SENTRI: Single-Particle Energy Transducer for Radionuclide Injections for Personalized Targeted Radionuclide Cancer Therapy.

PURPOSE: Targeted radionuclide therapy (TRT), whereby a tumor-targeted molecule is linked to a therapeutic beta- or alpha-emitting radioactive nuclide, is a promising treatment modality for patients with metastatic cancer, delivering radiation systemically. However, patients still progress due to suboptimal dosing, driven by the large patient-to-patient variability. Therefore, the ability to continuously monitor the real-time dose deposition in tumors and organs at risk provides an additional dimension of information during clinical trials that can enable insights into better strategies to personalize TRT. METHODS AND MATERIALS: Here, we present a single beta-particle sensitive dosimeter consisting of a 0.27-mm3 monolithic silicon chiplet directly implanted into the tumor. To maximize the sensitivity and have enough detection area, minimum-size diodes (1 µm2) are arrayed in 64 × 64. Signal amplifiers, buffers, and on-chip memories are all integrated in the chip. For verification, PC3-PIP (prostate-specific membrane antigen [PSMA]+) and PC3-flu (PSMA-) cell lines are injected into the left and right flanks of the mice, respectively. The devices are inserted into each tumor and measure activities at 5 different time points (0-2 hours, 7-9 hours, 12-14 hours, 24-26 hours, and 48-50 hours) after 177Lu-PSMA-617 injections. Single-photon emission computed tomography/computed tomography scans are used to verify measured data. RESULTS: With a wide detection range from 0.013 to 8.95 MBq/mL, the system is capable of detecting high tumor uptake as well as low doses delivered to organs at risk in real time. The measurement data are highly proportional (R2 > 0.99) to the 177Lu-PSMA-617 activity. The in vivo measurement data agree well with the single-photon emission computed tomography/computed tomography results within acceptable errors (±1.5%ID/mL). CONCLUSIONS: Given the recent advances in clinical use of TRT in prostate cancer, the proposed system is verified in a prostate cancer mouse model using 177Lu-PSMA-617.

Chemoenzymatic Syntheses of Fluorine-18-Labeled Disaccharides from [18F] FDG Yield Potent Sensors of Living Bacteria In Vivo.

Chemoenzymatic techniques have been applied extensively to pharmaceutical development, most effectively when routine synthetic methods fail. The regioselective and stereoselective construction of structurally complex glycans is an elegant application of this approach that is seldom applied to positron emission tomography (PET) tracers. We sought a method to dimerize 2-deoxy-[18F]-fluoro-d-glucose ([18F]FDG), the most common tracer used in clinical imaging, to form [18F]-labeled disaccharides for detecting microorganisms in vivo based on their bacteria-specific glycan incorporation. When [18F]FDG was reacted with ß-d-glucose-1-phosphate in the presence of maltose phosphorylase, the α-1,4- and α-1,3-linked products 2-deoxy-[18F]-fluoro-maltose ([18F]FDM) and 2-deoxy-2-[18F]-fluoro-sakebiose ([18F]FSK) were obtained. This method was further extended with the use of trehalose (α,α-1,1), laminaribiose (ß-1,3), and cellobiose (ß-1,4) phosphorylases to synthesize 2-deoxy-2-[18F]fluoro-trehalose ([18F]FDT), 2-deoxy-2-[18F]fluoro-laminaribiose ([18F]FDL), and 2-deoxy-2-[18F]fluoro-cellobiose ([18F]FDC). We subsequently tested [18F]FDM and [18F]FSK in vitro, showing accumulation by several clinically relevant pathogens including Staphylococcus aureus and Acinetobacter baumannii, and demonstrated their specific uptake in vivo. Both [18F]FDM and [18F]FSK were stable in human serum with high accumulation in preclinical infection models. The synthetic ease and high sensitivity of [18F]FDM and [18F]FSK to S. aureus including methicillin-resistant (MRSA) strains strongly justify clinical translation of these tracers to infected patients. Furthermore, this work suggests that chemoenzymatic radiosyntheses of complex [18F]FDG-derived oligomers will afford a wide array of PET radiotracers for infectious and oncologic applications.

Covalent Proteins as Targeted Radionuclide Therapies Enhance Antitumor Effects.

Molecularly targeted radionuclide therapies (TRTs) struggle with balancing efficacy and safety, as current strategies to increase tumor absorption often alter drug pharmacokinetics to prolong circulation and normal tissue irradiation. Here we report the first covalent protein TRT, which, through reacting with the target irreversibly, increases radioactive dose to the tumor without altering the drug's pharmacokinetic profile or normal tissue biodistribution. Through genetic code expansion, we engineered a latent bioreactive amino acid into a nanobody, which binds to its target protein and forms a covalent linkage via the proximity-enabled reactivity, cross-linking the target irreversibly in vitro, on cancer cells, and on tumors in vivo. The radiolabeled covalent nanobody markedly increases radioisotope levels in tumors and extends tumor residence time while maintaining rapid systemic clearance. Furthermore, the covalent nanobody conjugated to the α-emitter actinium-225 inhibits tumor growth more effectively than the noncovalent nanobody without causing tissue toxicity. Shifting the protein-based TRT from noncovalent to covalent mode, this chemical strategy improves tumor responses to TRTs and can be readily scaled to diverse protein radiopharmaceuticals engaging broad tumor targets.

Chemoenzymatic syntheses of fluorine-18-labeled disaccharides from [ 18 F]FDG yield potent sensors of living bacteria in vivo.

Chemoenzymatic techniques have been applied extensively to pharmaceutical development, most effectively when routine synthetic methods fail. The regioselective and stereoselective construction of structurally complex glycans is an elegant application of this approach, that is seldom applied to positron emission tomography (PET) tracers. We sought a method to dimerize 2-deoxy-[ 18 F]-fluoro-D-glucose ([ 18 F]FDG), the most common tracer used in clinical imaging, to form [ 18 F]-labeled disaccharides for detecting microorganisms in vivo based on their bacteria-specific glycan incorporation. When [ 18 F]FDG was reacted with ß-D-glucose-1-phosphate in the presence of maltose phosphorylase, both the α-1,4 and α-1,3-linked products 2-deoxy-[ 18 F]-fluoro-maltose ([ 18 F]FDM) and 2-deoxy-2-[ 18 F]-fluoro-sakebiose ([ 18 F]FSK) were obtained. This method was further extended with the use of trehalose (α,α-1,1), laminaribiose (ß-1,3), and cellobiose (ß-1,4) phosphorylases to synthesize 2-deoxy-2-[ 18 F]fluoro-trehalose ([ 18 F]FDT), 2-deoxy-2-[ 18 F]fluoro-laminaribiose ([ 18 F]FDL), and 2-deoxy-2-[ 18 F]fluoro-cellobiose ([ 18 F]FDC). We subsequently tested [ 18 F]FDM and [ 18 F]FSK in vitro, showing accumulation by several clinically relevant pathogens including Staphylococcus aureus and Acinetobacter baumannii, and demonstrated their specific uptake in vivo. The lead sakebiose-derived tracer [ 18 F]FSK was stable in human serum and showed high uptake in preclinical models of myositis and vertebral discitis-osteomyelitis. Both the synthetic ease, and high sensitivity of [ 18 F]FSK to S. aureus including methicillin-resistant (MRSA) strains strongly justify clinical translation of this tracer to infected patients. Furthermore, this work suggests that chemoenzymatic radiosyntheses of complex [ 18 F]FDG-derived oligomers will afford a wide array of PET radiotracers for infectious and oncologic applications.

Elevated labile iron in castration-resistant prostate cancer is targetable with ferrous iron-activatable antiandrogen therapy.

Clinical responses to second generation androgen signaling inhibitors (e.g., enzalutamide) in metastatic castration-resistant prostate cancer (mCRPC) are variable and transient, and are associated with dose limiting toxicities, including rare but severe CNS effects. We hypothesized that changes to iron metabolism coincident with more advanced disease might be leveraged for tumor-selective delivery of antiandrogen therapy. Using the recently described chemical probes SiRhoNox and 18F-TRX in mCRPC models, we found elevated Fe2+ to be a common feature of mCRPC in vitro and in vivo. We next synthesized ferrous-iron activatable drug conjugates of second and third-generation antiandrogens and found these conjugates possessed comparable or enhanced antiproliferative activity across mCRPC cell line models. Mouse pharmacokinetic studies showed that these prototype antiandrogen conjugates are stable in vivo and limited exposure to conjugate or free antiandrogen in the brain. Our results reveal elevated Fe2+ to be a feature of mCRPC that might be leveraged to improve the tolerability and efficacy of antiandrogen therapy.

Theranostic Targeting of CUB Domain-Containing Protein 1 (CDCP1) in Multiple Subtypes of Bladder Cancer.

PURPOSE: Despite recent approvals for checkpoint inhibitors and antibody-drug conjugates targeting NECTIN4 or TROP2, metastatic bladder cancer remains incurable and new treatment strategies are urgently needed. CUB domain-containing protein 1 (CDCP1) is a cell surface protein and promising drug target for many cancers. This study aimed to determine whether CDCP1 is expressed in bladder cancer and whether CDCP1 can be targeted for treatment with radiolabeled antibodies. EXPERIMENTAL DESIGN: CDCP1 expression was evaluated in four bladder cancer datasets (n = 1,047 biopsies). A tissue microarray of primary bladder cancer biopsies was probed for CDCP1 by IHC. CDCP1 expression was evaluated in patient-derived xenografts and cell lysates by immunoblot, flow cytometry, and saturation binding assays. Tumor detection in mouse bladder cancer models was tested using 89Zr-labeled 4A06, a monoclonal antibody targeting the ectodomain of CDCP1. 177Lu-4A06 was applied to mice bearing UMUC3 or HT-1376 xenografts to evaluate antitumor effects (CDCP1 expression in UMUC3 is 10-fold higher than HT-1376). RESULTS: CDCP1 was highest in the basal/squamous subtype, and CDCP1 was expressed in 53% of primary biopsies. CDCP1 was not correlated with pathologic or tumor stage, metastatic site, or NECTIN4 and TROP2 at the mRNA or protein level. CDCP1 ranged from 105 to 106 receptors per cell. Mechanism studies showed that RAS signaling induced CDCP1 expression. 89Zr-4A06 PET detected five human bladder cancer xenografts. 177Lu-4A06 inhibited the growth of UMUC3 and HT-1376 xenografts, models with high and moderate CDCP1 expression, respectively. CONCLUSIONS: These data establish that CDCP1 is expressed in bladder cancer, including TROP2 and NECTIN4-null disease, and suggest that bladder cancer can be treated with CDCP1-targeted radiotherapy.

Impact of Radiolabeling Strategies on the Pharmacokinetics and Distribution of an Anti-PD-L1 PET Ligand.

Molecular imaging with PET offers an alternative method to quantify programmed-death-ligand 1 (PD-L1) to accurately select patients for immunotherapies. More and more clinical and preclinical trials involve radiolabeling of antibody fragments for their desirably fast clearance and high tumor penetration. As the radiolabeling strategy can significantly impact pharmacokinetics and biodistribution, we explored in this work a site-specific radiofluorination strategy on an anti-PD-L1 fragment antigen-binding (Fab) and compared the pharmacokinetic and biodistribution properties with the same Fab labeled using stochastic radiolabeling chemistry. We applied an enzymatic bioconjugation mediated by a variant of the lipoic acid ligase (LplA) that promotes the formation of an amide bond between a short peptide cloned onto the C terminus of the Fab. A synthetic analogue of the enzyme natural substrate, lipoic acid, was radiolabeled with fluorine-18 for site-specific conjugation by LplA. We compared the biodistribution of the site-specifically labeled Fab with a stochastically labeled Fab on lysine side chains in tumor-bearing mice. The two methods of fluorination demonstrate a comparable whole-body biodistribution. The 89Zr-labeled Fab had different biodistribution compared to either 18F-labeled Fab. We attribute the difference to [89Zr] metabolism. Fab-LAP-[18F]FPyOctA therefore reflects better the true pharmacokinetic profile of the Fab.

Defining the Magnetic Resonance Features of Renal Lesions and Their Response to Everolimus in a Transgenic Mouse Model of Tuberous Sclerosis Complex.

Tuberous sclerosis complex (TSC) is an inherited genetic disorder characterized by mutations in TSC1 or TSC2 class of tumor suppressers which impact several organs including the kidney. The renal manifestations are usually in the form of angiomyolipoma (AML, in 80% of the cases) and cystadenomas. mTOR inhibitors such as rapamycin and everolimus have shown efficacy in reducing the renal tumor burden. Early treatment prevents the progression of AML; however, the tumors regrow upon cessation of therapy implying a lifelong need for monitoring and management of this morbid disease. There is a critical need for development of imaging strategies to monitor response to therapy and progression of disease which will also facilitate development of newer targeted therapy. In this study we evaluated the potential of multiparametric 1H magnetic resonance imaging (mpMRI) to monitor tumor response to therapy in a preclinical model of TSC, the transgenic mouse A/J Tsc2+/- . We found 2-dimensional T2-weighted sequence with 0.5 mm slice thickness to be optimal for detecting renal lesions as small as 0.016 mm3. Baseline characterization of lesions with MRI to assess physiological parameters such as cellularity and perfusion is critical for distinguishing between cystic and solid lesions. Everolimus treatment for three weeks maintained tumor growth at 36% from baseline, while control tumors displayed steady growth and were 70% larger than baseline at the end of therapy. Apparent diffusion coefficient, T1 values and normalized T2 intensity changes were also indictive of response to treatment. Our results indicate that standardization and implementation of improved MR imaging protocols will significantly enhance the utility of mpMRI in determining the severity and composition of renal lesions for better treatment planning.

CUB Domain-Containing Protein 1 (CDCP1) Is a Target for Radioligand Therapy in Castration-Resistant Prostate Cancer, including PSMA Null Disease.

PURPOSE: With the improvement in overall survival with 177Lu-PSMA 617, radioligand therapy (RLT) is now a viable option for patients with metastatic castration-resistant prostate cancer (mCRPC). However, responses are variable, in part due to low PSMA expression in 30% of patients. Herein, we evaluated whether the cell surface protein CUB domain-containing protein 1 (CDCP1) can be exploited to treat mCRPC with RLT, including in PSMA-low subsets. EXPERIMENTAL DESIGN: CDCP1 levels were evaluated using RNA sequencing from 119 mCRPC biopsies. CDCP1 levels were assessed in 17 post-enzalutamide- or abiraterone-treated mCRPC biopsies, 12 patient-derived xenografts (PDX), and prostate cancer cell lines. 4A06, a recombinant human antibody that targets the CDCP1 ectodomain, was labeled with Zr-89 or Lu-177 and tested in tumor-bearing mice. RESULTS: CDCP1 expression was observed in 90% of mCRPC biopsies, including small-cell neuroendocrine (SCNC) and adenocarcinomas with low FOLH1 (PSMA) levels. Fifteen of 17 evaluable mCRPC biopsies (85%) demonstrated membranous CDCP1 expression, and 4 of 17 (23%) had higher CDCP1 H-scores compared with PSMA. CDCP1 was expressed in 10 of 12 PDX samples. Bmax values of approximately 22,000, 6,200, and 2,800 fmol/mg were calculated for PC3, DU145, and C4-2B human prostate cancer cells, respectively. 89Zr-4A06 PET detected six human prostate cancer xenografts, including PSMA-low tumors. 177Lu-4A06 significantly suppressed growth of DU145 and C4-2B xenografts. CONCLUSIONS: The data provide the first evidence supporting CDCP1-directed RLT to treat mCRPC. Expanded studies are warranted to determine whether CDCP1 is a viable drug target for patients with mCPRC.

Ferrous iron-activatable drug conjugate achieves potent MAPK blockade in KRAS-driven tumors.

KRAS mutations drive a quarter of cancer mortality, and most are undruggable. Several inhibitors of the MAPK pathway are FDA approved but poorly tolerated at the doses needed to adequately extinguish RAS/RAF/MAPK signaling in the tumor cell. We found that oncogenic KRAS signaling induced ferrous iron (Fe2+) accumulation early in and throughout mutant KRAS-mediated transformation. We converted an FDA-approved MEK inhibitor into a ferrous iron-activatable drug conjugate (FeADC) and achieved potent MAPK blockade in tumor cells while sparing normal tissues. This innovation allowed sustainable, effective treatment of tumor-bearing animals, with tumor-selective drug activation, producing superior systemic tolerability. Ferrous iron accumulation is an exploitable feature of KRAS transformation, and FeADCs hold promise for improving the treatment of KRAS-driven solid tumors.

Switchable assembly and function of antibody complexes in vivo using a small molecule.

The antigen specificity and long serum half-life of monoclonal antibodies have made them a critical part of modern therapeutics. These properties have been coopted in a number of synthetic formats, such as antibody-drug conjugates, bispecific antibodies, or Fc-fusion proteins to generate novel biologic drug modalities. Historically, these new therapies have been generated by covalently linking multiple molecular moieties through chemical or genetic methods. This irreversible fusion of different components means that the function of the molecule is static, as determined by the structure. Here, we report the development of a technology for switchable assembly of functional antibody complexes using chemically induced dimerization domains. This approach enables control of the antibody's intended function in vivo by modulating the dose of a small molecule. We demonstrate this switchable assembly across three therapeutically relevant functionalities in vivo, including localization of a radionuclide-conjugated antibody to an antigen-positive tumor, extension of a cytokine's half-life, and activation of bispecific, T cell-engaging antibodies.

Targeting a proteolytic neoepitope on CUB domain containing protein 1 (CDCP1) for RAS-driven cancers.

Extracellular proteolysis is frequently dysregulated in disease and can generate proteoforms with unique neoepitopes not found in healthy tissue. Here, we demonstrate that Abs that selectively recognize a proteolytic neoepitope on CUB domain containing protein 1 (CDCP1) could enable more effective and safer treatments for solid tumors. CDCP1 is highly overexpressed in RAS-driven cancers, and its ectodomain is cleaved by extracellular proteases. Biochemical, biophysical, and structural characterization revealed that the 2 cleaved fragments of CDCP1 remain tightly associated with minimal proteolysis-induced conformational change. Using differential phage display, we generated recombinant Abs that are exquisitely selective to cleaved CDCP1 with no detectable binding to the uncleaved form. These Abs potently targeted cleaved CDCP1-expressing cancer cells as an Ab-drug conjugate, an Ab-radionuclide conjugate, and a bispecific T cell engager. In a syngeneic pancreatic tumor model, these cleaved-specific Abs showed tumor-specific localization and antitumor activity with superior safety profiles compared with a pan-CDCP1 approach. Targeting proteolytic neoepitopes could provide an orthogonal "AND" gate for improving the therapeutic index.

In Vivo Profiling with 18F-YJH08 Reveals Diverse Tissue Patterns of Antagonist/Glucocorticoid Receptor Interactions.

Demonstrating target engagement in vivo is an important milestone in drug development, both to establish on target, on tissue interactions but also to identify potentially undesirable off tissue binding. The glucocorticoid receptor (GR) is a long-studied yet vexing drug target that has recently re-emerged as a potential druggable driver of many solid tumor types including breast and prostate cancer, and several antagonists are currently in early phase clinical trials. Since GR is also ubiquitously expressed in normal tissues, understanding antagonist/GR interactions in normal tissues and tumor is crucial to defining a therapeutic index. Herein, we demonstrate that the GR radioligand 18F-YJH08 can map drug/GR engagement in vivo. Profiling target engagement in vivo showed that the GR antagonists RU486 (mifepristone) and CORT125281 engaged GR in fewer normal tissues compared to ORIC-101 or the agonist dexamethasone. Furthermore, 18F-YJH08 detected GR in human prostate cancer tumor models and measured receptor binding by RU486. In summary, these data show for the first time that antagonist/GR interactions can be measured in vivo with 18F-YJH08, a finding with clinical relevance as GR antagonists and 11C-YJH08 are currently in clinical trials.

Preparation of Radiolabeled Antibodies for Nuclear Medicine Applications in Immuno-Oncology.

The mixed patient responses to antibodies targeting immune checkpoint proteins (e.g., CTLA-4, PD-1, PD-L1) have generated tremendous interest in discovering biomarkers that predict which patients will best respond to these treatments. To complement molecular biomarkers obtained from biopsies, the nuclear medicine community has begun developing radiopharmaceuticals that may provide a more holistic assessment of the biological character of all disease sites in patients. On the leading edge of clinical translation are a spectrum of radiolabeled antibodies targeting immune checkpoint proteins or T cell-specific antigens. The adoption of these reagents requires development of efficient and versatile methods for antibody bioconjugation and radiochemistry. We report herein protocols for the preparation of an anti-PD-L1 IgG1 (termed C4) labeled with zirconium-89. The approach is time and cost economical, high yielding, and adaptable to numerous antibody clones and platforms of interest to the immune-oncology community. Included also are representative methods for characterizing the pharmacology of the antibody post bioconjugation, and conducting an in vivo assessment of radiotracer biodistribution in tumor bearing mouse models.

Optimizing Immuno-PET Imaging of Tumor PD-L1 Expression: Pharmacokinetic, Biodistribution, and Dosimetric Comparisons of 89Zr-Labeled Anti-PD-L1 Antibody Formats.

PET imaging of programmed cell death ligand 1 (PD-L1) may help to noninvasively predict and monitor responses to anti-programmed cell death 1/anti-PD-L1 immunotherapies. In this study, we compared the imaging characteristics of 3 radioligands derived from the anti-PD-L1 IgG1 complement 4 (C4). In addition to the IgG C4, we produced a fragment antigen-binding (Fab) C4, as well as a double-mutant IgG C4 (H310A/H435Q) with minimal affinity for the murine neonatal Fc receptor. Methods: The pharmacokinetics, biodistribution, and dosimetry of the 3 89Zr-labeled C4 ligands were compared by longitudinal PET/CT imaging in nude mice bearing subcutaneous human non-small cell lung cancer xenografts with positive (H1975 model) or negative (A549 model) endogenous PD-L1 expression. Results: The C4 radioligands substantially accumulated in PD-L1-positive tumors but not in PD-L1-negative tumors or in blocked PD-L1-positive tumors, confirming their PD-L1-specific tumor targeting. 89Zr-Fab C4 and 89Zr-IgG C4 (H310A/H435Q) were rapidly eliminated compared with 89Zr-IgG C4. Consequently, maximal tumor-to-muscle ratios were obtained earlier, at 4 h after injection for 89Zr-Fab C4 (ratio, ∼6) and 24 h after injection for 89Zr-IgG C4 (H310A/H435Q) (ratio, ∼9), versus 48 h after injection for 89Zr-IgG C4 (ratio, ∼8). Background activity in nontumor tissues was low, except for high kidney retention of 89Zr-Fab C4 and persistent liver accumulation of 89Zr-IgG C4 (H310A/H435Q) compared with 89Zr-IgG C4. Dosimetry estimates suggested that the C4 radioligands would yield organ-absorbed doses tolerable for repeated clinical PET imaging studies. Conclusion: This study highlights the potential of designing radioligands with shorter pharmacokinetics for PD-L1 immuno-PET imaging in a preclinical model and encourages further clinical translation of such radioligands.

Synthesis and Preliminary Biological Assessment of Carborane-Loaded Theranostic Nanoparticles to Target Prostate-Specific Membrane Antigen.

Boron neutron capture therapy (BNCT) is an encouraging therapeutic modality for cancer treatment. Prostate-specific membrane antigen (PSMA) is a cell membrane protein that is abundantly overexpressed in prostate cancer and can be targeted with radioligand therapies to stimulate clinical responses in patients. In principle, a spatially targeted neutron beam together with specifically targeted PSMA ligands could enable prostate cancer-targeted BNCT. Thus, we developed and tested PSMA-targeted poly(lactide-co-glycolide)-block-poly(ethylene glycol) (PLGA-b-PEG) nanoparticles (NPs) loaded with carborane and tethered to the radiometal chelator deferoxamine B (DFB) for simultaneous positron emission tomography (PET) imaging and selective delivery of boron to prostate cancer. Monomeric PLGA-b-PEGs were covalently functionalized with either DFB or the PSMA ligand ACUPA. Different nanoparticle formulations were generated by nanoemulsification of the corresponding unmodified and DFB- or ACUPA-modified monomers in varying percent fractions. The nanoparticles were efficiently labeled with 89Zr and were subjected to in vitro and in vivo evaluation. The optimized DFB(25)ACUPA(75) NPs exhibited strong in vitro binding to PSMA in direct binding and competition radioligand binding assays in PSMA(+) PC3-Pip cells. [89Zr]DFB(25) NPs and [89Zr]DFB(25)ACUPA(75) NPs were injected to mice with bilateral PSMA(-) PC3-Flu and PSMA(+) PC3-Pip dual xenografts. The NPs demonstrated twofold superior accumulation in PC3-Pip tumors to that of PC3-Flu tumors with a tumor/blood ratio of 25; however, no substantial effect of the ACUPA ligands was detected. Moreover, fast release of carborane from the NPs was observed, resulting in a low boron delivery to tumors in vivo. In summary, these data demonstrate the synthesis, characterization, and initial biological assessment of PSMA-targeted, carborane-loaded PLGA-b-PEG nanoparticles and establish the foundation for future efforts to enable their best use in vivo.

In Vivo Measurement of Granzyme Proteolysis from Activated Immune Cells with PET.

The biology of human granzymes remains enigmatic in part due to our inability to probe their functions outside of in vitro assays or animal models with divergent granzyme species. We hypothesize that the biology of human granzymes could be better elaborated with a translational imaging technology to reveal the contexts in which granzymes are secreted and biochemically active in vivo. Here, we advance toward this goal by engineering a Granzyme targeting Restricted Interaction Peptide specific to family member B (GRIP B) to measure secreted granzyme B (GZMB) biochemistry with positron emission tomography. A proteolytic cleavage of 64Cu-labeled GRIP B liberates a radiolabeled form of Temporin L, which sequesters the radioisotope by binding to adjacent phospholipid bilayers. Thus, at extended time points postinjection (i.e., hours, not seconds), tissue biodistribution of the radioisotope in vivo reflects relative units of the GZMB activity. As a proof of concept, we show in three syngeneic mouse cancer models that 64Cu-GRIP B detects GZMB from T cells activated with immune checkpoint inhibitors (CPI). Remarkably, the radiotracer detects the proteolysis within tumors but also in lymphoid tissue, where immune cells are activated by a systemic CPI. Control experiments with an uncleavable analogue of 64Cu-GRIP B and tumor imaging studies in germline GZMB knockout mice were applied to show that 64Cu-GRIP B is specific for GZMB proteolysis. Furthermore, we explored a potential noncytotoxic function for GZMB by applying 64Cu-GRIP B to a model of pulmonary inflammation. In summary, we demonstrate that granzyme biochemistry can be assessed in vivo using an imaging modality that can be scaled vertically into human subjects.

Quantitative Tissue Pharmacokinetics and EPR Effect of AGuIX Nanoparticles: A Multimodal Imaging Study in an Orthotopic Glioblastoma Rat Model and Healthy Macaque.

AGuIX are emerging radiosensitizing nanoparticles (NPs) for precision radiotherapy (RT) under clinical evaluation (Phase 2). Despite being accompanied by MRI thanks to the presence of gadolinium (Gd) at its surface, more sensitive and quantifiable imaging technique should further leverage the full potential of this technology. In this study, it is shown that 89 Zr can be labeled on such NPs directly for positron emission tomography (PET) imaging with a simple and scalable method. The stability of such complexes is remarkable in vitro and in vivo. Using a glioblastoma orthotopic rat model, it is shown that injected 89 Zr-AGuIX is detectable inside the tumor for at least 1 week. Interestingly, the particles seem to efficiently infiltrate the tumor even in necrotic areas, which places great hope for the treatment of radioresistant tumor. Lastly, the first PET/MR whole-body imaging is performed in non-human primate (NHP), which further demonstrates the translational potential of these bimodal NP.

Synthesis and Screening of α-Xylosides in Human Glioblastoma Cells.

Glycosaminoglycans (GAGs) such as heparan sulfate and chondroitin sulfate decorate all mammalian cell surfaces. These mucopolysaccharides act as coreceptors for extracellular ligands, regulating cell signaling, growth, proliferation, and adhesion. In glioblastoma, the most common type of primary malignant brain tumor, dysregulated GAG biosynthesis results in altered chain length, sulfation patterns, and the ratio of contributing monosaccharides. These events contribute to the loss of normal cellular function, initiating and sustaining malignant growth. Disruption of the aberrant cell surface GAGs with small molecule inhibitors of GAG biosynthetic enzymes is a potential therapeutic approach to blocking the rogue signaling and proliferation in glioma, including glioblastoma. Previously, 4-azido-xylose-α-UDP sugar inhibited both xylosyltransferase (XYLT-1) and ß-1,4-galactosyltransferase-7 (ß-GALT-7)-the first and second enzymes of GAG biosynthesis-when microinjected into a cell. In another study, 4-deoxy-4-fluoro-ß-xylosides inhibited ß-GALT-7 at 1 mM concentration in vitro. In this work, we seek to solve the enduring problem of drug delivery to human glioma cells at low concentrations. We developed a library of hydrophobic, presumed prodrugs 4-deoxy-4-fluoro-2,3-dibenzoyl-(α- or ß-) xylosides and their corresponding hydrophilic inhibitors of XYLT-1 and ß-GALT-7 enzymes. The prodrugs were designed to be activatable by carboxylesterase enzymes overexpressed in glioblastoma. Using a colorimetric MTT assay in human glioblastoma cell lines, we identified a prodrug-drug pair (4-nitrophenyl-α-xylosides) as lead drug candidates. The candidates arrest U251 cell growth at an IC50 = 380 nM (prodrug), 122 µM (drug), and U87 cells at IC50 = 10.57 µM (prodrug). Molecular docking studies were consistent with preferred binding of the α- versus ß-nitro xyloside conformer to XYLT-1 and ß-GALT-7 enzymes.

Molecular Imaging of Prostate Cancer Targeting CD46 Using ImmunoPET.

PURPOSE: We recently identified CD46 as a novel therapeutic target in prostate cancer. In this study, we developed a CD46-targeted PET radiopharmaceutical, [89Zr]DFO-YS5, and evaluated its performance for immunoPET imaging in murine prostate cancer models. EXPERIMENTAL DESIGN: [89Zr]DFO-YS5 was prepared and its in vitro binding affinity for CD46 was measured. ImmunoPET imaging was conducted in male athymic nu/nu mice bearing DU145 [AR-, CD46+, prostate-specific membrane antigen-negative (PSMA-)] or 22Rv1 (AR+, CD46+, PSMA+) tumors, and in NOD/SCID gamma mice bearing patient-derived adenocarcinoma xenograft, LTL-331, and neuroendocrine prostate cancers, LTL-331R and LTL-545. RESULTS: [89Zr]DFO-YS5 binds specifically to the CD46-positive human prostate cancer DU145 and 22Rv1 xenografts. In biodistribution studies, the tumor uptake of [89Zr]DFO-YS5 was 13.3 ± 3.9 and 11.2 ± 2.5 %ID/g, respectively, in DU145 and 22Rv1 xenografts, 4 days postinjection. Notably, [89Zr]DFO-YS5 demonstrated specific uptake in the PSMA- and AR-negative DU145 model. [89Zr]DFO-YS5 also showed uptake in the patient-derived LTL-331 and -331R models, with particularly high uptake in the LTL-545 neuroendocrine prostate cancer tumors (18.8 ± 5.3, 12.5 ± 1.8, and 32 ± 5.3 %ID/g in LTL-331, LTL-331R, and LTL-545, respectively, at 4 days postinjection). CONCLUSIONS: [89Zr]DFO-YS5 is an excellent PET imaging agent across a panel of prostate cancer models, including in both adenocarcinoma and neuroendocrine prostate cancer, both cell line- and patient-derived xenografts, and both PSMA-positive and -negative tumors. It demonstrates potential for clinical translation as an imaging agent, theranostic platform, and companion biomarker in prostate cancer.