Abstract Rationale Neuroblastoma is a devastating pediatric malignancy, for which surgical resection is a key factor in long-term survival. However, there are significant challenges in its resection, particularly in high-risk disease, as neuroblastoma encases surrounding critical structures, is often difficult to distinguish from desmoplastic or scar tissue, and can carry occult deposits of disease not readily identified on preoperative imaging or intraoperative visualization. Building on the principles of fluorescent and radio-guided surgery, in combination with the known overexpression of GD2 in neuroblastoma, we sought to develop and optimize 111 In-Dinutuximab-IRDye800, a dual-modality GD2-targeted intraoperative molecular imaging agent, for use in pediatric neuroblastoma to help enhance patient safety while facilitating a more complete resection. Methods Dinutuximab was conjugated to IRDye800 and DTPA, then radiolabeled with Indium-111 to yield 111 In-Dinutuximab-IRDye800. Optimization occurred through ELISA assay to assess binding affinity, fluorescence intensity analysis to determine the optimal fluorescent degree of labeling, and phototoxicity testing through flow cytometry. Rodent models of neuroblastoma were then generated through injection of SK-N-BE(2) human neuroblastoma cells into the left adrenal glands of nude mice or RNU rats. A series of fluorescent and gamma biodistributions was performed, varying the dose, timing, and specific activity of the tracer. Tumor and organ uptake of the tracer was compared with one- or two-way ANOVA as appropriate, with Sidak’s multiple comparison test to compare tumor uptake to individual organs. Once optimization was complete, a clinically significant events study modeled after human clinical trials was performed to evaluate the in vivo capabilities of 111 In-Dinutuximab-IRDye800. Results Increased ratios of IRDye800 per antibody led to decreased binding affinity for GD2 and was associated with formulation instability without significant return on fluorescence intensity. Specific activity of the tracer was not found to impact overall biodistribution of the tracer. A 45-50 µg dose of 111 In-Dinutuximab-IRDye800 with ratios around 1 DTPA and 1-1.5 IRDye800 per antibody imaged 4 days after tracer administration was found to be the optimal combination that maximized detectable tumor-specific signal. In the clinically significant events study mirroring human IMI clinical trials, fluorescent guidance identified additional malignant lesions not originally detected under white light in 64% of rodents. Conclusions 111 In-Dinutuximab-IRDye800 is a dual-modality GD2-targeted intraoperative imaging agent that is well-poised for clinical translation. As it preserves tumor specificity, yields clinically meaningful radiofluorescent signal, and is well-tolerated without adverse events after optimization was completed, it carries the potential to positively impact the safety and completeness of neuroblastoma resection.

Chimeric antigen receptor (CAR) T cell therapy has limited efficacy against solid tumors such as neuroblastoma (NB). Key obstacles include extensive tumor burden and the presence of an immunosuppressive tumor microenvironment (TME). We employ targeted radiopharmaceutical therapy (RPT) using [67Cu]Cu-LLP2A and show that it potentiated the anti-tumor activity of CAR T cells in radio-sensitive and radio-resistant NB models via distinct mechanisms. In radio-sensitive NB, RPT is directly tumoricidal while also enhancing CAR T cell efficacy through pro-immune pathways, most notably via the TNF-α pathway, leading to paracrine activation of T cells. In radio-resistant NB, RPT improves CAR T cells by remodeling the myeloid compartment in the TME and increasing the formation of immunological niches of cytotoxic CD8+ GZMB+ and CD4+ GZMB+ CAR T cells. While neither treatment modality alone can effectively treat NB, the combination of VLA-4-targeted RPT and GD2 or B7-H3 CAR T cells augments anti-tumor efficacy, resulting in marked tumor regression in preclinical NB models.

Tobacco smoke contains many known carcinogens, chief among which is nicotine-derived nitrosamine ketone (NNK). However, even among individuals with high exposure levels, only a minority develop lung cancer. Prior research highlights additional exogenous (e.g., air pollution) and endogenous (e.g., estrogen exposure) factors that may increase risk. Currently lacking in the literature is consideration of possible effects of psychological stress. To experimentally test stress effects, we utilized a well-established mouse lung adenocarcinoma model with NNK exposure of young female mice, along with a mouse stress protocol. We previously reported that repeated exposure to stress before, during and after 4 weeks of exposure to NNK (IP; 2x/week; 3 mg/injection) increased both overall lesion size and the total number of lung lesions compared to controls when assessed 19 weeks post completion of NNK exposure. The present study used the same protocol to test stress effects earlier in the process of NNK carcinogenesis. FVB/N female mice (4-6 wk) bought from a commercial supplier were acclimated for two weeks before study initiation. The repeated stress protocol was then begun (Week-0) for the Stress group (n=40) and continued throughout the study, while the Control group (n=40) remained in their home cages. The stress protocol included social disruption via changes in cage mates at the time of routine cage cleaning (2x/week) and 90-minute restraint stress prior to a cage mate change (1x/week). NNK treatment began on week-1 (IP; 2x/week; 3 mg/injection) and continued for 4 weeks. At 8 and 15 weeks after the end of NNK exposure, 20 mice in each study group were euthanized. Lungs were formalin inflated. Surface lung lesions were counted under a dissecting microscope and lesion sizes were objectively measured (blind) using Motic Images software. Lungs from mice euthanized at 15 weeks were then imbedded in paraffin and serially sectioned (4 µm) to allow H&E confirmation of lesion numbers and sizes by a veterinary pathologist and to provide slides for ongoing immunohistochemical characterization. Statistical analysis (ANOVA) revealed significant main effects of study group, with lungs from stressed mice having larger overall lesion sizes (p=.007), and total number of lesions (p=.012), compared to controls. As expected, those two outcomes were also significantly increased in the lungs from mice euthanized at 15- vs. 8-weeks (no interaction with group). Providing independent corroboration of the Motic imaging results, data from the histological evaluation of lesion sizes and numbers were significantly correlated (p's <.001). Conclusion: We replicated and extended our initial study demonstrating effects of repeated stress in an NNK model of lung cancer in young female mice to earlier phases of NNK-induced lung carcinogenesis. Dana H. Bovbjerg, Laura P. Stabile, Beatriz Kanterewicz, Jessica Manculich, Jessie R. Nedrow, Patrick Tarwater, Lora H. Rigatti, Frank J. Jenkins. Effects of psychological stress early in NNK-induced lung carcinogenesis in young female mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6344.

Background: The recruitment and accumulation of monocyte-derived macrophages (MDM) are increasingly recognized as key drivers of pulmonary fibrosis. Non-invasive monitoring of MDM accumulation offers a precision medicine approach to assess the inflammatory component of fibrotic lung diseases, allowing for personalized treatment plans. Recently, we demonstrated the potential of chemokine-like receptor 1 (CMKLR1)-targeted positron emission tomography (PET) for imaging newly recruited MDM in bleomycin-induced lung fibrosis, a model characterized by transient pneumonitis and non-progressive/resolving fibrosis. Here, we investigated whether this approach could monitor sustained MDM recruitment in progressive pulmonary fibrosis using an experimental silicosis model. Methods: Progressive fibrosis was induced in 12-16-week-old C57BL/6J mice via intratracheal administration of silicon dioxide (0.8 mg/g body weight). Healthy mice served as controls. MDM recruitment and CMKLR1 expression were assessed over time via flow cytometry and histology. Separate cohorts underwent PET/CT with a CMKLR1-targeting tracer, 64 Cu-NODAGA-CG34, on days 3, 7, 14, and 28 post-silica administration. Additionally, a longitudinal imaging cohort underwent 64 Cu-NODAGA-CG34 PET/CT on day 7 post-silica injection followed by high-resolution CT on day 28 to assess fibrosis severity. Results: Flow cytometry and histology revealed significant recruitment of CMKLR1-expressing MDM by day 3 post-silica injection, peaking on day 7 and persisting through days 14 and 28. This pattern was associated with progressive fibrosis over the 4-week period as detected by histology. PET demonstrated that lung uptake of 64 Cu-NODAGA-CG34 was highest on day 7 post-silica (>2-fold compared to controls) and, although declining on days 14 and 28, remained significantly elevated compared to controls, mirroring the dynamics of the recruitment of MDM as determined by flow cytometry. Notably, 64 Cu-NODAGA-CG34 uptake on day 7 strongly correlated with fibrosis severity on day 28, as determined by high-resolution CT, highlighting the potential of CMKLR1-targeted PET as a risk stratification tool. Conclusions: CMKLR1-targeted PET offers a promising non-invasive method for monitoring sustained MDM recruitment and assessing disease progression in a preclinical model progressive pulmonary fibrosis. This approach could serve as a valuable tool for predicting fibrosis severity and guiding risk stratification in pulmonary fibrosis.

Myeloid cells are key mediators of immunosuppression and treatment resistance in primary brain tumors, including glioblastoma (GBM). This study aims to eradicate CD11b+ immunosuppressive cells at the tumor site to enhance overall survival in a model of GBM using an α-emitting radiopharmaceutical therapy targeted to tumor-associated myeloid cells as a monotherapy or in combination with immune checkpoint inhibitors. An anti-CD11b (αCD11b) antibody was modified for radiolabeling with diagnostic (zirconium-89) or therapeutic (actinium-225) radioisotopes. Initial PET imaging and biodistribution studies using 89Zr-αCD11b found that an antibody concentration of ∼5 mg/kg of αCD11b (100 μg) was effective in saturating on-target/off-site sinks, such as the spleen, but effective in increasing tumor accumulation. The estimated maximum tolerable activity of [225Ac]Ac-DOTA-αCD11b (225Ac-αCD11b) was determined by biodistribution and dosimetry studies, including the free in vivo-generated decay daughters. The dose-limiting tissue was the bone marrow, and an estimated maximum tolerable activity (∼0.55 kBq, 100 μg) was determined. The therapeutic efficacy of 225Ac-αCD11b was evaluated by survival studies, both as a monotherapy and in combination with immune checkpoint inhibitors. Combination therapy resulted in increased survival in the GBM model compared with the monotherapy and controls; in addition, long-term survival was observed in 50% of the mice receiving combination therapy as well as in a single mouse receiving 225Ac-αCD11b alone. No long-term surviving mice were observed in the control groups. Long-term surviving mice were rechallenged, and potential antitumor immunity was observed, as no tumors developed over 120 days after rechallenge. Overall, these results validate the preclinical relevance of CD11b-targeted image-guided α-emitting radiopharmaceutical therapy.

Radiation treatment is delivered to the entire brain and spine to reduce tumor recurrence in children with medulloblastoma. Long term survivors suffer debilitating neurotoxicity from radiation to the healthy brain. Thus, more precise treatments are needed. We hypothesize that targeted radiopharmaceutical therapy will result in higher uptake within the tumor while sparing the surrounding healthy brain. A bio-informatics analysis of a large dataset (n=1641) was performed to identify a surface marker (SSTR2) that was enriched in medulloblastoma. Validation was done through immunohistochemical staining of patient tumor specimens (n=13). SSTR2 receptor number in medulloblastoma tumor cells was quantified by flow cytometry. Octreotate, a SSTR2-targeted peptide, was radiolabeled with copper-64 and in vitro uptake was assessed in patient-derived cell lines and in vivo uptake in a mouse with an intracranial tumor. In vivo uptake was assayed using positron emission tomography (PET). RNA-seq analysis revealed increased expression of several surface markers including PROM1, EPHA2, and SSTR2 in comparison to normal cerebellum. We elected to focus on SSTR2 given that it has a clinically validated targeting peptide (Octreotate). We observed increased SSTR2 expression in several medulloblastoma molecular subtypes with median z-scores of 0.45 for group 3, 0.47 for group 4, 0.47 for the WNT subtype. One way ANOVA followed by post-hoc testing demonstrated significant differences in SSTR2 expression between group 3, group 4, and WNT subtypes and normal cerebellum (p<0.001). Thirteen out of 14 tumor specimens had 3+ immunohistochemical staining as determined by a board-certified pathologist (D.M.). We determined that the D341 medulloblastoma cell line had the highest number of SSTR2 receptors with a mean and standard deviation of 10,082 +/- 5,934 when compared to our three other cell lines. We explored targeting the SSTR2 receptor using Octreotate radiolabeled with copper-64 (64Cu-DOTA-TATE) and found that specific uptake of 64Cu-DOTA-TATE was highest in D341 cells with a mean and standard deviation of 39,272 +/- 826 counts as compared to 280 +/- 73 counts in D341 cells blocked with excess peptide. Furthermore, 64Cu-DOTA-TATE uptake was increased in all our cells in comparison to cells blocked with excess peptide (p < 0.01). The mean standardized uptake value (SUV) following PET imaging of a mouse with an intracranial tumor was 0.31 in the tumor and 0.08 in the healthy brain at 1 hour, and 0.17 in the tumor and 0.08 in the healthy brain at 24 hours post-injection of 64Cu-DOTA-TATE. The data presented here validates the SSTR2 receptor as a target for selective delivery of radioactivity to intracranial medulloblastoma tumors with reduced radiation exposure to the normal healthy brain. This data supports further investigation of using SSTR2-radiopharmaceuticals to deliver therapeutic radionuclides for tumor-specific radiation with the potential to decrease long term neurotoxicity. Saad Sheikh, Mohamad Rahmdel, Daniel Marker, Kevin Hitchens, Jessie R. Nedrow. Somatostatin directed radiopharmaceutical therapy for medulloblastoma spares healthy brain radiation [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr C089.

Mesothelin (MSLN) is overexpressed in various malignancies, making it a promising target for molecular imaging and therapeutic strategies. Anti-MSLN VH-Fc fusion proteins show high tumor uptake as compared with monoclonal antibodies; however, elevated accumulation in Fc-rich organs (liver, spleen) can compromise tumor-to-background ratios and limit clinical applicability. To overcome this, we developed Fc mutant anti-MSLN VH-Fc fusion proteins incorporating G236R/L328R (GRLR) and L234A/L235A/P329G (LALAPG) mutations to eliminate FcγRs interactions. Engineered mutants exhibited high purity (>95%), retained strong MSLN binding (KD 2.2-3.7 nM), and effectively silenced FcγR binding by ex vivo and in vivo analyses. Following zirconium-89 radiolabeling, PET imaging was conducted across multiple xenograft models with varying MSLN expression. In HCT116 xenografts, [89Zr]Zr-2A10-VH-FcLALAPG demonstrated substantially higher uptake (13.0 ± 0.1%ID/g at 120 h p.i.) than [89Zr]Zr-2A10-VH-FcWT (4.2 ± 0.6%ID/g), while substantially reducing liver (LALAPG: 4.3 ± 0.6%ID/g vs WT: 19.8 ± 2.8%ID/g) and spleen (LALAPG: 9.3 ± 0.1%ID/g vs WT: 95.0 ± 39.3%ID/g) uptake. Biodistribution studies in additional xenograft models confirmed a high specific uptake for [89Zr]Zr-2A10-VH-FcLALAPG in tumors with moderate to high MSLN expression. Notably for the mutants, females exhibited higher renal retention than males, indicating sex-dependent pharmacokinetics. These findings highlight Fc-engineered VH-Fc fusion proteins, particularly the LALAPG, as promising agents with enhanced tumor specificity, improved pharmacokinetics, and significantly reduced off-target uptake, supporting their use in PET imaging-guided therapeutic applications.

Radiopharmaceutical therapy (RPT) is a promising approach to treating solid tumors, but therapeutic advances are impeded by the lack of broadly expressed targets and shared molecular vulnerability across different tumor types. Here, we evaluate VLA-4 (integrin α4β1) as a potential target for RPT in solid tumors and use radiolabeled copper-64 ([64Cu]Cu-) and copper-67 ([67Cu]Cu-CB-TE1A1P-PEG4-LLP2A) LLP2A, a peptidomimetic ligand of VLA-4, for preclinical imaging and RPT testing. Expression of ITGA4, the gene encoding the alpha 4 subunit (CD49d) of VLA-4, was evaluated in a variety of cancer tissues from publicly available datasets. VLA-4 protein expression was determined by flow cytometry in 22 different human and murine cancer cell lines. We used orthotopic syngeneic (i.e., B16-F10, B78, 4T1, GL261, TH-MYCN, and E2A-PBX1) and human (i.e., SK-MEL-37, 143B, and IMR-5) cancer models for in vivo PET/CT imaging and biodistribution studies. Selected models were used for dosimetry calculations with [64Cu]Cu-LLP2A. To assess in vivo tolerability and efficacy, we performed studies of [67Cu]Cu-LLP2A in tumor-free and B16-F10-bearing C57BL/6J mice (activity range, 0-74 MBq [0-2 mCi]), respectively. We found ITGA4 is overexpressed in hematological malignancies and a variety of solid tumors compared with healthy tissue. VLA-4 was expressed at medium to high levels in 17/22 (77%), at low levels in 4/22 (18%), and negative in 1/22 (5%) tested cell lines. PET/CT imaging with [64Cu]Cu-LLP2A showed tracer uptake in tumors and on-target off-tumor uptake in lymphoid tissues. [67Cu]Cu-LLP2A administered at an activity range of 37 to 74 MBq (1-2 mCi) was tolerated and did not cause long-term hematological or tissue toxicity, except for thymic atrophy. We observed tumor dose response to the activity administered to mice with B16-F10 melanoma. In summary, VLA-4 is broadly expressed across a variety of different cancer tissues and preclinical cancer cell lines, making it a promising target for [67Cu]Cu-LLP2A RPT. With proven on-target on-tumor effect, acceptable toxicity profile, and favorable dosimetry in preclinical models, further investigation of [67Cu]Cu-LLP2A as an RPT agent is warranted.

Supplementary Data - Results section

Abstract Lung cancer is the second most common cancer in both men and women in the US and the leading cause of cancer death. Among younger US women, lung cancer incidence rates have in recent years become higher than rates seen in men, despite comparable histories of tobacco use. The possible contribution of stress to higher lung cancer risk in young women has received little research attention despite preclinical evidence that exposure to stress-related neuroendocrine responses can cause DNA damage. The present study used a well-established lung cancer model in young female mice (in vivo exposure to NNK) along with a mouse stress model to explore their independent and combined effects on lung cancer development. In addition, we explored one possible biological pathway by chronic administration of a beta-adrenergic receptor blocker (propranolol). We also tested the use of in vivo imaging for repeated monitoring of tumor development over time. Cohort 1 (n=60) had 4 groups: Home cage control, NNK treatment alone, repeated stress (RS) alone and NNK+RS. Cohort 2 (n=60) had the same 4 groups with the addition of administration of propranolol in drinking water. Cohort 3 (n=48, imaging cohort) had 3 groups: Home cage control, NNK alone and NNK+RS. FVB/N (4-6 wk) were purchased from a commercial supplier and acclimated with gentle handling for two weeks. The repeated stress protocol began week-3 and continued for the duration of the study. It combined two well-established rodent models: social disruption stress via changes in cage mates at the time of cage cleaning (2x/week) and a 90-minute restraint stress immediately prior to cage change (1x/week). NNK or saline treatment began on week-4 (IP; 2x/week; 3 mg/injection) and continued for 4 weeks. 21 weeks after stress initiation, the mice in cohorts 1 and 2 were sacrificed, lungs removed, inflated and tumors counted and sized. Mice in cohort 3 were imaged by Hounsfield-calibrated micro-CT (μCT) at baseline, 14, 19 and 23 weeks. The entire lung space, excluding the heart, was defined as a volumetric region of interests (VOIs). The VOIs were subjected to thresholding to segment the lungs into air space and tissue space compartments. The volume of tissue space of each animal was ratioed against its own baseline tissue space volume. Results from Cohort 1 revealed significantly greater numbers and overall size of lung tumors in stressed mice that received NNK. No significant effects were seen in the absence of NNK. Results from Cohort 2 revealed no significant differences between stressed and unstressed groups treated with propranolol. Cohort 3 revealed a pattern of results with stressed mice generally showing the highest lung tumor volume levels (Hounsfield Units), which reached significance at 19 weeks in preliminary analyses. Conclusion: In this study, we found that repeated stress increased lung tumor burden in female mice treated with NNK, and that a beta-adrenergic pathway may be involved. Citation Format: Frank Jenkins, Laura P. Stabile, Jessie R. Nedrow, Kathryn Day, Joseph D. Latoche, Patrick M. Tarwater, Jessica Manculich, Dana H. Bovbjerg. Effects of repeated psychological stress on NNK-induced lung cancer development in young female mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3391.

Precision management of fibrotic lung diseases is challenging due to their diverse clinical trajectories and lack of reliable biomarkers for risk stratification and therapeutic monitoring. Here, we validated the accuracy of CMKLR1 as an imaging biomarker of the lung inflammation-fibrosis axis. By analyzing single-cell RNA sequencing datasets, we demonstrated CMKLR1 expression as a transient signature of monocyte-derived macrophages (MDMφ) enriched in patients with idiopathic pulmonary fibrosis (IPF). Consistently, we identified MDMφ as the major driver of the uptake of CMKLR1-targeting peptides in a murine model of bleomycin-induced lung fibrosis. Furthermore, CMKLR1-targeted positron emission tomography in the murine model enabled quantification and spatial mapping of inflamed lung regions infiltrated by CMKLR1-expressing macrophages and emerged as a robust predictor of subsequent lung fibrosis. Last, high CMKLR1 expression by bronchoalveolar lavage cells identified an inflammatory endotype of IPF with poor survival. Our investigation supports the potential of CMKLR1 as an imaging biomarker for endotyping and risk stratification of fibrotic lung diseases.

INTRODUCTION: Despite the advent of immunotherapy as a promising therapeutic, glioblastoma (GBM) remains resistant to using checkpoint blockade against programmed cell death protein 1 (PD-1) on T cells. The highly immunosuppressive tumor milieu of GBM prevents rescue of inactivated T cells due to decreased numbers of infiltrating T cells at the tumor site as well as increased recruitment of myeloid cells. Moreover, the current strategy of using antibodies against PD-1 (anti-PD-1) requires multiple intravenous infusions every two or three weeks with debilitating systemic effects. METHODS: Mice orthotopically implanted with GL261 glioma cells were injected with PCL:PEG:PCL hydrogel polymers loaded with anti-PD-1 in one of the following locations: cervical lymph nodes, inguinal lymph nodes, and the tumor site. Mice treated systemically with anti-PD-1 were used as comparative controls. Kaplan-Meier curves were generated for all arms, with subsequent ex vivo flow cytometric staining for CD4, CD8, IFN-y, TNF-a, Foxp3, and PD-1. RESULTS: Mice implanted with PCL:PEG:PCL hydrogels carrying anti-PD-1 at the site of their lymph nodes showed significantly improved survival outcomes compared to mice systemically treated with three doses of anti-PD-1 (p < 0.001). Flow cytometric analysis of lymph nodes and brain tissue in mice injected with this gel demonstrated increased levels of IFN-y, indicating greater reversal of immunosuppression compared to standard treatment. CONCLUSIONS: Our data demonstrates proof of principle of the advantages of using localized therapy that targets lymph nodes for GBM. We propose a paradigm shift for developing new sustained local treatments with immunotherapy that are able to eliminate the need for multiple systemic infusions and their off-target effects.

Patients with HER2-positive and triple negative breast cancer (TNBC) are associated with increased risk to develop metastatic disease including reoccurring disease that is resistant to standard and targeted therapies. The αVβ3 has been implicated in BC including metastatic disease. The aims of this study were to investigate the potential of αVβ3-targeted peptides to deliver radioactive payloads to BC tumors expressing αVβ3 on the tumor cells or limited to the tumors' neovascular. Additionally, we aimed to assess the pharmacokinetic profile of the targeted α-particle therapy (TAT) agent [225Ac]Ac-DOTA-cRGDfK dimer peptide and the in vivo generated decay daughters. The expression of αVβ3 in a HER2-positive and a TNBC cell line were evaluated using western blot analysis. The pharmacokinetics of [111In]In-DOTA-cRGDfK dimer, a surrogate for the TAT-agent, was evaluated in subcutaneous mouse tumor models. The pharmacokinetic of the TAT-agent [225Ac]Ac-DOTA-cRGDfK dimer and its decay daughters were evaluated in healthy mice. Selective uptake of [111In]In-DOTA-cRGDfK dimer was shown in subcutaneous tumor models using αVβ3-positive tumor cells as well as αVβ3-negative tumor cells where the expression is limited to the neovasculature. Pharmacokinetic studies demonstrated rapid accumulation in the tumors with clearance from non-target organs. Dosimetric analysis of [225Ac]Ac-DOTA-cRGDfK dimer showed the highest radiation absorbed dose to the kidneys, which included the contributions from the free in vivo generated decay daughters. This study shows the potential of delivering radioactive payloads to BC tumors that have αVβ3 expression on the tumor cells as well as limited expression to the neovascular of the tumor. Furthermore, this work determines the radiation absorbed doses to normal organs/tissues and identified key organs that act as suppliers and receivers of the actinium-225 free in vivo generated α-particle-emitting decay daughters.

Glioblastoma (GBM) is the most common primary malignant brain tumor. Currently, there are few effective treatment options for GBM beyond surgery and chemo-radiation, and even with these interventions, median patient survival remains poor. While immune checkpoint inhibitors (ICIs) have demonstrated therapeutic efficacy against non-central nervous system cancers, ICI trials for GBM have typically had poor outcomes. TIGIT is an immune checkpoint receptor that is expressed on activated T-cells and has a role in the suppression of T-cell and Natural Killer (NK) cell function. As TIGIT expression is reported as both prognostic and a biomarker for anti-TIGIT therapy, we constructed a molecular imaging agent, [89Zr]Zr-DFO-anti-TIGIT (89Zr-αTIGIT), to visualize TIGIT in preclinical GBM by immunoPET imaging. PET imaging and biodistribution analysis of 89Zr-αTIGIT demonstrated uptake in the tumor microenvironment of GBM-bearing mice. Blocking antibody and irrelevant antibody tracer studies demonstrated specificity of 89Zr-αTIGIT with significance at a late time point post-tracer injection. However, the magnitude of 89Zr-αTIGIT uptake in tumor, relative to the IgG tracer was minimal. These findings highlight the features and limitations of using 89Zr-αTIGIT to visualize TIGIT in the GBM microenvironment.

Abstract Background Surgical resection is integral for the treatment of neuroblastoma, the most common extracranial solid malignancy in children. Safely locating and resecting primary tumor and remote deposits of disease remains a significant challenge, resulting in high rates of complications and incomplete surgery, worsening outcomes. Intraoperative molecular imaging (IMI) uses targeted radioactive or fluorescent tracers to identify and visualize tumors intraoperatively. GD2 was selected as an IMI target, as it is highly overexpressed in neuroblastoma and minimally expressed in normal tissue. Methods GD2 expression in neuroblastoma cell lines was measured by flow cytometry. DTPA and IRDye® 800CW were conjugated to anti-GD2 antibody to generate DTPA-αGD2-IR800. Binding affinity (Kd) of the antibody and the non-radiolabeled tracer were then measured by ELISA assay. Human neuroblastoma SK-N-BE(2) cells were surgically injected into the left adrenal gland of 3.5-5-week-old nude mice and the orthotopic xenograft tumors grew for 5 weeks. 111In-αGD2-IR800 or isotype control tracer was administered via tail vein injection. After 4 and 6 days, mice were euthanized and gamma and fluorescence biodistributions were measured using a gamma counter and ImageJ analysis of acquired SPY-PHI fluorescence images of resected organs (including tumor, contralateral adrenal, kidneys, liver, muscle, blood, and others). Organ uptake was compared by one-way ANOVA (with a separate analysis for each tracer/day combination), and if significant, Sidak’s multiple comparison test was used to compare the uptake of each organ to the tumor. Handheld tools were also used to detect and visualize tumor in situ, and to assess for residual disease following non-guided resection. Results 111In-αGD2-IR800 was successfully synthesized with 0.75-2.0 DTPA and 2–3 IRDye® 800CW per antibody and retained adequate antigen-binding (Kd = 2.39 nM for aGD2 vs. 21.31 nM for DTPA-aGD2-IR800). The anti-GD2 tracer demonstrated antigen-specific uptake in mice with human neuroblastoma xenografts (gamma biodistribution tumor-to-blood ratios of 3.87 and 3.88 on days 4 and 6 with anti-GD2 tracer), while isotype control tracer did not accumulate (0.414 and 0.514 on days 4 and 6). Probe accumulation in xenografts was detected and visualized using widely available operative tools (Neoprobe® and SPY-PHI camera) and facilitated detection ofputative residual disease in the resection cavity following unguided resection. Conclusions We have developed a dual-labeled anti-GD2 antibody-based tracer that incorporates In-111 and IRDye® 800CW for radio- and fluorescence-guided surgery, respectively. The tracer adequately binds to GD2, specifically accumulates in GD2-expressing xenograft tumors, and enables tumor visualization with a hand-held NIR camera. These results encourage the development of 111In-αGD2-IR800 for future use in children with neuroblastoma, with the goal of improving patient safety, completeness of resection, and overall patient outcomes.