PODS radioconjugates exhibited high specific binding, internalization and intracellular trapping of radioactivity on HER2-positive BT474 breast carcinoma cells in vitro. Iso-[(211)At]AGMB-PODS-5F7GGC displayed considerably higher in vitro stability in the presence of cysteine and HSA compared with the maleimide analogue, [(211)At]MEAGMB-5F7GGC, excellent tumor uptake and high in vivo stability. Superior tumor-to-kidney activity ratios were seen for iso-[(211)At]AGMB-PODS-5F7GGC and its radioiodinated analogue compared with [(177)Lu]Lu-DOTA-PODS-5F7GGC. These results suggest that iso-[(211)At]AGMB-PODS-5F7GGC warrants further evaluation as a targeted α-particle emitting agent for the treatment of HER2-expressing malignancies.

VlsE (variable major protein-like sequence, expressed) is an outer surface protein of the Lyme disease pathogen (

The synthesis of radioimmunoconjugates via the stochastic attachment of bifunctional chelators to lysines can yield heterogeneous products with suboptimaliin vitro/iandiin vivo/ibehavior. 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-bisNsub3/sub) 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 [sup89/supZr]Zr-supSSK/supDFO-pertuzumab, a radioimmunoconjugate of the HER2-targeting mAb pertuzumab labeled with desferrioxamine (DFO) and the positron-emitting radiometal zirconium-89 (sup89/supZr). [sup89/supZr]Zr-supSSK/supDFO-pertuzumab was compared to a pair of analogous probes: one synthesized via random lysine modification ([sup89/supZr]Zr-DFO-pertuzumab) and another via thiol-maleimide chemistry ([sup89/supZr]Zr-supmal/supDFO-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), andsup89/supZr-labeled variants of each were synthesized ingt;99% radiochemical yield and molar activities of up to ∼55.5 GBq/μmol (10 mCi/mg). Subsequently, theiin vivo/ibehavior of this trio ofsup89/supZr-immunoPET probes was interrogated in athymic nude mice bearing subcutaneous HER2-expressing BT-474 human breast cancer xenografts. [sup89/supZr]Zr-supSSK/supDFO-pertuzumab, [sup89/supZr]Zr-supmal/supDFO-pertuzumab, and [sup89/supZr]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 [sup89/supZr]Zr-supSSK/supDFO-pertuzumab, [sup89/supZr]Zr-supmal/supDFO-pertuzumab, and [sup89/supZr]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 excellentiin vitro/iandiin vivo/iperformance. 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.

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.

VlsE (variable major protein-likesequence,expressed) is an outer surface protein of the Lyme disease pathogen (Borreliellaspecies) and a key diagnostic biomarker of Lyme disease. However, the high sequence variability of VlsE poses a challenge to the development of consistent VlsE-based diagnostics and therapeutics. In addition, the standard diagnostic protocols detect immunoglobins elicited by the Lyme pathogen, not the presence of pathogen or its derived antigens. Here we describe the development of recombinant monoclonal antibodies (rMAbs) that bind specifically to conserved epitopes on VlsE. We first quantified amino-acid sequence variability encoded by thevlsgenes from thirteenB. burgdorferigenomes by evolutionary analyses. We showed broad inconsistencies of the sequence phylogeny with the genome phylogeny, indicating rapid gene duplications, losses, and recombination at thevlslocus. To identify conserved epitopes, we synthesized peptides representing five long conserved invariant regions (IRs) on VlsE. We tested the antigenicity of these five IR peptides using sera from three mammalian host species including human patients, the natural reservoir white-footed mouse (Peromyscus leucopus), and VlsE-immunized New Zealand rabbits (Oryctolagus cuniculus). The IR4 and IR6 peptides emerged as the most antigenic and reacted strongly with both the human and rabbit sera, while all IR peptides reacted poorly with sera from natural hosts. Four rMAbs binding specifically to the IR4 and IR6 peptides were identified, cloned, and purified. Given their specific recognition of the conserved epitopes on VlsE, these IR-specific rMAbs are promising diagnostic and theragnostic agents for direct detection of Lyme disease pathogens regardless of strain heterogeneity.

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 re-bridging 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–PEG(4)-NH(2)). Subsequently, the modification of a HER2-targeting Fab with a fluorescein-conjugated variant of DiPODS (DiPODS-PEG(4)-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.

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.

Human epidermal growth factor receptor 2 (HER2)-targeted zirconium 89–pertuzumab PET/CT successfully helped to identify HER2-positive metastases in women with HER2-negative primary breast cancer.

Ovarian cancer is the fifth leading cause of cancer deaths amongst 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: (ss)B43.13-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 (ss)B43.13-IR800 to CA125-expressing OVCAR3 human ovarian cancer cells. NIRF imaging studies demonstrated that (ss)B43.13-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 (ss)B43.13-IR800 can bind and identify CA125-expressing cells in primary tumor and metastatic lymph node samples from human patients with HGSOC.

Purpose: Glioblastoma multiforme is a highly aggressive form of brain cancer whose location, tendency to infiltrate healthy surrounding tissue, and heterogeneity significantly limit survival, with scant progress having been made in recent decades. Experimental Design: 123I-MAPi (Iodine-123 Meitner-Auger PARP1 inhibitor) is a precise therapeutic tool composed of a PARP1 inhibitor radiolabeled with an Auger- and gamma-emitting iodine isotope. Here, the PARP inhibitor, which binds to the DNA repair enzyme PARP1, specifically targets cancer cells, sparing healthy tissue, and carries a radioactive payload within reach of the cancer cells' DNA. Results: The high relative biological efficacy of Auger electrons within their short range of action is leveraged to inflict DNA damage and cell death with high precision. The gamma ray emission of 123I-MAPi allows for the imaging of tumor progression and therapy response, and for patient dosimetry calculation. Here we demonstrated the efficacy and specificity of this small-molecule radiotheranostic in a complex preclinical model. In vitro and in vivo studies demonstrate high tumor uptake and a prolonged survival in mice treated with 123I-MAPi when compared with vehicle controls. Different methods of drug delivery were investigated to develop this technology for clinical applications, including convection enhanced delivery and intrathecal injection. Conclusions: Taken together, these results represent the first full characterization of an Auger-emitting PARP inhibitor which demonstrate a survival benefit in mouse models of GBM and confirm the high potential of 123I-MAPi for clinical translation.

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 (89)Zr-radiolabeled CD8-specific murine mAb (anti-CD8(wt), clone 2.43), a well-known depleting mAb, and its deglycosylated counterpart (anti-CD8(degly)). Deglycosylation was achieved via enzymatic treatment with the peptide: N-glycosidase F (PNGaseF). Both anti-CD8(wt) and anti-CD8(degly) mAbs were conjugated to p-SCN-Bn-desferrioxamine (DFO) and labeled with (89)Zr. 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-CD8(wt) was confirmed via SDS-PAGE. A reduction in FcγR interaction was exhibited by DFO-anti-CD8(degly), while its binding to CD8 remained unchanged. Tissue distribution showed similar pharmacokinetics of [(89)Zr]Zr-DFO-anti-CD8(degly) 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-CD8(wt), whereas an increase in CD8(+) T cells was shown with DFO-anti-CD8(degly). 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 [(89)Zr]Zr-DFO-anti-CD8(degly) compared to mice imaged with [(89)Zr]Zr-DFO-anti-CD8(wt). 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.

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-βGal). 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-βGal (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.

Radiochemical conversion is an important term to be included in the "Consensus nomenclature rules for radiopharmaceutical chemistry". Radiochemical conversion should be used to define reaction efficiency by measuring the transformation of components in a crude reaction mixture at a given time, whereas radiochemical yield is better suited to define the efficiency of an entire reaction process including, for example, separation, isolation, filtration, and formulation. (C) 2020 Elsevier Inc. All rights reserved.

This approach leverages the rapid, bio-orthogonal inverse electron demand Diels-Alder reaction between a radiolabeled tetrazine and a trans-cyclooctene-bearing antibody to enable pretargeted positron emission tomography imaging and endoradiotherapy in a murine model of cancer. Radiolabeled antibodies have shown promise as tools for both the nuclear imaging and endoradiotherapy of cancer, but the protracted circulation time of radioimmunoconjugates can lead to high radiation doses to healthy tissues. To circumvent this issue, we have developed an approach to positron emission tomography (PET) imaging and radioimmunotherapy (RIT) predicated on radiolabeling the antibody after it has reached its target within the body. This in vivo pretargeting strategy is based on the rapid and bio-orthogonal inverse electron demand Diels-Alder reaction between tetrazine (Tz) and trans-cyclooctene (TCO). Pretargeted PET imaging and RIT using TCO-modified antibodies in conjunction with Tz-bearing radioligands produce high activity concentrations in target tissues as well as reduced radiation doses to healthy organs compared to directly labeled radioimmunoconjugates. Herein, we describe how to prepare a TCO-modified antibody (humanized A33-TCO) as well as how to synthesize two Tz-bearing radioligands: one labeled with the positron-emitting radiometal copper-64 ([Cu-64]Cu-SarAr-Tz) and one labeled with the beta-emitting radiolanthanide lutetium-177 ([Lu-177]Lu-DOTA-PEG(7)-Tz). We also provide a detailed description of pretargeted PET and pretargeted RIT experiments in a murine model of human colorectal carcinoma. Proper training in both radiation safety and the handling of laboratory mice is required for the successful execution of this protocol.

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 ...

Purpose: Interest in targeted alpha-therapy has surged due to α-particles' high cytotoxicity. However, the widespread clinical use of this approach could be limited by on-/off-target toxicities. Here, we investigated the inverse electron-demand Diels–Alder ligation between an 225Ac-labeled tetrazine radioligand and a trans-cyclooctene–bearing anti-CA19.9 antibody (5B1) for pretargeted α-radioimmunotherapy (PRIT) of pancreatic ductal adenocarcinoma (PDAC). This alternative strategy is expected to reduce nonspecific toxicities as compared with conventional radioimmunotherapy (RIT). Experimental Design: A side-by-side comparison of 225Ac-PRIT and conventional RIT using a directly 225Ac-radiolabeled immunoconjugate evaluates the therapeutic efficacy and toxicity of both methodologies in PDAC murine models. Results: A comparative biodistribution study of the PRIT versus RIT methodology underscored the improved pharmacokinetic properties (e.g., prolonged tumor uptake and increased tumor-to-tissue ratios) of the PRIT approach. Cerenkov imaging coupled to PRIT confirmed the in vivo biodistribution of 225Ac-radioimmunoconjugate but—importantly—further allowed for the ex vivo monitoring of 225Ac's radioactive daughters' redistribution. Human dosimetry was extrapolated from the mouse biodistribution and confirms the clinical translatability of 225Ac-PRIT. Furthermore, longitudinal therapy studies performed in subcutaneous and orthotopic PDAC models confirm the therapeutic efficacy of 225Ac-PRIT with the observation of prolonged median survival compared with control cohorts. Finally, a comparison with conventional RIT highlighted the potential of 225Ac-PRIT to reduce hematotoxicity while maintaining therapeutic effectiveness. Conclusions: The ability of 225Ac-PRIT to deliver a radiotherapeutic payload while simultaneously reducing the off-target toxicity normally associated with RIT suggests that the clinical translation of this approach will have a profound impact on PDAC therapy.

Purpose: Advances in our understanding of the contribution of aberrant glycosylation to the pro-oncogenic signaling and metastasis of tumor cells have reinvigorated the development of mucin-targeted therapies. Here, we validate the tumor-targeting ability of a novel monoclonal antibody (mAb), AR9.6, that binds MUC16 and abrogates downstream oncogenic signaling to confer a therapeutic response. Experimental Design: The in vitro and ex vivo validation of the binding of AR9.6 to MUC16 was achieved via flow cytometry, radioligand binding assay (RBA), and immunohistochemistry (IHC). The in vivo MUC16 targeting of AR9.6 was validated by creating a 89Zr-labeled radioimmunoconjugate of the mAb and utilizing immunoPET and ex vivo biodistribution studies in xenograft models of human ovarian and pancreatic cancer. Results: Flow cytometry, RBA, and IHC revealed that AR9.6 binds to ovarian and pancreatic cancer cells in an MUC16-dependent manner. The in vivo radiopharmacologic profile of 89Zr-labeled AR9.6 in mice bearing ovarian and pancreatic cancer xenografts confirmed the MUC16-dependent tumor targeting by the radioimmunoconjugate. Radioactivity uptake was also observed in the distant lymph nodes (LNs) of mice bearing xenografts with high levels of MUC16 expression (i.e., OVCAR3 and Capan-2). IHC analyses of these PET-positive LNs highlighted the presence of shed antigen as well as necrotic, phagocytized, and actively infiltrating neoplastic cells. The humanization of AR9.6 did not compromise its ability to target MUC16-expressing tumors. Conclusions: The unique therapeutic mechanism of AR9.6 combined with its excellent in vivo tumor targeting makes it a highly promising theranostic agent. huAR9.6 is poised for clinical translation to impact the management of metastatic ovarian and pancreatic cancers.

Funding Information: Financial support from the Finnish Cultural Foundation (grant no. 00190375), the Academy of Finland (decision nos. 318422, 278056, 298481, 302102, and 331659), and the University of Helsinki Research Funds are gratefully acknowledged. Finnish Center for Laboratory Animal Pathology (FCLAP) at HiLIFE, University of Helsinki, is acknowledged for the tissue preparation and staining services. The authors also thank Dr. Jaakko Sarparanta for help with phospho‐MEK western blot, Ms. Karina Moslova for assistance with the elemental analysis, Ms. Alexandra Correia for assistance with cell culture, and Ms. Phetcharada Imlimthan for the assistance with the images. Publisher Copyright: © 2021 The Authors. Small published by Wiley-VCH GmbH Copyright: Copyright 2021 Elsevier B.V., All rights reserved. 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 (177Lu) enables simultaneous tracking of tracer accumulation with single-photon emission computed tomography and radiotherapy. Therefore, the codelivery of 177Lu 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, 177Lu-labeled CNC NPs loaded with vemurafenib ([177Lu]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 [177Lu]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 [177Lu]Lu-CNC-V NPs in the lung, liver, and spleen. Ultimately, the median survival time of animals is doubly increased after treatment with [177Lu]Lu-CNC-V NPs compared to control groups. The enhanced therapeutic efficacy of [177Lu]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.

The proliferation of plastics in the environment continues at an alarming rate. Plastic particles have been found to be persistent and ubiquitous pollutants in a variety of environments, including sea water, fresh water, soil, and air. In light of this phenomenon, the scientific and medical communities have become increasingly wary of the dangers posed to human health by chronic exposure to microplastics (89Zr; t1/2 ~ 3.3 d). Subsequently, positron emission tomography (PET) was used to visualize the biodistribution of these radioplastics in C57BL/6J mice at 6, 12, 24, and 48 h after ingestion. The imaging data reveal that the majority of the radioplastics remain in the gastrointestinal tract and are eliminated through the feces by 48 h post-ingestion, a result reinforced by acute biodistribution studies. Ultimately, this work suggests that nuclear imaging—and PET in particular—can be a sensitive and effective tool in the urgent and rapidly growing effort to study the in vivo behavior and potential toxicity of micro- and nanoplastics.

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.

Significance Radioimmunotherapy is predicated on harnessing the exquisite specificity of antibodies to deliver cytotoxic radiation to tumors. Yet the long circulation time of radioimmunoconjugates leads to high radiation doses to healthy tissues. Pretargeted radioimmunotherapy (PRIT) circumvents this problem by decoupling the antibody and radionuclide, injecting the former prior to the latter, and empowering the two components to combine at the tumor. We have leveraged bioorthogonal click chemistry, a colorectal cancer-targeting antibody, and a pair radioligands bearing radioisotopes of copper—positron-emitting copper-64 and beta particle-emitting copper-67—to create a pretargeting system that enables both theranostic positron emission tomography and PRIT. We believe that the incorporation of theranostic imaging into PRIT regimens could be instrumental in the clinical success of the modality., 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.

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 (