Targeting both GD2 and B7-H3 using bispecific antibody improves tumor selectivity for GD2-positive tumors.

Objectives: Disialoganglioside 2 (GD2), overexpressed by cancers such as melanoma and neuroblastoma, is a tumor antigen for targeted therapy. The delivery of conventional IgG antibody technologies targeting GD2 is limited clinically by its co-expression on nerves that contributes to toxicity presenting as severe neuropathic pain. To improve the tumor selectivity of current GD2-targeting approaches, a next-generation bispecific antibody targeting GD2 and B7-H3 (CD276) was generated. Methods: Differential expression of human B7-H3 (hB7-H3) was transduced into GD2+ B78 murine melanoma cells and confirmed by flow cytometry. We assessed the avidity and selectivity of our GD2-B7-H3 targeting bispecific antibodies (INV34-6, INV33-2, and INV36-6) towards GD2+/hB7-H3- B78 cells relative to GD2+/hB7-H3+ B78 cells using flow cytometry and competition binding assays, comparing results an anti-GD2 antibody (dinutuximab, DINU). The bispecific antibodies, DINU, and a non-targeted bispecific control (bsAb CTRL) were conjugated with deferoxamine for radiolabeling with Zr-89 (t1/2 = 78.4 h). Using positron emission tomography (PET) studies, we evaluated the in vivo avidity and selectivity of the GD2-B7-H3 targeting bispecific compared to bsAb CTRL and DINU using GD2+/hB7-H3+ and GD2+/hB7-H3- B78 tumor models. Results: Flow cytometry and competition binding assays showed that INV34-6 bound with high avidity to GD2+/hB7-H3+ B78 cells with high avidity but not GD2+/hB7-H3+ B78 cells. In comparison, no selectivity between cell types was observed for DINU. PET in mice bearing the GD2+/hB7-H3- and GD2+/hB7-H3+ B78 murine tumor showed similar biodistribution in normal tissues for [89Zr]Zr-Df-INV34-6, [89Zr]Zr-Df-bsAb CTRL, and [89Zr]Zr-Df-DINU. Importantly, [89Zr]Zr-Df-INV34-6 tumor uptake was selective to GD2+/hB7-H3+ B78 over GD2+/hB7-H3- B78 tumors, and substantially higher to GD2+/hB7-H3+ B78 than the non-targeted [89Zr]Zr-Df-bsAb CTRL control. [89Zr]Zr-Df-DINU displayed similar uptake in both GD2+ tumor models, with uptake comparable to [89Zr]Zr-Df-INV34-6 in the GD2+/hB7-H3+ B78 model. Conclusion: The GD2-B7-H3 targeting bispecific antibodies successfully improved selectivity to cells expressing both antigens. This approach should address the severe toxicities associated with GD2-targeting therapies by reducing off-tumor GD2 binding in nerves. Continued improvements in bispecific antibody technologies will continue to transform the therapeutic biologics landscape.

Synthesis of 64Cu-, 55Co-, and 68Ga-Labeled Radiopharmaceuticals Targeting Neurotensin Receptor-1 for Theranostics: Adjusting In Vivo Distribution Using Multiamine Macrocycles.

The development of theranostic radiotracers relies on their binding to specific molecular markers of a particular disease and the use of corresponding radiopharmaceutical pairs thereafter. This study reports the use of multiamine macrocyclic moieties (MAs), as linkers or chelators, in tracers targeting the neurotensin receptor-1 (NTSR-1). The goal is to achieve elevated tumor uptake, minimal background interference, and prolonged tumor retention in NTSR-1-positive tumors. Methods: We synthesized a series of neurotensin antagonists bearing MA linkers and metal chelators. The MA unit is hypothesized to establish a strong interaction with the cell membrane, and the addition of a second chelator may enhance water solubility, consequently reducing liver uptake. Small-animal PET/CT imaging of [64Cu]Cu-DOTA-SR-3MA, [64Cu]Cu-NT-CB-NOTA, [68Ga]Ga-NT-CB-NOTA, [64Cu]Cu-NT-CB-DOTA, and [64Cu]Cu-NT-Sarcage was acquired at 1, 4, 24, and 48 h after injection using H1299 tumor models. [55Co]Co-NT-CB-NOTA was also tested in HT29 (high NTSR-1 expression) and Caco2 (low NTSR-1 expression) colorectal adenocarcinoma tumor models. Saturation binding assay and internalization of [55Co]Co-NT-CB-NOTA were used to test tracer specificity and internalization in HT29 cells. Results: In vivo PET imaging with [64Cu]Cu-NT-CB-NOTA, [68Ga]Ga-NT-CB-NOTA, and [55Co]Co-NT-CB-NOTA revealed high tumor uptake, high tumor-to-background contrast, and sustained tumor retention (≤48 h after injection) in NTSR-1-positive tumors. Tumor uptake of [64Cu]Cu-NT-CB-NOTA remained at 76.9% at 48 h after injection compared with uptake 1 h after injection in H1299 tumor models, and [55Co]Co-NT-CB-NOTA was retained at 60.2% at 24 h compared with uptake 1 h after injection in HT29 tumor models. [64Cu]Cu-NT-Sarcage also showed high tumor uptake with low background and high tumor retention 48 h after injection Conclusion: Tumor uptake and pharmacokinetic properties of NTSR-1-targeting radiopharmaceuticals were greatly improved when attached with different nitrogen-containing macrocyclic moieties. The study results suggest that NT-CB-NOTA labeled with either 64Cu/67Cu, 55Co/58mCo, or 68Ga (effect of 177Lu in tumor to be determined in future studies) and NT-Sarcage labeled with 64Cu/67Cu or 55Co/58mCo may be excellent diagnostic and therapeutic radiopharmaceuticals targeting NTSR-1-positive cancers. Also, the introduction of MA units to other ligands is warranted in future studies to test the generality of this approach.

"Off-Label Use" of the Siderophore Enterobactin Enables Targeted Imaging of Cancer with Radioactive Ti(IV).

The development of inert, biocompatible chelation methods is required to harness the emerging positron emitting radionuclide 45Ti for radiopharmaceutical applications. Herein, we evaluate the Ti(IV)-coordination chemistry of four catechol-based, hexacoordinate chelators using synthetic, structural, computational, and radiochemical approaches. The siderophore enterobactin (Ent) and its synthetic mimic TREN-CAM readily form mononuclear Ti(IV) species in aqueous solution at neutral pH. Radiolabeling studies reveal that Ent and TREN-CAM form mononuclear complexes with the short-lived, positron-emitting radionuclide 45Ti(IV), and do not transchelate to plasma proteins in vitro and exhibit rapid renal clearance in naïve mice. These features guide efforts to target the 45Ti isotope to prostate cancer tissue through the design, synthesis, and evaluation of Ent-DUPA, a small molecule conjugate composed of a prostate specific membrane antigen (PSMA) targeting peptide and a monofunctionalized Ent scaffold. The [45Ti][Ti(Ent-DUPA)]2- complex forms readily at room temperature. In a tumor xenograft model in mice, selective tumor tissue accumulation (8±5 %, n=5), and low off-target uptake in other organs is observed. Overall, this work demonstrates targeted imaging with 45Ti(IV), provides a foundation for advancing the application of 45Ti in nuclear medicine, and reveals that Ent can be repurposed as a 45Ti-complexing cargo for targeted nuclear imaging applications.

Radiolabeling Diaminosarcophagine with Cyclotron-Produced Cobalt-55 and [55Co]Co-NT-Sarcage as a Proof of Concept in a Murine Xenograft Model.

Cobalt-sarcophagine complexes exhibit high kinetic inertness under various stringent conditions, but there is limited literature on radiolabeling and in vivo positron emission tomography (PET) imaging using no carrier added 55Co. To fill this gap, this study first investigates the radiolabeling of DiAmSar (DSar) with 55Co, followed by stability evaluation in human serum and EDTA, pharmacokinetics in mice, and a direct comparison with [55Co]CoCl2 to assess differences in pharmacokinetics. Furthermore, the radiolabeling process was successfully used to generate the NTSR1-targeted PET agent [55Co]Co-NT-Sarcage (a DSar-functionalized SR142948 derivative) and administered to HT29 tumor xenografted mice. The [55Co]Co-DSar complex can be formed at 37 °C with purity and stability suitable for preclinical in vivo radiopharmaceutical applications, and [55Co]Co-NT-Sarcage demonstrated prominent tumor uptake with a low background signal. In a direct comparison with [64Cu]Cu-NT-Sarcage, [55Co]Co-NT-Sarcage achieved a higher tumor-to-liver ratio but with overall similar biodistribution profile. These results demonstrate that Sar would be a promising chelator for constructing Co-based radiopharmaceuticals including 55Co for PET and 58mCo for therapeutic applications.

Amplification of Cerenkov luminescence using semiconducting polymers for cancer theranostics.

The therapeutic efficacy of photodynamic therapy is limited by the ability of light to penetrate tissues. Due to this limitation, Cerenkov luminescence (CL) from radionuclides has recently been proposed as an alternative light source in a strategy referred to as Cerenkov radiation induced therapy (CRIT). Semiconducting polymer nanoparticles (SPNs) have ideal optical properties, such as large absorption cross-sections and broad absorbance, which can be utilized to harness the relatively weak CL produced by radionuclides. SPNs can be doped with photosensitizers and have nearly 100% energy transfer efficiency by multiple energy transfer mechanisms. Herein, we investigated an optimized photosensitizer doped SPN as a nanosystem to harness and amplify CL for cancer theranostics. We found that semiconducting polymers significantly amplified CL energy transfer efficiency. Bimodal PET and optical imaging studies showed high tumor uptake and retention of the optimized SPNs when administered intravenously or intratumorally. Lastly, we found that photosensitizer doped SPNs have excellent potential as a cancer theranostics nanosystem in an in vivo tumor therapy study. Our study shows that SPNs are ideally suited to harness and amplify CL for cancer theranostics, which may provide a significant advancement for CRIT that are unabated by tissue penetration limits.

Separation of cyclotron-produced cobalt-55/58m from iron targets using cation exchange chromatography with non-aqueous solvents and extraction chromatography.

Cobalt-55 and -58m form a theranostic pair that has relevant properties for cancer research. We report a cation exchange chromatography/extraction chromatography method that separates cyclotron-produced 55/58mCo from 54/57Fe in <1.5 h, recovers >85% Co and achieves [55Co]Co-NOTA and -DOTA AMA 89 ± 48 and 35 ± 7 MBq/nmol (EOB), respectively. Cobalt-55 and -58m were quantitatively labeled to functionalized NOTA at 106 and 50 MBq/nmol (EOB), respectively, corroborating measured AMA. This method is faster than previously published methods and achieves better [55/58mCo]Co-NOTA and -DOTA AMA.

Copper-Mediated Radiobromination of (Hetero)Aryl Boronic Pinacol Esters.

A copper-mediated radiobromination of (hetero)aryl boronic pinacol esters is described. Cyclotron-produced [76/77Br]bromide was isolated using an anion exchange cartridge, wherein the pre-equilibration and elution solutions played a critical role in downstream deboro-bromination. The bromination tolerates a broad range of functional groups, labeling molecules with ranging electronic and steric effects. Bologically active radiopharmaceuticals were synthesized, including two radiobrominated inhibitors of poly ADP ribose polymerase, a clinically relevant chemotherapeutic target for ovarian, breast, and prostate cancers.

Theranostic cobalt-55/58m for neurotensin receptor-mediated radiotherapy in vivo: A pilot study with dosimetry.

Neurotensin receptor 1 (NTSR1) can stimulate tumor proliferation through neurotensin (NTS) activation and are overexpressed by a variety of cancers. The high binding affinity of NTS/NTSR1 makes radiolabeled NTS derivatives interesting for cancer diagnosis and staging. Internalization of NTS/NTSR1 also suggests therapeutic application with high LET alpha particles and low energy electrons. We investigated the therapeutic efficacy of [58mCo]Co-NOTA-NT-20.3 in vivo using murine models xenografted with NTSR1-positive HT29 human colorectal adenocarcinoma cells, and utilized [55Co]Co-NOTA-NT-20.3 for dosimetry. METHODS: Targeting properties and cytotoxicity of [55/58mCo]Co-NOTA-NT-20.3 were assessed with HT29 cells. Female nude mice were xenografted with HT29 tumors and administered [55Co or 58mCo]Co-NOTA-NT-20.3 to evaluate pharmacokinetics or for therapy, respectively. Dosimetry calculations followed the Medical Internal Radiation Dose (MIRD) formalism and human absorbed dose rate per unit activity were obtained from OpenDose. The pilot therapy study consisted of two groups (each N = 3) receiving 110 ± 15 MBq and 26 ± 6 MBq [58mCo]Co-NOTA-NT-20.3 one week after tumor inoculation, and control (N = 3). Tumor sizes and masses were measured twice a week after therapy. Complete blood count and kidney histology were also performed to assess toxicity. RESULTS: HPLC measured radiochemical purity of [55,58mCo]Co-NOTA-NT-20.3 > 99 %. Labeled compounds retained NTS targeting properties. [58mCo]Co-NOTA-NT-20.3 exhibited cytotoxicity for HT29 cells and was >15× more potent than [58mCo]CoCl2. Xenografted tumors responded modestly to administered doses, but mice showed no signs of radiotoxicity. Absorbed dose to tumor and kidney with 110 MBq [58mCo]Co-NOTA-NT-20.3 were 0.6 Gy and 0.8 Gy, respectively, and other organs received less than half of the absorbed dose to tumor. Off-target radiation dose from cobalt-58g was small but reduces the therapeutic window. CONCLUSION: The enhanced in vitro cytotoxicity and high tumor-to-background led us to investigate the therapeutic efficacy of [58mCo]Co-NOTA-NT-20.3 in vivo. Although we were unable to induce tumor response commensurate with [177Lu]Lu-NT127 (NLys-Lys-Pro-Tyr-Tle-Leu) studies involving similar time-integrated activity, the absence of observed toxicity may constitute an opportunity for targeting vectors with improved uptake and/or retention to avoid the aftereffects of other high-LET radioactive emissions. Future studies with higher uptake, activity and/or multiple dosing regimens are warranted. The theranostic approach employed in this work was crucial for dosimetry analysis.

Spleen-Targeted Glabridin-Loaded Nanoparticles Regulate Polarization of Monocyte/Macrophage (Mo /Mφ ) for the Treatment of Cerebral Ischemia-Reperfusion Injury.

During cerebral ischemia-reperfusion (I-R) injury, the infiltration of monocyte/macrophages (Mo /Mφ ) into the ischemic penumbra causes inflammatory damage but also regulates tissue repair in the penumbra. The regulation and balance of Mo /Mφ polarization is considered as a potential therapeutic target for treating cerebral I-R injury. Herein, these findings demonstrate that glabridin (Gla)-loaded nanoparticles (i.e., NPGla -5k) can effectively inhibit M1-polarization and enhance M2-polarization of Mo /Mφ . Positron emission tomography (PET) imaging shows that NPGla -5k can selectively accumulate in the spleen following intravenous injection. Spleen-targeted Cy5-NPGla -5k can co-localize with peripheral macrophages in the penumbra at 24 h after tail-vein injection. Interestingly, NPGla -5k treatment can reduce inflammatory damage, protect dying neurons, and improve nervous system function. The protective effect of spleen-targeted NPGla -5k against cerebral I-R injury in mice encourages an exploration of their use for clinical treatment of patients with cerebral I-R injury.

ImmunoPET of trophoblast cell-surface antigen 2 (Trop-2) expression in pancreatic cancer.

PURPOSE: Without a standard test for pancreatic carcinomas, this highly lethal disease is normally diagnosed at its advanced stage, leading to a low survival rate of patients. Trophoblast cell-surface antigen 2 (Trop-2), a transmembrane glycoprotein, is associated with cell proliferation and highly expressed in most of solid epithelial tumors, including pancreatic cancer. A non-invasive method of imaging Trop-2 would greatly benefit clinical diagnosis and monitoring of pancreatic cancer. In the current study, 89Zr-labeled anti-Trop-2 antibody (AF650) was recruited for the systemic evaluation of Trop-2 as an immunoPET target for pancreatic cancer imaging. METHODS: AF650 was conjugated with desferrioxamine (DFO) and then radiolabeled with 89Zr. Trop-2 expression levels were determined in three pancreatic cancer cell lines (BxPC-3, MIA PaCa-2, and AsPC-1) via western blot, flow cytometry, saturation binding assay, and immunofluorescence staining. The targeting capacity of 89Zr-DFO-AF650 was evaluated in mouse models with subcutaneous xenograft of pancreatic cancers via PET imaging and bio-distribution studies. In addition, a Trop-2-positive orthotopic cancer model was recruited for further validating the targeting specificity of 89Zr-DFO-AF650. RESULTS: BxPC-3 cells expressed high levels of Trop-2, while AsPC-1 and MIA PaCa-2 cells expressed low levels of Trop-2. Additionally, 89Zr-DFO-AF650 exhibited high specificity to Trop-2 in BxPC-3 cells (Kd = 22.34 ± 2.509 nM). In subcutaneous xenograft models, about 28.8 ± 7.63%ID/g tracer accumulated in the BxPC-3 tumors at 120 h post injection, which was much higher than those reaching MIA PaCa-2 (6.76 ± 2.08%ID/g) and AsPC-1 (3.51 ± 0.69%ID/g) tumors (n = 4). More importantly, 89Zr-DFO-AF650 could efficiently distinguish primary tumors in the orthotopic BxPC-3 cancer model, showing high correlation between PET imaging and bio-distribution and sensitivity. CONCLUSIONS: 89Zr-DFO-AF650 can be effectively used to detect pancreatic cancer via Trop-2-mediated immunoPET in vivo, clearly revealing the great potential of Trop-2-based non-invasive imaging in pancreatic cancer detection and treatment monitoring.

A High Separation Factor for 165Er from Ho for Targeted Radionuclide Therapy.

Background: Radionuclides emitting Auger electrons (AEs) with low (0.02-50 keV) energy, short (0.0007-40 µm) range, and high (1-10 keV/µm) linear energy transfer may have an important role in the targeted radionuclide therapy of metastatic and disseminated disease. Erbium-165 is a pure AE-emitting radionuclide that is chemically matched to clinical therapeutic radionuclide 177Lu, making it a useful tool for fundamental studies on the biological effects of AEs. This work develops new biomedical cyclotron irradiation and radiochemical isolation methods to produce 165Er suitable for targeted radionuclide therapeutic studies and characterizes a new such agent targeting prostate-specific membrane antigen. Methods: Biomedical cyclotrons proton-irradiated spot-welded Ho(m) targets to produce 165Er, which was isolated via cation exchange chromatography (AG 50W-X8, 200-400 mesh, 20 mL) using alpha-hydroxyisobutyrate (70 mM, pH 4.7) followed by LN2 (20-50 µm, 1.3 mL) and bDGA (50-100 µm, 0.2 mL) extraction chromatography. The purified 165Er was radiolabeled with standard radiometal chelators and used to produce and characterize a new AE-emitting radiopharmaceutical, [165Er]PSMA-617. Results: Irradiation of 80-180 mg natHo targets with 40 µA of 11-12.5 MeV protons produced 165Er at 20-30 MBq·µA-1·h-1. The 4.9 ± 0.7 h radiochemical isolation yielded 165Er in 0.01 M HCl (400 µL) with decay-corrected (DC) yield of 64 ± 2% and a Ho/165Er separation factor of (2.8 ± 1.1) · 105. Radiolabeling experiments synthesized [165Er]PSMA-617 at DC molar activities of 37-130 GBq·µmol-1. Conclusions: A 2 h biomedical cyclotron irradiation and 5 h radiochemical separation produced GBq-scale 165Er suitable for producing radiopharmaceuticals at molar activities satisfactory for investigations of targeted radionuclide therapeutics. This will enable fundamental radiation biology experiments of pure AE-emitting therapeutic radiopharmaceuticals such as [165Er]PSMA-617, which will be used to understand the impact of AEs in PSMA-targeted radionuclide therapy of prostate cancer.

CD38-Targeted Theranostics of Lymphoma with 89Zr/177Lu-Labeled Daratumumab.

Lymphoma is a heterogeneous disease with varying clinical manifestations and outcomes. Many subtypes of lymphoma, such as Burkitt's lymphoma and diffuse large B cell lymphoma, are highly aggressive with dismal prognosis even after conventional chemotherapy and radiotherapy. As such, exploring specific biomarkers for lymphoma is of high clinical significance. Herein, a potential marker, CD38, is investigated for differentiating lymphoma. A CD38-targeting monoclonal antibody (mAb, daratumumab) is then radiolabeled with Zr-89 and Lu-177 for theranostic applications. As the diagnostic component, the Zr-89-labeled mAb is highly specific in delineating CD38-positive lymphoma via positron emission tomography (PET) imaging, while the Lu-177-labeled mAb serves well as the therapeutic component to suppress tumor growth after a one-time administration. These results strongly suggest that CD38 is a lymphoma-specific marker and prove that 89Zr/177Lu-labeled daratumumab facilitates immunoPET imaging and radioimmunotherapy of lymphoma in preclinical models. Further clinical evaluation and translation of this CD38-targeted theranostics may be of significant help in lymphoma patient stratification and management.

Temporal analysis of type 1 interferon activation in tumor cells following external beam radiotherapy or targeted radionuclide therapy.

Rationale: Clinical interest in combining targeted radionuclide therapies (TRT) with immunotherapies is growing. External beam radiation therapy (EBRT) activates a type 1 interferon (IFN1) response mediated via stimulator of interferon genes (STING), and this is critical to its therapeutic interaction with immune checkpoint blockade. However, little is known about the time course of IFN1 activation after EBRT or whether this may be induced by decay of a TRT source. Methods: We examined the IFN1 response and expression of immune susceptibility markers in B78 and B16 melanomas and MOC2 head and neck cancer murine models using qPCR and western blot. For TRT, we used 90Y chelated to NM600, an alkylphosphocholine analog that exhibits selective uptake and retention in tumor cells including B78 and MOC2. Results: We observed significant IFN1 activation in all cell lines, with peak activation in B78, B16, and MOC2 cell lines occurring 7, 7, and 1 days, respectively, following RT for all doses. This effect was STING-dependent. Select IFN response genes remained upregulated at 14 days following RT. IFN1 activation following STING agonist treatment in vitro was identical to RT suggesting time course differences between cell lines were mediated by STING pathway kinetics and not DNA damage susceptibility. In vivo delivery of EBRT and TRT to B78 and MOC2 tumors resulted in a comparable time course and magnitude of IFN1 activation. In the MOC2 model, the combination of 90Y-NM600 and dual checkpoint blockade therapy reduced tumor growth and prolonged survival compared to single agent therapy and cumulative dose equivalent combination EBRT and dual checkpoint blockade therapy. Conclusions: We report the time course of the STING-dependent IFN1 response following radiation in multiple murine tumor models. We show the potential of TRT to stimulate IFN1 activation that is comparable to that observed with EBRT and this may be critical to the therapeutic integration of TRT with immunotherapies.

Low-Dose Radiation Potentiates the Propagation of Anti-Tumor Immunity against Melanoma Tumor in the Brain after In Situ Vaccination at a Tumor outside the Brain.

Brain metastases develop in over 60% of advanced melanoma patients and negatively impact quality of life and prognosis. In a murine melanoma model, we previously showed that an in situ vaccination (ISV) regimen, combining radiation treatment and intratumoral (IT) injection of immunocytokine (IC: anti-GD2 antibody fused to IL2), along with the immune checkpoint inhibitor anti-CTLA-4, robustly eliminates peripheral flank tumors but only has modest effects on co-occurring intracranial tumors. In this study, we investigated the ability of low-dose radiation to the brain to potentiate anti-tumor immunity against a brain tumor when combined with ISV + anti-CTLA-4. B78 (GD2+, immunologically "cold") melanoma tumor cells were implanted into the flank and the right striatum of the brain in C57BL/6 mice. Flank tumors (50-150 mm3) were treated following a previously optimized ISV regimen [radiation (12 Gy × 1, treatment day 1), IT-IC (50 µg daily, treatment days 6-10), and anti-CTLA-4 (100 µg, treatment days 3, 6, 9)]. Mice that additionally received whole-brain radiation treatment (WBRT, 4 Gy × 1) on day 15 demonstrated significantly increased survival compared to animals that received ISV + anti-CTLA-4 alone, WBRT alone or no treatment (control) (P < 0.001, log-rank test). Timing of WBRT was critical, as WBRT administration on day 1 did not significantly enhance survival compared to ISV + anti-CTLA-4, suggesting that the effect of WBRT on survival might be mediated through immune modulation and not just direct tumor cell cytotoxicity. Modest increases in T cells (CD8+ and CD4+) and monocytes/macrophages (F4/80+) but no changes in FOXP3+ regulatory T cells (Tregs), were observed in brain melanoma tumors with addition of WBRT (on day 15) to ISV + anti-CTLA-4. Cytokine multiplex immunoassay revealed distinct changes in both intracranial melanoma and contralateral normal brain with addition of WBRT (day 15) to ISV + anti-CTLA-4, with notable significant changes in pro-inflammatory (e.g., IFNγ, TNFα and LIX/CXCL5) and suppressive (e.g., IL10, IL13) cytokines as well as chemokines (e.g., IP-10/CXCL10 and MIG/CXCL9). We tested the ability of the alkylphosphocholine analog, NM600, to deliver immunomodulatory radiation to melanoma brain tumors as a targeted radionuclide therapy (TRT). Yttrium-86 (86Y) chelated to NM600 was delivered intravenously by tail vein to mice harboring flank and brain melanoma tumors, and PET imaging demonstrated specific accumulation up to 72 h at each tumor site (∼12:1 brain tumor/brain and ∼8:1 flank tumor/muscle). When NM600 was chelated to therapeutic ß-particle-emitting 90Y and administered on treatment day 13, T-cell infiltration and cytokine profiles were altered in melanoma brain tumor, like that observed for WBRT. Overall, our results demonstrate that addition of low-dose radiation, timed appropriately with ISV administration to tumors outside the brain, significantly increases survival in animals co-harboring melanoma brain tumors. This observation has potentially important translational implications as a treatment strategy for increasing the response of tumors in the brain to systemically administered immunotherapies.

Autologous transplant therapy alleviates motor and depressive behaviors in parkinsonian monkeys.

Degeneration of dopamine (DA) neurons in the midbrain underlies the pathogenesis of Parkinson's disease (PD). Supplement of DA via L-DOPA alleviates motor symptoms but does not prevent the progressive loss of DA neurons. A large body of experimental studies, including those in nonhuman primates, demonstrates that transplantation of fetal mesencephalic tissues improves motor symptoms in animals, which culminated in open-label and double-blinded clinical trials of fetal tissue transplantation for PD1. Unfortunately, the outcomes are mixed, primarily due to the undefined and unstandardized donor tissues1,2. Generation of induced pluripotent stem cells enables standardized and autologous transplantation therapy for PD. However, its efficacy, especially in primates, remains unclear. Here we show that over a 2-year period without immunosuppression, PD monkeys receiving autologous, but not allogenic, transplantation exhibited recovery from motor and depressive signs. These behavioral improvements were accompanied by robust grafts with extensive DA neuron axon growth as well as strong DA activity in positron emission tomography (PET). Mathematical modeling reveals correlations between the number of surviving DA neurons with PET signal intensity and behavior recovery regardless autologous or allogeneic transplant, suggesting a predictive power of PET and motor behaviors for surviving DA neuron number.

ImmunoPET/NIRF/Cerenkov multimodality imaging of ICAM-1 in pancreatic ductal adenocarcinoma.

PURPOSE: We dual-labeled an intercellular adhesion molecule-1 (ICAM-1) monoclonal antibody (mAb) and evaluated its effectiveness for lesion detection and surgical navigation in pancreatic ductal adenocarcinoma (PDAC) via multiple noninvasive imaging approaches, including positron emission tomography (PET), near-infrared fluorescence (NIRF), and Cerenkov luminescence imaging (CLI). METHODS: ICAM-1 expression in PDAC cell lines (BxPC-3 and AsPC-1) was assessed via flow cytometry and immunofluorescent staining. An ICAM-1 mAb labeled by IRDye 800CW and radionuclide zirconium-89 (denoted as [89Zr]Zr-DFO-ICAM-1-IR800) was synthesized. Its performance was validated via in vivo comparative PET/NIRF/CLI and biodistribution (Bio-D) studies in nude mice bearing subcutaneous BxPC-3/AsPC-1 tumors or orthotopic BxPC-3 tumor models using nonspecific IgG as an isotype control tracer. RESULTS: ICAM-1 expression was strong in the BxPC-3 and minimal in the AsPC-1 cell line. Both multimodality imaging and Bio-D data exhibited more prominent uptake of [89Zr]Zr-DFO-ICAM-1-IR800 in BxPC-3 tumors than in AsPC-1 tumors. The uptake of [89Zr]Zr-DFO-IgG-IR800 in BxPC-3 tumors was similar to that of [89Zr]Zr-DFO-ICAM-1-IR800 in AsPC-1 tumors. These results demonstrate the desirable affinity and specificity of [89Zr]Zr-DFO-ICAM-1-IR800 compared to [89Zr]Zr-DFO-IgG-IR800. Orthotopic BxPC-3 tumor foci could also be clearly delineated by [89Zr]Zr-DFO-ICAM-1-IR800. An intermodal match was achieved in the ICAM-1-targeted immunoPET/NIRF/CLI. The positive expression levels of ICAM-1 in BxPC-3 tumor tissue were further confirmed by immunohistopathology. CONCLUSION: We successfully developed a dual-labeled ICAM-1-targeted tracer for PET/NIRF/CLI of PDAC that can facilitate better diagnosis and intervention of PDAC upon clinical translation.

Cyclotron-Produced 132La as a PET Imaging Surrogate for Therapeutic 225Ac.

The aim of this work was to explore 132La as a PET imaging surrogate for 225Ac using a DOTA-based, tumor-targeting alkylphosphocholine (NM600). Methods:132La was produced on a biomedical cyclotron. For in vivo experiments, mice bearing 4T1 tumors were administered 132La-NM600, and PET/CT scans were acquired up to 24 h after injection. After the last time point, the ex vivo tissue distribution was measured to corroborate the in vivo PET data. The ex vivo tissue distribution in mice was determined at 4 and 24 h after injection of 225Ac-NM600. Results: PET/CT images showed elevated, persistent 132La-NM600 uptake in the tumor. Low bone accumulation confirmed the in vivo stability of the conjugate. Ex vivo biodistribution studies validated the image-derived quantitative data, and the comparison of the 132La-NM600 and 225Ac-NM600 tissue distributions revealed a similar biodistribution for the 2 radiotracers. Conclusion: These findings suggest that 132La is a suitable imaging surrogate to probe the in vivo biodistribution of 225Ac radiotherapeutics.

In vivo characterization of PD-L1 expression in breast cancer by immuno-PET with 89Zr-labeled avelumab.

Programmed death protein 1 and programmed death-ligand 1 (PD-1/PD-L1) have been widely studied as one of the most critical immune check-point pairs in the cancer microenvironment. In breast cancer (BrCa), the expression of PD-L1 is regarded as a determinant biomarker for patient stratification and prediction of inhibition response. Quantitative positron emission tomography (PET) imaging of PD-L1 expression in tumors using a therapeutic antibody in the clinic seems to be a promising approach that can complement conventional histopathological methods and overcome several issues, such as the tumor heterogeneities, sampling representativeness and clear differentiation of positive and negative results. In this study, we synthesized and evaluated 89Zr-labeled avelumab (Ave) for the in vivo characterization of PD-L1 expression in BrCa. Confocal imaging of BrCa cells and flow cytometry were employed to evaluate PD-L1 expression in MDA-MB-231 cells. The intact human monoclonal antibody targeting PD-L1, i.e., Ave, was conjugated to p-SCN-Deferoxamine (Df) and labeled with 89Zr. After intravenous injection of 89Zr-Df-avelumab (89Zr-Df-Ave), PET imaging of MDA-MB-231 tumor-bearing mice, with or without blocking, was performed. High PD-L1 expression of MDA-MB-231 cells was confirmed by in vitro immuno-fluorescent staining and flow cytometry. PET imaging indicated the peak uptake of 89Zr-Df-Ave in the tumor (6.4±1.0 %ID/g), spleen (10.2±0.7 %ID/g) and lymph nodes (6.9±1.0 %ID/g) at 48 h after injection (n=4). Blocking study using unlabeled Ave could reduce the tracer uptake in these tissues (5.2±1.0 %ID/g in the tumor, 4.9±0.5 %ID/g in the spleen and 5.8±1.1 %ID/g in lymph nodes at 48 h, n=4), which demonstrated the specificity of 89Zr-Df-Ave. Biodistribution study and immuno-fluorescent staining were consistent with the quantitative data from PET imaging. Herein, we offer the evidence supporting the value of immuno-PET imaging using 89Zr-Df-Ave for non-invasive characterization of PD-L1 expression in BrCa and the applicability of this tracer in BrCa for treatment evaluation after immunotherapy.

Selenium-Doped Carbon Quantum Dots Act as Broad-Spectrum Antioxidants for Acute Kidney Injury Management.

The manifestation of acute kidney injury (AKI) is associated with poor patient outcomes, with treatment options limited to hydration or renal replacement therapies. The onset of AKI is often associated with a surfeit of reactive oxygen species. Here, it is shown that selenium-doped carbon quantum dots (SeCQDs) have broad-spectrum antioxidant properties and prominent renal accumulation in both healthy and AKI mice. Due to these properties, SeCQDs treat or prevent two clinically relevant cases of AKI induced in murine models by either rhabdomyolysis or cisplatin using only 1 or 50 µg per mouse, respectively. The attenuation of AKI in both models is confirmed by blood serum measurements, kidney tissue staining, and relevant biomarkers. The therapeutic efficacy of SeCQDs exceeds amifostine, a drug approved by the Food and Drug Administration that also acts by scavenging free radicals. The findings indicate that SeCQDs show great potential as a treatment option for AKI and possibly other ROS-related diseases.

HPMA-based star polymer biomaterials with tuneable structure and biodegradability tailored for advanced drug delivery to solid tumours.

Design, controlled synthesis, physico-chemical and biological characteristics of novel well-defined biodegradable star-shaped copolymers intended for advanced drug delivery is described. These new biocompatible star copolymers were synthesised by grafting monodispersed semitelechelic linear (sL) N-(2-hydroxypropyl)methacrylamide copolymers onto a 2,2-bis(hydroxymethyl)propionic acid (bisMPA)-based polyester dendritic core of various structures. The hydrodynamic diameter of the star copolymer biomaterials can be tuned from 13 to 31 nm and could be adjusted to a given purpose by proper selection of the bisMPA dendritic core type and generation and by considering the sL copolymer molecular weight and polymer-to-core molar ratio. The hydrolytic degradation was proved for both the star copolymers containing either dendron or dendrimer core, showing the spontaneous hydrolysis in duration of few weeks. Finally, it was shown that the therapy with the biodegradable star conjugate with attached doxorubicin strongly suppresses the tumour growth in mice and is fully curative in most of the treated animals at dose corresponding approximately to one fourth of maximum tolerated dose (MTD) value. Both new biodegradable systems show superior efficacy and tumour accumulation over the first generation of star copolymers containing non-degradable PAMAM core.