The MET receptor tyrosine kinase is mutated or amplified in ~6% of non-small cell lung cancer (NSCLC) and overexpressed in ~80% of all NSCLC cases. A theranostic agent that can both see and treat MET-altered NSCLC has never been described before in the literature. Here, we report a shark-derived single-domain variable new antigen receptor (VNAR) for MET with theranostic applications. Following the immunization of a juvenile nurse shark (Ginglymostoma cirratum) with the extracellular domain of human MET, we identified a VNAR clone that specifically engaged MET with high affinity. Engineering the lead VNAR into a bivalent human Fc, vMET1-Fc, yielded a construct that selectively targeted and was internalized by MET-positive cells without affecting cell viability or downstream MET signaling. When radiolabeled with the positron emitting isotope Zr-89, [89Zr]Zr-vMET1-Fc enabled longitudinal PET/CT imaging. High tumor uptake with low background was observed in MET-positive NSCLC xenografts administered [89Zr]Zr-vMET1-Fc. As a targeted beta-particle radiotherapy, [177Lu]Lu-vMET1-Fc resulted in marked tumor-growth delay and exhibited a favorable toxicity profile, collectively improving progression-free survival in NSCLC mouse models. Non-human primate PET/CT imaging studies with ([89Zr]Zr-vMET1-Fc in healthy rhesus macaques confirmed favorable biodistribution and dosimetry, predictable clearance, and minimal off-target uptake. Additional blood chemistry analysis found no significant immune response or cytotoxicity. Together, these findings establish vMET1-Fc as a theranostic agent for imaging and treating MET-altered NSCLC.

Radioluminescence imaging (RLI) using nanoscintillators offers great potential for biomedical applications, yet remains constrained by low quantum efficiency and the reliance of Cerenkov imaging on high-energy radionuclides. The rational design of core-shell nano-transducers overcomes these constraints by enhancing X-ray absorption and energy confinement, thereby enabling efficient γ-ray excited radioluminescence. We engineered NaGdF₄:15

Acute kidney injury (AKI) is driven by a vicious interplay among excessive reactive oxygen species (ROS) accumulation and persistent renal hypoxia, which collectively disrupt redox homeostasis and exacerbate tubular injury. However, current therapeutic strategies primarily provide supportive care and fail to simultaneously modulate these interconnected pathological stressors. Herein, we report a cerium-doped rosmarinic acid nanosystem (RA/Ce NPs) formed via self-polymerization that catalytically eliminates ROS while simultaneously generating oxygen to reprogram the pathological renal microenvironment. The ultrasmall RA/Ce NPs enable efficient chelator-free 89Zr radiolabeling for positron emission tomography (PET) imaging. Redox-switchable Ce3+/Ce4+ centers confer robust catalytic activity and ROS-responsive biodegradation, ensuring potent antioxidation with safe clearance. RA/Ce NPs suppress intracellular ROS, preserve mitochondrial membrane potential, and protect against oxidative injury in vitro. In rhabdomyolysis-induced AKI mice, PET imaging reveals pronounced renal accumulation, while treatment markedly alleviates tubular damage, oxidative stress and hypoxia, leading to restored renal function with excellent biocompatibility. This work presents a natural-product-derived catalytic nanoplatform integrating antioxidation, oxygenation, imaging and biodegradability, offering a promising strategy for precision therapy of AKI.

Targeted α-therapy (TAT) is a promising therapeutic strategy for advanced cancers, but redistribution of short-lived daughter radionuclides remains a key challenge. This is particularly true for 225Ac and 212Pb, whose decay chains both include α-emitting bismuth isotopes that are difficult to image directly. Production and subsequent radiochemical isolation of 206Bi (t1/2 = 6.24 d, 100% EC+β+) was investigated as a SPECT-imaging surrogate for 212/213Bi, alpha-emitting daughters of 225Ac and 212Pb. Isotopically enriched 206PbO targets were irradiated with 11.9 MeV protons to access the 206Pb(p,n)206Bi nuclear reaction on a 16 MeV GE PETtrace cyclotron. Following target dissolution in nitric acid, 206Bi separates from 206Pb using branched-DGA resin, achieving efficient Pb/Bi separation and recovery of enriched 206Pb for subsequent irradiation. The irradiation yielded 1.9 ± 0.2 MBq/μAh of 206Bi (n = 4), with a decay-corrected radiochemical yield of 96.6 ± 2.6% (n = 4), 206Pb recycling efficiency of 95.3 ± 1.5% (n = 8), and high chemical and radionuclidic purity (>99.88%). Preclinical SPECT/CT imaging of a Derenzo phantom documents a 1.03 mm spatial resolution, and first in vivo SPECT/CT imaging records preferential renal uptake of unchelated 206Bi in ICR mice. These results establish a practical production route for 206Bi and demonstrate its potential as preclinical SPECT tracer for investigating radiobismuth biodistribution relevant to alpha-emitter daughters redistribution.

Cuproptosis, a unique form of programmed cell death triggered by copper overload and subsequent proteotoxic stress, represents a promising avenue for cancer therapy. Radiotherapy may influence cuproptosis-related pathways in tumors, highlighting the possibility of combining radiotherapy to enhance cuproptosisis. However, how copper delivered in a radioactive form affects these processes remains unclear. Here, we doped radioactive 64Cu into elesclomol (ES-Cu[II]) and encapsulated by an RGD-modified nanoparticle, named ES-64Cu[II] @Nanoparticle-RGD (ES-64Cu@NP). This agent demonstrated high tumor accumulation (7.66 ± 0.93

The dynamic field of radiopharmaceuticals is currently experiencing an explosion of growth due in part to excitement over the emerging field of theranostics (therapy and diagnostics). Radiopharmaceuticals use physiological targeting methods to deliver radionuclides with medically relevant decay properties to disease biomarkers for diagnosis and treatment, offering opportunities for early disease imaging and radiation therapy treatment in disease pathologies that are inoperable or refractory to other forms of radiotherapy. Sustaining this rapidly growing field depends heavily on the continued design and production of novel, effective radiopharmaceuticals. Effective therapeutic radiopharmaceuticals cause complex and varied cellular responses, and to choose radionuclides that maximize therapeutic response, researchers must understand radiation biology. Cellular radiation response depends heavily on factors including linear energy transfer (LET), dose, dose rate, targeted location, direct or indirect energy deposition mechanisms, the broader cellular matrix, cellular stress signaling pathways, and endogenous radiation protection mechanisms. Because of the extensive application of low-LET external beam radiation on clinical cancer treatments, biological responses to low-LET form the basis of radiation biology and are generally considered transferable to high-LET radiopharmaceuticals. However, increased focus on high-LET, radiopharmaceutical therapy-specific radiation biology is motivated by differences between low- and high-LET radiation, external beam versus radiopharmaceutical therapy-induced biological response, and the observed varied clinical responses to radiopharmaceutical therapies. This review article summarizes historical understanding of low- and high-LET radiation responses within cells, with emphasis on radiopharmaceutical-specific responses when available, and discusses current gaps in understanding in the radiation biology of radiotheranostic pharmaceuticals.

Cell-based therapies have transformed the treatment landscape for cancer, yet their clinical translation remains limited by unpredictable in vivo behavior and variable patient responses. Accurate, noninvasive image-based tracking of therapeutic cells, such as PET/CT, is essential for understanding biodistribution, improving safety, and optimizing the design of next-generation treatments. However, existing radiolabeling strategies for cell tracking using PET/CT lack the stability and sensitivity required for reliable long-term imaging. Here, we present a direct radiolabeling strategy that oxidizes cell surface sialic acids to conjugate aminooxy-DFO (AOD) and subsequently radiolabels cells with 89Zr under biocompatible conditions. We radiolabeled five human and nonhuman primate immune cell types with high radiochemical incorporation (17-194 μCi per million cells) and purity (∼90%), while preserving cell viability. Serial PET/CT imaging over 7-8 days revealed conserved biodistribution patterns across all cell types tested. This approach provides a robust, applicable platform for longitudinal PET/CT tracking of therapeutic cells.

Abstract Background Bromine-76, a positron emitting radionuclide for positron emission tomography (PET) imaging, and bromine-77, an Auger electron (Ae) emitting radionuclide, make a unique halogen theranostic pair. These isotopes have previously been used to synthesize a rucaparib-derived, poly-ADP-ribose polymerase inhibitor ([ 76/77 Br]RD1). As [ 77 Br]bromide activity in [ 76/77 Br]RD1 radiosyntheses increased, coincident with solid target hardware changes in our facility, radiopharmaceutical yield became less reproducible. This work describes the modifications to this hardware, distillation procedures, and [ 76/77 Br]bromide solution chemistry to enable reproducible production of preclinical quantities of [ 76/77 Br]RD1. Results Isotopically enriched cobalt selenide targets had production yields of 50.7 ± 8.7 MBq/µAh for bromine-76 and 14.5 ± 3.3 MBq/µAh for bromine-77, consistent with previous reports. Adoption of the GE PETtrace Solid Target Platform (STP) increased target backing diameter to 22 mm. Increasing dry distillation duration from 5 to 6 min to 8–9 min and furnace temperature from 1050 to 1055 °C released 94 ± 5% ( n = 17) of activity, in agreement with previous smaller targets (92 ± 13%, n = 35). Rinsing glassware and loading the quaternary methyl ammonium (QMA) anion exchange cartridge in 1 mM sodium bicarbonate reduced [ 76/77 Br]bromide elution volume from 2.9 to 0.9 mL. Across all distillations, when [ 77 Br]bromide was isolated in less than 1 mL of QMA eluant, copper-mediated bromodeborylation of a BPin precursor molecule formed [ 76/77 Br]RD1 with a radiochemical conversion of 98 ± 3% ( n = 17) and overall radiopharmaceutical yield of 70 ± 10% ( n = 15) following purification and final formulation. Up to 580 MBq of purified [ 77 Br]RD1 have been produced with this system. Conclusion The new target production technique made targets suitable for long-term use with the GE PETtrace STP. Following modifications to the distillation process, [ 76/77 Br]bromide production yields from GE PETtrace STP targets agreed with those previously published. The changes described in this work not only aimed to maximize [ 76/77 Br]bromide recovery after distillation, but also chemical reactivity in large-scale [ 76/77 Br]RD1 radiopharmaceutical syntheses. This balancing act resulted in a slightly lower [ 76/77 Br]bromide recovery yield but more consistently [ 76/77 Br]RD1 product yield.

Titanium-45 is a positron-emitting radiometal with decay characteristics attractive for clinical positron emission tomography (PET) imaging (t1/2 = 3.08 h, β+ = 84.8%, β+ = .439 MeV). We report methods for producing 45Ti at commercial scale via 13 MeV proton irradiation of spot-welded natSc foil cyclotron targets that are thick to 13 MeV protons and robust to beam currents up to 100 µA, producing tens of GBq (>1 Ci) of 45Ti. Titanium-45 is extracted from bulk natSc via ZR resin and recovered in 0.1 M oxalic acid with radiochemical yields of 72.7 ± 0.6% (n = 3) and 78 ± 3% (n = 3) from 0.25 and 0.5 mm thick natSc targets, respectively. Separation conditions are optimized for rapid and facile radiopharmaceutical incorporation by coordination using siderophore chelators which have previously demonstrated compatibility with 45Ti. Purified 45Ti contains <1 µg natFe and achieves apparent molar activity (AMA) of 29 - 440 GBq/µmol (0.8 – 11.9 Ci/µmol) at EoB for productions ranging from 35 – 122 GBq (0.096 – 3300 Ci). AMAs were determined utilizing the catechol based chelator tren-2,3-dihydroxy-benzamide (TREN-CAM), forming [45Ti][Ti(TREN-CAM)]2-. We observed an increase of AMA in correlation with production size and a decrease of AMA in correlation with increased Sc target mass, indicating that stable contaminants in the target material likely limit currently achievable AMA. The methods described herein establish and validate a robust 45Ti isotope production framework which enables commercial scale production and clinical translation of 45Ti PET agents.

Calcium influx is a fundamental component of neuronal signaling and is markedly altered during neuropathic pain. Voltage-gated calcium channels (VGCCs) play a central role in nociceptive transmission; however, multiple calcium-permeable pathways contribute to calcium-associated neural activity. Because Mn2+ shares similar ionic properties and cellular transport mechanisms with Ca2+, positron-emitting [51Mn]MnCl2 has potential as a molecular imaging probe for visualizing pain-associated calcium activity in vivo. This study evaluated [51Mn]MnCl2 PET/MR for detecting neuropathic pain-associated neural activity in a rat spared nerve injury (SNI) model before and after analgesic intervention. Male Sprague–Dawley rats underwent SNI surgery or sham surgery and were evaluated using von Frey behavioral testing, in vivo [51Mn]MnCl2 PET/MR imaging, before and after sustained-release buprenorphine administration. Ex vivo biodistribution was completed after all imaging studies were completed. Tracer uptake within the spinal cord and peripheral nervous system was compared between SNI and sham animals. SNI animals demonstrated significantly increased mechanical hypersensitivity accompanied by elevated [51Mn]MnCl2 uptake within the spinal cord regions associated with sciatic nerve innervation before analgesic intervention. Following buprenorphine administration, behavioral hypersensitivity and spinal cord tracer uptake were reduced, while ex vivo biodistribution demonstrated significantly greater tracer accumulation within injured sciatic nerves compared to sham controls. These findings provide proof-of-concept evidence that positron-emitting manganese can detect neuropathic pain-associated calcium-related neural activity in the SNI model. While additional studies are needed to establish mechanistic specificity, improve quantitative assessment of peripheral nerves, and validate findings in larger cohorts, [51Mn]MnCl2 PET/MR shows promise as a molecular imaging approach for investigating neuropathic pain.

With the emission of both Auger and conversion electrons (AE and CE) together with some x-rays and a few low intensity gamma-rays, palladium-103 (103Pd, T1/2 17 d) and its daughter rhodium-103m (103mRh, T1/2 56 min) are interesting radionuclides for applications in targeted radionuclide therapy. 103Pd's x-ray emissions have been applied in brachytherapy implants, but the unavailability of non-carrier-added (n.c.a.) 103Pd has limited the development of 103Pd-radiolabeled compounds that exploit its AE and CE emissions. Through a collaboration between UWM and DTU, we have developed a procedure to produce n.c.a. 103Pd from Rh foils and electroplated Rh targets on graphite backings. The targets were irradiated on 16 MeV cyclotrons at 20-40 μA with a production yield of 1.6-2.2 MBq/μAh. Rh dissolution was performed in HCl by applying alternating current. The commercially available AG-1X8, TK200, normal DGA, and branched DGA resins were tested for 103Pd purification from the Rh bulk target material. The DGA resins were found to be the most convenient with high separation factors and quantitative 103Pd elution in HCl solutions. The Rh could be recycled and used for electroplating new targets. Recycled Rh targets improved the molar activity of the 103Pd produced. After two branched DGA columns, 87.2 ± 6.3 % of 103Pd activity was recovered. ICP-OES analysis of the purified batches showed the presence of only few remaining impurities: 1.2 ± 0.3 nmol Pd, 0.4 ± 0.3 nmol Rh and traces of Al, Fe, and Zn. Finally, n.c.a. 103Pd radiolabeling was demonstrated with the TETA chelator in acetate buffered solutions.

Cancer-associated fibroblasts (CAFs) in the stroma of solid tumors promote an immunosuppressive tumor microenvironment (TME) that drives resistance to therapies. The expression of the protease fibroblast activation protein (FAP) on the surface of CAFs has made FAP a target for development of therapies to dampen immunosuppression. Relatively few biologics have been developed for FAP and none have been developed that exploit the unique engagement properties of Variable New Antigen Receptors (VNARs) from shark antibodies. As the smallest binding domain in nature, VNARs cleverage unique geometries and recognize epitopes conventional antibodies cannot. By directly immunizing a nurse shark with FAP, we created a large anti-FAP VNAR phage display library. This library allowed us to identify a suite of anti-FAP VNARs through traditional biopanning and also by an in silico approach that did not require any prior affinity-based enrichment in vitro. We investigated four VNAR-Fc fusion proteins for theranostic properties and found that all four recognized FAP with high affinity and were rapidly internalized by FAP-positive cells. As a result, the VNAR-Fc constructs were effective antibody-drug conjugates in vitro and were able to localize to FAP-positive xenografts in vivo. Our findings establish VNAR-Fc constructs as a versatile platform for theranostic development that could yield innovative cancer therapies targeting the TME.

Disialoganglioside 2 (GD2) is overexpressed in multiple cancers, such as melanoma and neuroblastoma, but also in peripheral nerves. To improve current GD2-targeting approaches, next-generation heterodimeric bispecific human IgG antibodies were created, each with one antibody binding fragment (Fab) arm specific for GD2 and the other Fab arm specific for B7-H3 (CD276) to drive tumor selectivity. The avidity and selectivity of our GD2-B7-H3 targeting bispecific antibodies (INV34-6, INV33-2, and INV36-6) were determined by flow cytometry and competition binding assays in GD2+/hB7-H3+ B78 cells. INV34-6 showed high avidity for GD2+/hB7-H3+ but not GD2+/hB7-H3- B78 cells, contrasting with the similar cell binding to these cells observed with the anti-GD2 antibody Dinutuximab (DINU). The bispecific antibodies, DINU, and a nontargeted bispecific control (bsAb CTRL) were conjugated with deferoxamine for radiolabeling with Zr-89 (t1/2 = 78.4 h). Positron emission tomography (PET) corroborated the in vivo avidity and selectivity of the GD2-B7-H3 targeting bispecific compared to bsAb CTRL and DINU in GD2+/hB7-H3+ and GD2+/hB7-H3- B78 tumor models. PET in mice bearing the GD2+/hB7-H3- and GD2+/hB7-H3+ B78 murine xenografts 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, unlike [89Zr]Zr-Df-DINU, which displayed elevated tumor uptake, irrespective of hB7-H3 expression. Nontargeted [89Zr]Zr-Df-bsAb CTRL isotype control showed markedly lower uptake in all tested tumor models. Overall, bispecific antibodies binding GD2 and B7-H3 showed improved selectivity for targeting tumor cells expressing both antigens. This approach may enhance antitumor efficacy while addressing the toxicity limitations of current GD2-targeting therapies by reducing off-tumor GD2 binding in nerves.

Chelation approaches that are compatible with a multitude of isotopes are an important area of development. Here, we introduce the design, synthesis, and evaluation of 2,3-dihydroxyterephthalate/catechol chelator conjugates compatible with the positron emission tomography (PET) isotopes 68Ga3+ and 45Ti4+, targeting the prostate-specific membrane antigen (PSMA). The conjugates are made in a multistep organic synthesis incorporating 2,3-dihydroxyterephthalate, linked to the amino hexanoic acid-extended, urea-dipeptides EuE or KuE (substrates of the PSMA active site). The radiochemical complexes, [45Ti][Ti(TREN-CAM-hex-EuE)]2-, [45Ti][Ti(TREN-CAM-hex-KuE)]2-, and [68Ga][Ga(TREN-CAM-hex-KuE)]3- form readily at room temperature within 15 min with a molar activity of 24-29 mCi/μmol. The corresponding chelates are stable in phosphate-buffered saline (PBS) solution prior to injection. Subsequent in vivo studies in a bilateral tumor xenograft mouse model were conducted, including 90- and 270-min PET, followed by biodistribution and metabolite analysis at 2 or 5 h postinjection. These studies demonstrated selective uptake of the radiochemical complexes in the PSMA-expressing tumor (17.25 ± 4.15, 13.84 ± 3.85, 15.64 ± 6.37% ID/g for [45Ti][Ti(TREN-CAM-hex-EuE)]2-, [45Ti][Ti(TREN-CAM-hex-KuE)]2- and [68Ga][Ga(TREN-CAM-hex-KuE)]3- respectively), with pharmacokinetics dominated by renal clearance. Delayed clearance of the [45Ti][Ti(TREN-CAM-hex-KuE)]2- complex is observed when compared with that of [68Ga][Ga(TREN-CAM-hex-KuE)]3- as indicated by elevated activity retention in the blood, which we attribute to the charge difference and partial complex dissociation. Urine metabolite analysis shows that [68Ga][Ga(TREN-CAM-hex-KuE)]3- is excreted >98% intact, while [45Ti][Ti(TREN-CAM-hex-KuE)]2- exhibited signs of dechelation. Conclusively, our data support further investigation of bifunctional TREN-CAM derivatives as a synthetically accessible bifunctional chelator class for 68Ga3+ and 45Ti4+ isotopes.

The Trop2-targeting antibody-drug conjugate (ADC) sacituzumab govitecan (Trodelvy) has demonstrated remarkable efficacy in patients with metastatic triple-negative breast cancer (TNBC). ImmunoPET imaging offers a noninvasive method to visualize the expression and distribution of target antigens in vivo. In this study, we developed F(ab’)2 fragments of Trodelvy for immunoPET imaging to detect Trop2 expression in TNBC models, aiming to achieve a shorter imaging window. Trodelvy-F(ab’)2 was prepared using the IdeS protease kit and purified with Magne Protein A beads and MagneHis™ Ni Particles. The products were characterized by non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography. Trodelvy-F(ab’)2 was subsequently conjugated with p-SCN-Bn-NOTA (NOTA) for radiolabeling with 64Cu. ImmunoPET imaging using [64Cu]Cu-NOTA-Trodelvy-F(ab’)2 was conducted at multiple time points to assess its in vivo targeting capability. Immunohistochemical and immunofluorescence analyses were performed on tumor tissues obtained from tumor-bearing mice. The radiochemical yield of [64Cu]Cu-NOTA-Trodelvy-F(ab’)2 exceeded 90

Triple-negative breast cancer (TNBC) and non-small cell lung cancer (NSCLC) are aggressive solid tumors with limited treatment options. Nectin cell adhesion molecule 4 (Nectin4) is a tumor-associated antigen frequently overexpressed in these cancers, making it a promising therapeutic and imaging target. Here, we report the development and evaluation of [89Zr]Zr-desferrioxamine (DFO)-Padcev, a radiolabeled antibody-drug conjugate targeting Nectin4, for immuno-positron emission tomography (ImmunoPET) imaging. [89Zr]Zr-DFO-Padcev is synthesized with a radiochemical yield of 88.87% ± 2.59% and a radiochemical purity above 99%. ImmunoPET imaging successfully visualizes Nectin4-positive tumors in TNBC (MDA-MB-468) and NSCLC (H1975) models as early as 6 h post-injection, with uptake progressively increasing and peaking at 48 h (14.57 ± 1.94 and 9.50 ± 0.76 %ID/g, respectively). Minimal tumor uptake is observed in blocking and Nectin4-negative controls, confirming specificity. Complementary fluorescence imaging further reveals the in vivo distribution of Padcev, providing valuable insights into optimal therapeutic time windows.

The development of matched diagnostic and therapeutic radiopharmaceuticals─theranostic pairs─has emerged as a promising strategy to advance personalized nuclear medicine. However, many current systems rely on chemically distinct elements such as the 68Ga3+/177Lu3+ pair, leading to inconsistencies in the pharmacokinetics. Here, we evaluate bifunctional chelator platforms derived of triazamacrocycle picolinates, capable of stably incorporating three clinically relevant isotopes 18F-, 44Sc3+, and 177Lu3+. mpatcn supported the formation of [18F][ScF], [44Sc][Sc], and [177Lu][Lu] complexes when conjugated to a PSMA-targeting peptide (picaga-Met-hex-KuE). picaga-Met-hex-KuE displayed quantitative radiochemical yields and >95% formulation stability after 2 h. Biodistribution and metabolite analysis confirmed PSMA-targeting, minimal off-target uptake, and renal clearance for all picaga-Met-hex-KuE systems. Additionally, we demonstrate that a cartridge-based purification method formulates [18F][ScF(picaga-Met-hex-KuE)] in >95% radiochemical purity with nondecay corrected yields of 32% in under 110 min. These results establish picaga-Met-hex-KuE as a lead scaffold for the 18F/44Sc/177Lu triad, enabling single-kit radiopharmaceutical preparation for theranostic applications.

Progressive forms of interstitial lung diseases, including idiopathic pulmonary fibrosis (IPF), are deadly disorders lacking non-invasive biomarkers for assessment of early disease activity, which presents a major obstacle in disease management. Excessive extracellular matrix (ECM) deposition is a hallmark of these disorders, with fibronectin being an abundant ECM glycoprotein that is highly upregulated in early fibrosis and serves as a scaffold for the deposition of other matrix proteins. Due to its role in active fibrosis, we are targeting fibronectin as a biomarker of early lung fibrosis disease activity via the PEGylated fibronectin-binding polypeptide (PEG-FUD). In this work, we demonstrate the binding of PEG-FUD to the fibrotic lung throughout the course of bleomycin-induced murine model of pulmonary fibrosis. We first analyzed the binding of radiolabeled PEG-FUD following direct incubation to precision cut lung slices from mice at different stages of experimental lung fibrosis. Then, we administered fluorescently labeled PEG-FUD subcutaneously to mice over the course of bleomycin-induced pulmonary fibrosis and assessed peptide uptake 24 h later through ex vivo tissue imaging. Using both methods, we found that peptide targeting to the fibrotic lung is increased during the fibrogenic phase of the single dose bleomycin lung fibrosis model (days 7 and 14 post-bleomycin). At these timepoints we found a correlative relationship between peptide uptake and fibrotic burden. These data suggest that PEG-FUD targets fibronectin associated with active fibrogenesis in this model, making it a promising candidate for a clinically translatable molecular imaging probe to non-invasively determine pulmonary fibrosis disease activity, enabling accelerated therapeutic decision-making.

CD70 is an emerging biomarker for both solid tumors and hematologic malignancies, highlighting the urgent need for a molecular imaging tracer capable of visualizing CD70 with favorable pharmacokinetics. Methods: ABDB6 was prepared by fusing the albumin-binding domain ABD035 with the CD70-targeting single-domain antibody RCCB6, which we previously reported. The resulting ABDB6 was then conjugated to the bifunctional chelator p-SCN-NOTA and labeled with 64Cu to produce [64Cu]Cu-NOTA-ABDB6. Flow cytometry was used to screen 6 lymphoma cell lines with varying CD70 expression levels. Cell uptake and in vivo immuno-PET imaging studies were conducted to fully evaluate the pharmacokinetic properties and tumor-targeting efficacy of [64Cu]Cu-NOTA-ABDB6. An ABDB6 blocking study was performed to validate the targeting specificity of [64Cu]Cu-NOTA-ABDB6, followed by immunohistochemistry and fluorescent immunostaining studies to correlate tracer uptake with CD70 expression. Results: 64Cu labeling of ABDB6 achieved a high radiochemical yield and specific activity. Significant CD70 expression was observed in 5 lymphoma cell lines (TMD8, HBL1, OCI-LY10, LCL-EBV, and type III latency Burkitt lymphoma [BL] cells) but not in type I latency BL cells, which served as the negative control. [64Cu]Cu-NOTA-ABDB6 exhibited good affinity for CD70 protein at the nanomolar level (inhibitory concentration of 50%, 91.57 nM) and specificity in binding to human CD70. Immuno-PET imaging of [64Cu]Cu-NOTA-ABDB6 demonstrated excellent tumor uptake and retention in various CD70-positive lymphoma models (TMD8, type III latency BL, and LCL-EBV), with the highest tumor uptake values recorded as 24.67 ± 1.36, 18.02 ± 4.29, and 14.68 ± 1.20 percentage injected dose per gram of tissue (%ID/g) at 48 h after injection, respectively. These tumor uptake values were significantly higher than that of the CD70-negative type I latency BL tumor, which had an uptake of 3.59 ± 0.28 %ID/g at the same scanning time point (P < 0.05). In the TMD8 blocking group, tumor uptake was 5.99 ± 1.20 %ID/g at 48 h after injection, significantly lower than in the TMD8 control group (P < 0.01). Both biodistribution and histology results corroborated these imaging findings. Conclusion: [64Cu]Cu-NOTA-ABDB6 immuno-PET effectively visualized varying levels of CD70 in different lymphoma models. Its clinical potential may provide insights into CD70 expression in lymphoma patients.

Representative images of γH2AX foci over time in (A) MOC2 (12 Gy) and (B) B78 (4 Gy) cells following EBRT, 90Y, 177Lu, and 225Ac.