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PURPOSE: The lack of effective molecular biomarkers to monitor idiopathic pulmonary fibrosis (IPF) activity or treatment response remains an unmet clinical need. Herein, we determined the utility of fibroblast activation protein inhibitor for positron emission tomography (FAPI PET) imaging in a mouse model of pulmonary fibrosis. METHODS: Pulmonary fibrosis was induced by intratracheal administration of bleomycin (1 U/kg) while intratracheal saline was administered to control mice. Subgroups from each cohort (n=3–5) underwent dynamic 1 h PET/CT after intravenously injecting FAPI-46 radiolabeled with gallium-68 ([(68)Ga]Ga-FAPI-46) at 7 d and 14 d following disease induction. Animals were sacrificed following imaging for ex vivo gamma counting and histologic correlation. [(68)Ga]Ga-FAPI-46 uptake was quantified and reported as percent injected activity per cc (%IA/cc) or percent injected activity (%IA). Lung CT density in Hounsfield units (HU) was also correlated with histologic examinations of lung fibrosis. RESULTS: CT only detected differences in the fibrotic response at 14 d post-bleomycin administration. [(68)Ga]Ga-FAPI-46 lung uptake was significantly higher in the bleomycin group than in control subjects at 7 d and 14 d. Significantly (P=0.0012) increased [(68)Ga]Ga-FAPI-46 lung uptake in the bleomycin groups at 14 d (1.01±0.12 %IA/cc) vs. 7 d (0.33±0.09 %IA/cc) at 60 min post-injection of the tracer was observed. These findings were consistent with an increase in both fibrinogenesis and FAP expression as seen in histology. CONCLUSION: CT was unable to assess disease activity in a murine model of IPF. Conversely, FAPI PET detected both the presence and activity of lung fibrogenesis, making it a promising tool for assessing early disease activity and evaluating the efficacy of therapeutic interventions in lung fibrosis patients.

Movie S3 from Dual Targeting of Tissue Factor and CD105 for Preclinical PET Imaging of Pancreatic Cancer

Determination of NOTA to Fab Fragment Ratio

Supplementary Figure 1. Purification of heterodimer after click chemistry conjugation of ALT-836-Fab and TRC105-Fab Supplementary Figure 2. MALDI-TOF mass spectra of ALT-836-Fab: [M+H]+ = 47266.0, TRC105-Fab: [M+H]+ = 47454.7, and heterodimer: exact [M+H]+ = 103504.0. Supplementary Figure 3. Flow cytometry analysis in BXPC-3 after 1 h incubation of FITC-Fab conjugates for the comparison of their in vitro targeting Supplementary Figure 4. Radiolabeling efficiency at different copper: immunoconjugate ratios were used to determine the number of NOTA per antibody fragment molecule Supplementary Figure 5. PET imaging and biodistribution of 64CuCl2 in BXPC-3 tumor-bearing mice. Supplementary Figure 6. PET imaging and quantitative ROI analysis of 64Cu-NOTA-heterodimer with TF and CD105 dual-blocking in BXPC-3 tumor-bearing mice. Supplementary Figure 7. Coronal images and transverse PET/CT images of normal nude mice at 3, 15, 24 and 30 h following injection of 64Cu-NOTA-heterodimer Supplementary Figure 8. PET ROI-derived quantification of 64Cu-NOTA-heterodimer uptake in the tumor, normal pancreas, blood pool, liver, kidney and muscle at 3 h, 15 h, 24 h and 30 h p.i. of the tracer

Purpose: Pancreatic adenocarcinoma is a highly aggressive cancer, currently treated with limited success and dismal outcomes. New diagnostic and treatment strategies offer the potential to reduce cancer mortality. Developing highly specific noninvasive imaging probes for pancreatic cancer is essential to improving diagnostic accuracy and monitoring therapeutic intervention.Experimental Design: A bispecific heterodimer was synthesized by conjugating an anti-tissue factor (TF) Fab with an anti-CD105 Fab, via the bio-orthogonal “click” reaction between tetrazine (Tz) and trans-cyclooctene (TCO). The heterodimer was labeled with 64Cu for PET imaging of nude mice bearing BXPC-3 xenograft and orthotopic pancreatic tumors.Results: PET imaging of BXPC-3 (TF/CD105+/+) xenograft tumors with 64Cu-labeled heterodimer displayed significantly enhanced tumor uptake (28.8 ± 3.2 %ID/g; n = 4; SD) at 30 hours postinjection, as compared with each of their monospecific Fab tracers (12.5 ± 1.4 and 7.1 ± 2.6 %ID/g; n = 3; SD). In addition, the activity–concentration ratio allowed for effective tumor visualization (tumor/muscle ratio 75.2 ± 9.4 at 30 hours postinjection.; n = 4; SD). Furthermore, 64Cu-NOTA-heterodimer enabled sensitive detection of orthotopic pancreatic tumor lesions with an uptake of 17.1 ± 4.9 %ID/g at 30 hours postinjection and tumor/muscle ratio of 72.3 ± 46.7.Conclusions: This study demonstrates that dual targeting of TF and CD105 provided synergistic improvements in binding affinity and tumor localization of the heterodimer. Dual-targeted imaging agents of pancreatic and other cancers may assist in diagnosing pancreatic malignancies as well as reliable monitoring of therapeutic response. Clin Cancer Res; 22(15); 3821–30. ©2016 AACR.

An immunosuppressive tumor microenvironment has hampered the efficacy of immunotherapy in prostate cancer. However, radiation-induced immunological effects can partly mediate anti-tumor effects by promoting a pro-inflammatory environment potentially responsive to immunotherapy. Herein, we examined the immunomodulatory properties of a radiopharmaceutical therapy (RPT) with NM600 radiolabeled with either a beta or alpha emitter in two prostate cancer models. 225Ac-NM600, but not 177Lu-NM600, promoted significant anti-tumor effects and improved overall survival. Immunomodulatory effects were dose, radionuclide, and tumor type-dependent. 225Ac-NM600 elicited an array of immunomodulatory effects such as increased CD8/Treg ratio, activation of effector and memory T cells, abrogation of infiltrating suppressor cells (e.g., Tregs and MDSCs), and increased levels of Th1 cytokine and pro-inflammatory chemokines. Importantly, we demonstrate the need to carefully characterize the immune responses elicited by RPT both pre-clinically and clinically to maximize tumor control and avoid potential counterproductive immunosuppressive effects.TeaserTargeted alpha therapy can create a pro-inflammatory tumor micro-environment that partly explains stronger anti-tumor responses in prostate cancer

Most shared resource flow cytometry facilities do not permit analysis of radioactive samples. We are investigating low-dose molecular targeted radionuclide therapy (MTRT) as an immunomodulator in combination with in situ tumor vaccines and need to analyze radioactive samples from MTRT-treated mice using flow cytometry. Further, the sudden shutdown of core facilities in response to the COVID-19 pandemic has created an unprecedented work stoppage. In these and other research settings, a robust and reliable means of cryopreservation of immune samples is required. We evaluated different fixation and cryopreservation protocols of disaggregated tumor cells with the aim of identifying a protocol for subsequent flow cytometry of the thawed sample, which most accurately reflects the flow cytometric analysis of the tumor immune microenvironment of a freshly disaggregated and analyzed sample. Cohorts of C57BL/6 mice bearing B78 melanoma tumors were evaluated using dual lymphoid and myeloid immunophenotyping panels involving fixation and cryopreservation at three distinct points during the workflow. Results demonstrate that freezing samples after all staining and fixation are completed most accurately matches the results from noncryopreserved equivalent samples. We observed that cryopreservation of living, unfixed cells introduces a nonuniform alteration to PD1 expression. We confirm the utility of our cryopreservation protocol by comparing tumors treated with in situ tumor vaccines, analyzing both fresh and cryopreserved tumor samples with similar results. Last, we use this cryopreservation protocol with radioactive specimens to demonstrate potentially beneficial effector cell changes to the tumor immune microenvironment following administration of a novel MTRT in a dose- and time-dependent manner.

Systemic radiation treatments that preferentially irradiate cancer cells over normal tissue, known as targeted radionuclide therapy (TRT), have shown significant potential for treating metastatic prostate cancer. Preclinical studies have demonstrated the ability of external beam radiation therapy (EBRT) to sensitize tumors to T cell checkpoint blockade. Combining TRT approaches with immunotherapy may be more feasible than combining with EBRT to treat widely metastatic disease, however the effects of TRT on the prostate tumor microenvironment alone and in combinfation with checkpoint blockade have not yet been studied.C57BL/6 mice-bearing TRAMP-C1 tumors and FVB/NJ mice-bearing Myc-CaP tumors were treated with a single intravenous administration of either low-dose or high-doseOur data suggest that the combination of

Background: Targeted radionuclide therapy (TRT) delivers radiation treatment systemically to tumor sites via a therapeutic radionuclide-linked tumor-selective targeting vector. NM600 is an alkylphosphocholine analog selectively taken up and retained in murine and human tumor cells. We previously showed that low dose radiation delivery with 90Y-NM600 improves tumor response to immune checkpoint inhibitors (ICIs). Understanding the effect of different radionuclide physical properties (emission type, linear energy transfer (LET), half-life, and tissue range) on immunomodulation of metastatic cancers may guide therapy development. Here, we evaluated the type 1 interferon (IFN1) response elicited by 90Y-, 177Lu-, and 225Ac-NM600 in an immunologically cold syngeneic murine tumor model, B78 melanoma. We hypothesized that the unique physical properties of radionuclides will differentially impact immunomodulation by TRT. Methods: Mice bearing B78 WT or Tmem173 -/- CRISPR deletion B78 (STING KO) tumors were randomized to receive 1.5 Gy external beam radiation (EBRT), an equivalent tumor dose of 90Y-, 177Lu-, or 225Ac-NM600 determined by the Monte Carlo-based RAPID platform, or no radiation on day 1. Tumors were harvested on days 4, 7, and 10 for RT-qPCR. Mice bearing two B78 WT or Tmem173 -/- CRISPR deletion B78 (STING KO) tumors were randomized to receive 4 Gy external beam radiation therapy (EBRT), an equivalent tumor dose of 90Y- or 177Lu-NM600, 0.5 μCi 225Ac-NM600, or no radiation +/- dual ICI (anti-CTLA4 and anti-PDL1). Mice were monitored for tumor growth and survival following these treatments. Results: Both EBRT and TRT upregulated expression of IFN1 response-associated genes (Ifnβ1, Mx1) in B78 WT tumors. Only TRT induced upregulation of Ifnβ1 and Mx1 in STING KO B78 tumors. Ddx58, which encodes RIG-I, integral to an alternative IFN1 pathway, was upregulated in both B78 WT and STING KO tumors following 225Ac-NM600, but not other treatments. TRT in B78 STING KO tumors had earlier expression of IFN1 response-associated genes than B78 WT, 225Ac-NM600 in combination with dual ICI improved overall survival over 90Y- or 177Lu-NM600 + ICI and 225Ac-NM600 monotherapy. Conclusions: The distinct physical properties of TRT radiation, γ, β or α, affect the timing, magnitude, and molecular pathways leading to this IFN1 response. Understanding TRT effects on the tumor microenvironment may optimize TRT and immunotherapy. Citation Format: Thanh Phuong T. Nguyen, Caroline P. Kerr, Joseph J. Grudzinski, Carolina A. Ferreira, Julia Sheehan-Klenk, Ohyun Kwon, Maria Powers, Paul A. Clark, Raghava N. Sriramaneni, Reinier Hernandez, Bryan Bednarz, Jamey P. Weichert, Zachary S. Morris. Radionuclide-specific effects of90Y-,177Lu-, or225Ac-NM600 targeted radionuclide therapy on tumor immunomodulation and enhanced immunotherapy response in syngeneic murine tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6407.

Background: Sequencing of immune checkpoint inhibitors (ICI) and external beam radiation therapy (EBRT) for cancer treatment has been studied, but the optimal sequencing has yet to be determined. While some studies have noted therapeutic advantages of priming the tumor immune microenvironment with EBRT prior to ICI, others have described the benefit of modulating the tumor infiltrating lymphocyte (TIL) population with ICI before EBRT. Targeted radionuclide therapy (TRT) approaches allow investigation of how irradiation by a tumor-targeted radionuclide and differences in emission type, linear energy transfer, and dose rate affect optimal timing of ICI administration. NM600 is an alkylphosphocholine analog selectively taken up by tumors capable of chelating numerous radionuclides for comparative studies. Objective: We use two immunologically cold tumor models, MOC2 head and neck squamous cell carcinoma and B78 melanoma, to describe the influence of dose rate on type I interferon (IFN1) signaling and the effect of ICI and 90Y-, 177Lu-, and 225Ac-NM600 TRT sequences on tumor response. Methods: 90Y, 177Lu, or 225Ac were added to culture media in activities estimated using GEANT4 Monte Carlo to deliver 12 Gy to the cell monolayer. qPCR was performed on cDNA from cells irradiated with EBRT, 90Y, 177Lu, or 225Ac, and harvested on days 1, 3, or 7. In vivo dosimetry was performed using the Monte Carlo-based RAPID platform utilizing serial PET/CT or SPECT/CT imaging and/or longitudinal biodistribution. Differences over time (days 4, 7, 14, 21, 28 after RT) in TIL and systemic immune cell populations were measured by flow cytometry following no treatment, 12 Gy EBRT, or 90Y-, 177Lu-, or 225Ac-NM600 in MOC2 tumors. Mice bearing B78 tumors received 1.5 Gy 90Y-, 177Lu-, or 225Ac-NM600, or no radiation on day 1 +/- ICI (anti-CTLA4 + anti-PDL1) on days -3/0/3 (early), 4/7/10 (middle), or 11/14/17 (late). Mice were monitored for tumor growth and survival. Results: TRT and EBRT induced IFN1 responses in MOC2 cells. MOC2 cells treated every 24h with EBRT-matched 90Y/225Ac dose rates led to upregulation of IFN1-associated Ifnb1 and Mx1, mimicking radionuclide-induced responses. Increased tumor CD8/Treg ratios and decreased Tregs were observed at day 7 following all RT forms in MOC2 tumors. Long half-life 225Ac-NM600 (90Y: 65h; 177Lu: 161h; 225Ac: 240h) induced similar TIL changes at day 21. For 1.5 Gy 90Y-, 177Lu-, and 225Ac-NM600, B78 tumor growth delay and statistically significant overall survival benefit over respective TRT monotherapy and control groups was observed with early (day -3/0/3) dual ICI administration. Conclusions: These studies demonstrate novel immunomodulatory effects of α- and β- emitting TRT and the capacity to achieve substantial antitumor responses with appropriate TRT + ICI sequencing. These results may inform clinical trial design of TRT + ICI regimens for patients with metastatic cancers. Citation Format: Caroline P. Kerr, Joseph J. Grudzinski, Carolina A. Ferreira, David Adam, Julia Sheehan-Klenk, Amber M. Bates, Won Jong Jin, Ohyun Kwon, Justin C. Jagodinsky, Maria Powers, Raghava N. Sriramaneni, Paul A. Clark, Luke Zangl, Thanh Phuong T. Nguyen, Anatoly N. Pinchuk, Cynthia Choi, Christopher F. Massey, Reinier Hernandez, Bryan Bednarz, Jamey P. Weichert, Zachary S. Morris. Impact of sequencing of immune checkpoint blockade and targeted radionuclide therapy on murine tumor response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2828.

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.

In breast cancer, the extracellular matrix (ECM) undergoes remodeling and changes the tumor microenvironment to support tumor progression and metastasis. Fibronectin (FN) assembly is an important step in the regulation of the tumor microenvironment since the FN matrix precedes the deposition of various other ECM proteins, controls immune cell infiltration, and serves as a reservoir for cytokines and growth factors. Therefore, FN is an attractive target for breast cancer therapy and imaging. Functional Upstream Domain (FUD) is a 6-kDa peptide targeting the N-terminal 70-kDa domain of FN, which is critical for fibrillogenesis. FUD has previously been shown to function as an anti-fibrotic peptide both in vitro and in vivo. In this work, we conjugated the FUD peptide with 20-kDa of PEG (PEG-FUD) and demonstrated its improved tumor exposure compared to non-PEGylated FUD in a murine breast cancer model via multiple imaging modalities. Importantly, PEG-FUD peptide retained a nanomolar binding affinity for FN and maintained in vitro plasma stability for up to 48 h. Cy5-labeled PEG-FUD bound to exogenous or endogenous FN assembled by fibroblasts. The in vivo fluorescence imaging with Cy5-labeled FUD and FUD conjugates demonstrated that PEGylation of the FUD peptide enhanced blood exposure after subcutaneous (SC) injection and significantly increased accumulation of FUD peptide in 4T1 mammary tumors. Intravital microscopy confirmed that Cy5-labeled PEG-FUD deposited mostly in the extravascular region of the tumor microenvironment after SC administration. Lastly, positron emission tomography/computed tomography imaging showed that (64)Cu-labeled PEG-FUD preferentially accumulated in the 4T1 tumors with improved tumor uptake compared to (64)Cu-labeled FUD (48 h: 1.35 ± 0.05 vs. 0.59 ± 0.03 %IA/g, P< 0.001) when injected intravenously (IV). The results indicate that PEG-FUD targets 4T1 breast cancer with enhanced tumor retention compared to non-PEGylated FUD, and biodistribution profiles of PEG-FUD after SC and IV injection may guide the optimization of PEG-FUD as a therapeutic and/or imaging agent for use in vivo.

BackgroundThe antitumor effects of external beam radiation therapy (EBRT) are mediated, in part, by an immune response. We have reported that a single fraction of 12 Gy EBRT combined with intratumoral anti-GD2 hu14.18-IL2 immunocytokine (IC) generates an effective in situ vaccine (ISV) against GD2-positive murine tumors. This ISV is effective in eradicating single tumors with sustained immune memory; however, it does not generate an adequate abscopal response against macroscopic distant tumors. Given the immune-stimulatory capacity of radiation therapy (RT), we hypothesized that delivering RT toallsites of disease would augment systemic antitumor responses to ISV.MethodsWe used a syngeneic B78 murine melanoma model consisting of a ‘primary’ flank tumor and a contralateral smaller ‘secondary’ flank tumor, treated with 12 Gy EBRT and intratumoral IC immunotherapy to the primary and additional EBRT to the secondary tumor. As a means of delivering RT to all sites of disease, both known and occult, we also used a novel alkylphosphocholine analog, NM600, conjugated to90Y as a targeted radionuclide therapy (TRT). Tumor growth, overall survival, and cause of death were measured. Flow cytometry was used to evaluate immune population changes in both tumors.ResultsAbscopal effects of local ISV were amplified by delivering as little as 2–6 Gy of EBRT to the secondary tumor. When the primary tumor ISV regimen was delivered in mice receiving 12 Gy EBRT to the secondary tumor, we observed improved overall survival and more disease-free mice with immune memory compared with either ISV or 12 Gy EBRT alone. Similarly, TRT combined with ISV resulted in improved overall survival and a trend towards reduced tumor growth rates when compared with either treatment alone. Using flow cytometry, we identified an influx of CD8+T cells with a less exhausted phenotype in both the ISV-targeted primary and the distant secondary tumor following the combination of secondary tumor EBRT or TRT with primary tumor ISV.ConclusionsWe report a novel use for low-dose RT, not as a direct antitumor modality but as an immunomodulator capable of driving and expanding antitumor immunity against metastatic tumor sites following ISV.

There is a clinically unmet need for effective treatments for triple-negative breast cancer (TNBC), as it remains the most aggressive subtype of breast cancer. Herein, we demonstrate a promising strategy using a tumor-targeting alkylphosphocholine (NM600) for targeted radionuclide therapy of TNBC. Methods: NM600 was radiolabeled with 86Y for PET imaging and 177Lu for targeted radionuclide therapy. 86Y-NM600 PET imaging was performed on female BALB/C mice bearing syngeneic 4T07 (nonmetastatic) and 4T1 (metastatic) TNBC tumor grafts (n = 3–5). Quantitative data derived from a PET-image region-of-interest analysis, which was corroborated by ex vivo biodistribution, were used to estimate the dosimetry of 177Lu-NM600 treatments. Weight measurement, complete blood counts, and histopathology analysis were performed to determine 177Lu-NM600 toxicity in naïve BALB/C mice administered 9.25 or 18.5 MBq. Groups of mice bearing 4T07 or 4T1 grafts (n = 5–6) received excipient or 9.25 or 18.5 MBq of 177Lu-NM600 as a single or fractionated schedule, and tumor growth and overall survival were monitored. Results: Excellent tumor targeting and rapid normal-tissue clearance of 86Y-NM600 were noted in both 4T07 and 4T1 murine models. Ex vivo biodistribution corroborated the accuracy of the PET data and validated 86Y-NM600 as a surrogate for 177Lu-NM600. 177Lu-NM600 dosimetry showed absorbed doses of 2.04 ± 0.32 and 1.68 ± 0.06 Gy/MBq to 4T07 and 4T1 tumors, respectively, which were larger than those delivered to liver (1.28 ± 0.09 Gy/MBq) and to bone marrow (0.31 ± 0.05 Gy/MBq). The 177Lu-NM600 injected activities used for treatment were well tolerated and resulted in significant tumor growth inhibition and prolonged overall survival in both tested TNBC models. A complete response was attained in 60% of treated mice bearing 4T07 grafts. Conclusion: Overall, our results suggest that 177Lu-NM600 targeted radionuclide therapy has potential for TNBC and merits further exploration in a clinical setting.

Noninvasive imaging is a powerful tool for understanding the in vivo behavior of drug delivery systems and successfully translating promising platforms into the clinic. Extracellular vesicles (EVs), nano-sized vesicles with a lipid bilayer produced by nearly all cell types, are emerging platforms for drug delivery. To date, the biodistribution of EVs has been insufficiently investigated, particularly using nuclear imaging-based modalities such as positron emission tomography (PET). Herein, we developed positron-emitting radiotracers to investigate the biodistribution of EVs isolated from various human cell sources using PET imaging. Chelator conjugation did not impact EVs size and subsequent radiolabeling was found to be highly efficient and stable with Zr-89 (t1/2 = 78.4 h). In vivo tracking of EVs isolated from bone marrow-derived mesenchymal stromal cells (BMSCs EVs), primary human macrophages (Mϕ EVs), and a melanoma cell line (A375 EVs) were performed in immunocompetent ICR mice. Imaging studies revealed excellent in vivo circulation for all EVs, with a half-life of approximately 12 h. Significantly higher liver uptake was observed for Mϕ EVs, evidencing the tissue tropism of EV and highlighting the importance of carefully choosing EVs cell sources for drug delivery applications. Conversely, the liver, spleen, and lung uptake of the BMSC EVs and A375 EVs was relatively low. We also investigated the impact of immunodeficiency on the biodistribution of BMSC EVs using NSG mice. The spleen uptake drastically increased in NSG mice, which could confound results of therapeutic studies employing this mouse models. Lastly, PET imaging studies in a melanoma tumor model demonstrated efficient tumor uptake of BMSC EVs following intravenous injection. Overall, these imaging studies evidenced the potential of EVs as carriers to treat a variety of diseases, such as cancer or in regenerative medicine applications, and the necessity to understand EVs tropism to optimize their therapeutic deployment.

Rationale Murine syngeneic tumor models have revealed efficacious systemic antitumor responses following primary tumor in situ vaccination combined with targeted radionuclide therapy to secondary or metastatic tumors. Here we present studies on the safety and feasibility of this approach in a relevant translational companion dog model (n = 17 dogs) with advanced cancer. Methods The three component of the combination immuno-radiotherapy approach were employed either separately or in combination in companion dogs with advanced stage cancer. In situ vaccination was achieved through the administration of hypofractionated external beam radiotherapy and intratumoral hu14.18-IL2 fusion immunocytokine injections to the index tumor. In situ vaccination was subsequently combined with targeted radionuclide therapy using a theranostic pairing of IV 86Y-NM600 (for PET imaging and subject-specific dosimetry) and IV 90Y-NM600 (therapeutic radionuclide) prescribed to deliver an immunomodulatory 2 Gy dose to all metastatic sites in companion dogs with metastatic melanoma or osteosarcoma. In a subset of dogs, immunologic parameters preliminarily assessed. Results The components of the immuno-radiotherapy combination were well tolerated either alone or in combination, resulting in only transient low grade (1 or 2) adverse events with no dose-limiting events observed. In subject-specific dosimetry analyses, we observed 86Y-NM600 tumor:bone marrow absorbed-dose differential uptakes ≥2 in 4 of 5 dogs receiving the combination, which allowed subsequent safe delivery of at least 2 Gy 90Y-NM600 TRT to tumors. NanoString gene expression profiling and immunohistochemistry from pre- and post-treatment biopsy specimens provide evidence of tumor microenvironment immunomodulation by 90Y-NM600 TRT. Conclusions The combination of external beam radiotherapy, intratumoral immunocytokine, and targeted radionuclide immuno-radiotherapy known to have activity against syngeneic melanoma in murine models is feasible and well tolerated in companion dogs with advanced stage, spontaneously arising melanoma or osteosarcoma and has immunomodulatory potential. Further studies evaluating the dose-dependent immunomodulatory effects of this immuno-radiotherapy combination are currently ongoing.

BackgroundSystemic radiation treatments that preferentially irradiate cancer cells over normal tissue, known as targeted radionuclide therapy (TRT), have shown significant potential for treating metastatic prostate cancer. Preclinical studies have demonstrated the ability of external beam radiation therapy (EBRT) to sensitize tumors to T cell checkpoint blockade. Combining TRT approaches with immunotherapy may be more feasible than combining with EBRT to treat widely metastatic disease, however the effects of TRT on the prostate tumor microenvironment alone and in combinfation with checkpoint blockade have not yet been studied.MethodsC57BL/6 mice-bearing TRAMP-C1 tumors and FVB/NJ mice-bearing Myc-CaP tumors were treated with a single intravenous administration of either low-dose or high-dose 90Y-NM600 TRT, and with or without anti-PD-1 therapy. Groups of mice were followed for tumor growth while others were used for tissue collection and immunophenotyping of the tumors via flow cytometry.Results90Y-NM600 TRT was safe at doses that elicited a moderate antitumor response. TRT had multiple effects on the tumor microenvironment including increasing CD8 +T cell infiltration, increasing checkpoint molecule expression on CD8 +T cells, and increasing PD-L1 expression on myeloid cells. However, PD-1 blockade with TRT treatment did not improve antitumor efficacy. Tregs remained functional up to 1 week following TRT, but CD8 +T cells were not, and the suppressive function of Tregs increased when anti-PD-1 was present in in vitro studies. The combination of anti-PD-1 and TRT was only effective in vivo when Tregs were depleted.ConclusionsOur data suggest that the combination of 90Y-NM600 TRT and PD-1 blockade therapy is ineffective in these prostate cancer models due to the activating effect of anti-PD-1 on Tregs. This finding underscores the importance of thorough understanding of the effects of TRT and immunotherapy combinations on the tumor immune microenvironment prior to clinical investigation.

Molecular and cellular effects of radiotherapy on tumor microenvironment (TME) can help prime and propagate antitumor immunity. We hypothesized that delivering radiation to all tumor sites could augment response to immunotherapies. We tested an approach to enhance response to immune checkpoint inhibitors (ICIs) by using targeted radionuclide therapy (TRT) to deliver radiation semiselectively to tumors. NM600, an alkylphosphocholine analog that preferentially accumulates in most tumor types, chelates a radioisotope and semiselectively delivers it to the TME for therapeutic or diagnostic applications. Using serial 86Y-NM600 positron emission tomography (PET) imaging, we estimated the dosimetry of 90Y-NM600 in immunologically cold syngeneic murine models that do not respond to ICIs alone. We observed strong therapeutic efficacy and reported optimal dose (2.5 to 5 gray) and sequence for 90Y-NM600 in combination with ICIs. After combined treatment, 45 to 66% of mice exhibited complete response and tumor-specific T cell memory, compared to 0% with 90Y-NM600 or ICI alone. This required expression of STING in tumor cells. Combined TRT and ICI activated production of proinflammatory cytokines in the TME, promoted tumor infiltration by and clonal expansion of CD8+ T cells, and reduced metastases. In mice bearing multiple tumors, combining TRT with moderate-dose (12 gray) external beam radiotherapy (EBRT) targeting a single tumor augmented response to ICIs compared to combination of ICIs with either TRT or EBRT alone. The safety of TRT was confirmed in a companion canine study. Low-dose TRT represents a translatable approach to promote response to ICIs for many tumor types, regardless of location.

Alkylphosphocholine (APC) analogs are a novel class of broad-spectrum tumor-targeting agents that can be used for both diagnosis and treatment of cancer. The potential for clinical translation for APC analogs will strongly depend on their pharmacokinetic (PK) profiles. The aim of this work was to understand how the chemical structures of various APC analogs impact binding and PK. To achieve this aim, we performed in silico docking analysis, in vitro and in vivo partitioning experiments, and in vivo PK studies. Our results have identified 7 potential high-affinity binding sites of these compounds on human serum albumin (HSA) and suggest that the size of the functional group directly influences the albumin binding, partitioning, and PK. Namely, the bulkier the functional groups, the weaker the agent binds to albumin, the more the agent partitions onto lipoproteins, and the less time the agent spends in circulation. The results of these experiments provide novel molecular insights into the binding, partitioning, and PK of this class of compounds and similar molecules, as well as suggests pharmacological strategies to alter their PK profiles. Importantly, our methodology may provide a way to design better drugs by better characterizing PK profile for lead compound optimization.

We characterize the in vivo biodistribution and tumor selectivity of 86Y-NM600, a theranostic alkylphosphocholine radiometal chelate with broad tumor selectivity, in a variety of preclinical cancer models. Methods: Mice bearing flank tumors (representative of lung, pancreatic, prostate, liver, skin, and lymphoid cancers) were injected intravenously (IV) with 9.25 MBq of 86Y-NM600 and imaged longitudinally over 4-5 days using microPET/CT. Percent injected activity per gram (%IA/g) for each volume-of-interest (VOI) was measured at each time point for the organs of interest. Mice were euthanized after the final time point and the tumor and organs of interest were counted with an automatic gamma counter. Absorbed doses delivered by 90Y-NM600 per injected activity (Gy/MBq) were estimated. Mice bearing B78 flank tumors were injected with a prescription of 90Y-NM600 that delivered 2.5 Gy of absorbed tumor dose and was compared to an equivalent absorbed dose delivered via external beam radiotherapy (XRT) using tumor volume as a measure of response. Histology and complete blood counts (CBC) were analyzed in naive C57BL/6 mice who were injected with 9.25 MBq of 90Y-NM600 at 5, 10, and 28 days post injection (p.i.). Results: PET imaging showed consistent tumor accumulation and retention across all tumor models investigated with little off-target retention of NM600 except for in the liver, which is characteristic of hepatobiliary metabolism. The tumor uptake was highest in the pancreatic and lymphoid cancer models, reaching peak concentrations of 9.34 ± 2.66%IA/g (n = 3) and 9.10 ± 0.13 %IA/g (n = 3), respectively, at approximately 40-48 hr post injection. These corresponded to tumor dose estimates of 2.72 ± 0.33 Gy/MBq and 2.67 ± 0.32 Gy/MBq, respectively. In the toxicity study, there were no visible signs of acute toxicity by histology, and perturbation of hematological parameters was transient when observed, returning to pre-therapy levels after 28 days. Conclusion: NM600 is a theranostic agent with a unique ability to selectively target a variety of cancer types, presenting a unique opportunity for PET image guided TRT and combination with immunotherapies.

Introduction: GD2 is expressed on neuroblastoma, melanomas, small cell lung cancers and sarcomas. While it is expressed minimally on normal tissues, it is expressed on nerve cells and, anti-GD2 (dinutuximab) treatment can cause neuropathic pain. To increase tumor specificity, we developed a bispecific SNIPER antibody to simultaneously target two tumor antigens (GD2 and B7H3). B7H3 is overexpressed on multiple tumor types, with minimal expression on most normal cells and is absent on nerve cells. The goal of this SNIPER is to enhance tumor-specificity, while reducing toxicity, ultimately improving efficacy for cancers expressing both targets. Experimental Procedures: SNIPER specificity was tested by flow cytometry for binding to GD2 +/- B7H3 expressing cells. Using an Incucyte S3, we tested antibody internalization properties [compared to anti-B7H3 and dinutuximab monoclonal antibodies (mAbs)] and antibody dependent cellular-cytotoxicity (ADCC) capabilities. Mice bearing variants of GD2/B7H3-expressing tumors were intravenously injected with 89Zr- radiolabeled SNIPER and its longitudinal in vivo biodistribution was monitored via positron emission tomography imaging. In vivo efficacy studies of SNIPER were tested against mice bearing either melanoma or neuroblastoma tumors that express both GD2 and B7H3. Summary of Data: In vitro and in vivo tumor specificity testing confirmed that SNIPER specifically targets B7H3+/GD2+ cells, but it does not bind to GD2+/B7H3- cells (which simulate nerve cells). We observed high internalization of anti-GD2 mAbs; we did not observe antibody internalization of either the SNIPER or anti-B7H3 mAbs. SNIPER was as effective at ADCC as the dinutuximab, but we saw minimal ADCC with the anti-B7H3 mAbs. An afucosylated version of SNIPER showed significantly enhanced ADCC compared to dinutuximab. Our in vivo efficacy studies found that SNIPER was as as dinutuximab when given at the same dose. Conclusions: The Fab arms of SNIPER targeting GD2 and B7H3 each have low-to-moderate affinity. SNIPER binds with strong avidity when both arms bind to their antigens on the same cell. Stronger avidity through both arms binding results in high-tumor specificity. Because SNIPER should not bind to nerves, it may be possible to administer increased doses of SNIPER beyond the tolerable dose of dinutuximab, which could further improve efficacy. Ongoing studies include antitumor efficacy testing, nerve binding assays and assessing reduction of pain in vivo with SNIPER. Citation Format: Amy K. Erbe, Arika Feils, Zack Rosenkrans, Jessica Wiwczar, Daniel Gerhardt, Bonnie Hammer, Mildred Felder, Mark Bercher, Alina Hampton, Lizzie Frankel, Dan Spiegelman, Noah Tsarovsky, Alexander Rakhmilevich, Jacquelyn Hank, Bryan Glaser, Reinier Hernandez, Roland Green, Paul M. Sondel. Tumor-targeting and efficacy of B7H3/GD2 bispecific SNIPER antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2019.

Purpose: In preclinical studies, we have demonstrated that delivering low dose radiation to all tumor sites utilizing 90Y-NM600 improves the response to immune checkpoint inhibitors (ICIs). NM600 is an alkylphosphocholine analog that is selectively taken up and retained in murine and human tumors. In this study, the immunomodulatory capacities of three distinct radionuclides (90Y, 177Lu, 225Ac) were compared using immunologically cold syngeneic murine tumor models: MOC2 head and neck squamous cell carcinoma (HNSCC) and B78 melanoma. We hypothesized that physical properties of radionuclides (e.g. emission type, linear energy transfer (LET), half-life, dose rate) would differentially impact immunomodulation by TRT and ICI treatment response. Methods: Dosimetry performed using the Monte Carlo-based RAPID platform utilizing serial 86Y-NM600 PET/CT imaging determined that 100 μCi 90Y-NM600 or 200 μCi 177Lu-NM600 delivered ~12Gy to the MOC2 tumor and ~4Gy to the B78 tumor. 225Ac-NM600 was dosed at activities ≤0.5 μCi, as determined to be safe and effective previously. Mice bearing two MOC2, B78 WT, or Tmem173 -/- CRISPR deletion B78 (STING KO) tumors were randomized to receive external beam radiation therapy (EBRT), an equivalent tumor dose of 90Y- or 177Lu-NM600, 0.25 μCi 225Ac-NM600, or no radiation +/- dual ICI (anti-CTLA4 and anti-PDL1). Tumors, blood, bone marrow, and spleen were harvested at days 4, 7, 14, and 21 post-treatment for flow cytometry and RT-qPCR. Additional mice were monitored for tumor growth and survival following these treatments. Results: TRT and EBRT induced favorable tumor-specific immune cell infiltration (increased CD8/Treg ratio) at day 7. 225Ac-NM600 additionally induced similar changes at day 21, consistent with the longer half-life radioisotope (90Y: 65h; 177Lu: 161h; 225Ac: 240h). Expression of immune susceptibility markers (Mhc1, Pdl1) and type I interferon (IFN1) response-associated genes (Ifnβ1, Mx1) was upregulated following 12 Gy EBRT or TRT both in vitro and in vivo compared to non-irradiated controls in MOC2 cells and tumors. The timing and magnitude of these effects correlated with radionuclide half-life and LET. 225Ac-NM600 + ICI improved overall survival in B78 WT mice over 90Y- or 177Lu-NM600 + ICI, 225Ac-NM600 or ICI monotherapy, and non-irradiated controls. In the B78 STING KO melanoma cell line, this 225Ac-NM600 + ICI survival benefit was decreased. Conclusions: These studies demonstrate the capacity to deliver immunomodulatory radiation to tumors using gamma-, beta- or alpha-emitting TRT. The physical properties of the delivered radionuclide dictate timing and magnitude of the IFN1 response stimulated by TRT. Understanding these effects may be critical to safely and effectively integrating TRT and immunotherapies to enhance anti-tumor immunity in cancer patients. Citation Format: Caroline P. Kerr, Amber M. Bates, Joseph J. Grudzinski, Carolina A. Ferreira, Julia Sheehan-Klenk, David Adam, Maria Powers, Wonjong Jin, Justin C. Jagodinsky, Raghava N. Sriramaneni, Paul A. Clark, Luke Zangl, Anatoly N. Pinchuk, Alejandro J. Onate, Ria Kumari, Cynthia Choi, Christopher F. Massey, Bryan Bednarz, Reinier Hernandez, Jamey P. Weichert, Zachary S. Morris. Radionuclide-specific effects of 90Y-, 177Lu-, or 225Ac-NM600 targeted radionuclide therapy on tumor immunomodulation and enhancing immunotherapy response in murine tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1306.

We present a simplified, automatable single-column radiochemical separation method using the extraction chromatographic branched-DGA resin for the production of no-carrier-added 86Y with a radiochemical yield higher than 95%, an apparent molar activity of 1.4 ± 0.4 Ci/μmol (DOTA) and 2.3 ± 0.7 Ci/μmol (DTPA), and a run-to-run recycling efficiency of the isotopically-enriched target of 98 ± 1%. These results enable the preparation of 86Y radiopharmaceuticals for 86Y/90Y-based cancer theranostic applications.

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 mm(3)) were treated following a previously optimized ISV regimen [radiation (12 Gy × 1, treatment day 1), IT-IC (50 lg daily, treatment days 6–10), and + anti-CTLA-4 (100 lg, 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 ((86)Y) 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 β-particle-emitting (90)Y 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.

Background Disialoganglioside 2 (GD2) is expressed on neuroblastomas as well as melanomas, small cell lung cancers, and sarcomas. Anti-GD2 mAb (Dinutuximab) can be used to treat these cancers and is part of the standard care for neuroblastoma. While GD2 is expressed minimally on most normal tissues, it is expressed on some nerve cells, and anti-GD2 treatment can cause neuropathic pain. A separate tumor-antigen, B7H3, is overexpressed on multiple tumor types, including those listed above, with minimal expression on most normal cells and no expression on nerve cells. We developed a bispecific SNIPER antibody, INV721, to simultaneously target these 2 tumor antigens, with one arm specific to GD2 and the other arm to B7H3. The individual Fab arms targeting GD2 and B7H3 are each low to moderate affinity, such that INV721 will only bind with high affinity when both arms bind to their antigens on the same cell, resulting in high-specificity of the SNIPER to tumor cells. Methods INV721 binding to GD2/B7H3-expressing tumors was confirmed by flow cytometry, as well as in tumor-bearing mice injected with 89Zr-labeled to monitor in vivo biodistribution via positron emission tomography imaging. Antibody-dependent cellular cytotoxicity (ADCC) testing of INV721 was performed on human neuroblastoma and melanoma cell lines with an Incucyte spheroid-killing-assay. In vivo efficacy studies were carried out in mice bearing GD2/B7H3-expressing melanoma tumors to test our in situ vaccine (ISV) regimen, which included testing combinations of external beam radiation therapy (RT, 12Gy) ± INV721 (40 ug/dose) ± IL2 (75K U/dose). Results INV721 showed binding by flow cytometry to tumors that express both GD2 and B7H3 but minimal binding to cells that don’t express both antigens. 89Zr-INV721 showed elevated and persistent accumulation in the tumor with minimal uptake in normal tissues. Incucyte spheroid-killing assays revealed that INV721 was capable of ADCC. The ISV combination of RT+INV721+IL2 was capable of curing mice bearing ~57 mm3 melanoma tumors (12/12 mice tumor free), with >70% of these mice exhibiting long-term immune memory. Conclusions INV721 binds to cells that express both GD2 and B7H3, and these preliminary studies show that INV721 is effective in our ISV regimen at curing mice bearing tumors that express these antigens. We are continuing our efforts to determine if INV721 is associated with less pain than Dinutuximab. The goal of this SNIPER-antibody is to enhance the tumor-specific delivery of therapeutic mAbs, which may decrease toxicity and improve efficacy for cancers expressing both GD2 and B7H3.

The aim of this work was to explore (132)La as a PET imaging surrogate for (225)Ac using a DOTA-based, tumor-targeting alkylphosphocholine (NM600). Methods: (132)La was produced on a biomedical cyclotron. For in vivo experiments, mice bearing 4T1 tumors were administered (132)La-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 (225)Ac-NM600. Results: PET/CT images showed elevated, persistent (132)La-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 (132)La-NM600 and (225)Ac-NM600 tissue distributions revealed a similar biodistribution for the 2 radiotracers. Conclusion: These findings suggest that (132)La is a suitable imaging surrogate to probe the in vivo biodistribution of (225)Ac radiotherapeutics.

INTRODUCTION: The remarkable stability of the (89)Zr-DOTA complex has been shown in recent literature. The formation of this complex appears to require (89)Zr-chloride as the complexation precursor rather than the more conventional (89)Zr-oxalate. In this work we present a method for the direct isolation of (89)Zr-chloride from irradiated (nat)Y foils. METHODS: (89)Zr, (88)Zr, and (88)Y were prepared by 16 MeV proton irradiation of (nat)Y foils and used for batch-extraction based equilibrium coefficient measurements for TBP and UTEVA resin. Radionuclidically pure (89)Zr was prepared by 14 MeV proton-irradiation of (nat)Y foils. These foils were dissolved in concentrated HCl, trapped on columns of TBP or UTEVA resin, and (89)Zr-chloride was eluted in < 1 mL of 0.1 M HCl. For purposes of comparison, conventionally-isolated (89)Zr-oxalate was converted to (89)Zr-chloride by trapping, rinsing, and elution from a QMA cartridge into 1 M HCl. Trace metal analysis was performed on the resulting (89)Zr products. RESULTS: Equilibrium coefficients for Y and Zr were similar between UTEVA and TBP resins across all HCl concentrations. K(d) values of less than 10(−1) mL/g were observed for Y across all HCl concentrations. K(d) values of greater than 10(3) mL/g were observed at HCl concentrations greater than 9 M for Zr, falling to K(d) values of less than 10° mL/g at low HCl concentrations. (89)Zr-chloride was recovered from small columns of TBP in

This work presents the production with a cyclotron of the positron emitter 55Co via the 54Fe(d,n) and 58Ni(p,α) reactions and the Auger electron emitter 58mCo via the 57Fe(d,n) reaction after high current (40μA p and 60μA d) irradiation on electroplated targets. High specific activity radionuclides (up to 55.6 GBq/μmol 55Co and 31.8GBq/μmol 58mCo) with high radionuclidic purity (99.995% 55Co from 54Fe, 98.8% 55Co from 58Ni, and 98.7% 58mCo from 57Fe at end of bombardment, EoB), in high activity concentration (final separated radionuclide in 92%) were obtained after processing of the irradiated targets with novel radiochemical separation methods based on HCl dissolution and the resin N,N,N',N'-tetrakis-2-ethylhexyldiglycolamide (DGA, branched). One hour long irradiations using 38-65, 110-214 and 59-78mg of enriched 54Fe (99.93%), 58Ni (99.48%) and 57Fe (95.06%), respectively, electroplated over a 1.0cm2 surface, yielded 582 ± 66MBq 55Co, 372 ± 14MBq 55Co and 810 ± 186MBq 58mCo, respectively, decay corrected to EoB. The separation methods allow for the recovery of the costly enriched target materials, which were reconstituted into metallic targets after novel electroplating methods, with an overall recycling efficiency of 93 ± 4% for iron. The produced radionuclides were used to radiolabel the angiogenesis marker antibody TRC105 conjugated to the chelator NOTA as a demonstration of their quality.

Background: Radiation (RT) activates a type 1 interferon (IFN1) response and this is critical to the effect of RT in priming a response to immune checkpoint blockade (ie anti-CTLA4). However, little is known about the time course of this effect. Clinical interest in utilizing systemically administered targeted radionuclide therapy agents (TRT) is growing as these agents can be used to target multiple sites of disease including micro-metastases. It is unclear how IFN1 activation induced by continuous delivery of RT during exponential decay of a TRT source will compare to that induced following instantaneous external beam RT (EBRT). Here we report the time course of IFN1 response following RT in vitro and in vivo. Methods: For in vitro studies, we utilized murine models of melanoma (B16, B16 STING knockout, B78), and head and neck squamous cell carcinoma (MOC2). EBRT was prescribed to 2.5 Gy, 12 Gy, or 3 fractions of 8 Gy. For in vivo studies, syngeneic C57BL/6 mice were engrafted with either B78 or MOC2 cells on the flank and RT was delivered when mean tumor size was 100-150 mm3. EBRT was prescribed to 2.5 Gy or 12 Gy. For TRT, we used 90Y conjugated to NM600, an alkylphosphocholine analog that exhibits selective uptake and retention in tumor cells of nearly any type, including B78 and MOC2. Tumor-specific dosimetry for 90Y-NM600 was determined using sequential 86Y-NM600 PET/CT imaging (3h, 24h, 48h, 72h) and a Monte Carlo based dose calculation platform. TRT was prescribed to a cumulative absorbed dose of 2.5 Gy or 12 Gy. Following delivery of RT, cells or tumors were harvested at 1d, 7d, and 14d post RT and RNA was isolated. Gene expression of Ifn-β and IFN response elements was quantified by qPCR and normalized to untreated controls. 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 anti-CTLA-4 therapy reduced tumor growth and prolonged survival compared to single agent therapy. Conclusions: We report the time course of the STING-dependent IFN1 response following RT in multiple murine tumor models. We show the potential of TRT to stimulate IFN1 activation that is comparable to that observed with EBRT of equivalent cumulative dose. Further evaluation of the timing and magnitude of IFN1 response following EBRT and TRT may be critical to the optimal integration with immunotherapies. Citation Format: Justin C. Jagodinsky, Amber M. Bates, Reinier Hernandez, Joseph J. Grudzinski, Ian R. Marsh, Ishan Chakravarty, Ian S. Arthur, Luke M. Zangl, Ryan J. Brown, Erin J. Nystuen, Sarah E. Emma, Caroline Kerr, Won Jong Jin, Peter M. Carlson, Jonathan W. Engle, Eduardo Aluicio-Sarduy, Todd E. Barnhart, Trang Le, KyungMann Kim, Bryan P. Bednarz, Jamey P. Weichert, Ravi B. Patel, Zachary S. Morris. Temporal analysis of type 1 interferon activation in tumor cells following external beam radiotherapy or targeted radionuclide therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3060.

We have observed in preclinical studies that the delivery of low dose targeted radionuclide therapy (TRT) therapy to sites of primary and metastatic cancer can improve the anti-tumor immune response to immune checkpoint inhibition (ICI) with anti-CTLA4 or anti-PDL1. NM600, an alkylphosphocholine that can be radiolabeled with 90Y, is taken up and retained in most cancer cells following intravenous injection. Bempegaldesleukin (BEMPEG) is a first in class, CD122-preferential IL2 pathway agonist that can selectively stimulate an immune response. We hypothesized that combining low dose TRT with BEMPEG would increase immune activation to enhance the response to ICIs in immunologically “cold” murine cancer models. C57Bl/6 female mice were subcutaneously engrafted in the flank with the murine head and neck squamous cell carcinoma (HNSCC) cell line, MOC2. In vivo dosimetry performed using the Monte Carlo based RAPID platform following serial 86Y-NM600 PET/CT imaging demonstrated that 100 µCi of 90Y-NM600 would deliver ~8 Gy to the MOC2 tumor. Mice bearing MOC2 tumors (mean volume ~100 mm3) received combinations of 90Y-NM600 (100 µCi, day 1 IV), BEMPEG (16 µg, days 6, 15, and 24 IV), and anti-CTLA4 (200 µg, days 4, 7, and 10 IP) using a 2x2x2 study design. Tumor growth and survival were monitored. Blood was collected weekly and analyzed on the Abaxis VetScan HM5 to evaluate for toxicity. In a parallel 2x2x2 survival study, an orthotopic model generated by engrafting MOC2 tumors in the cheek was used, and mice were treated with combinations of BEMPEG, 90Y-NM600, and anti-PDL1 (200 µg, days 4, 7, and 10, IP). Mice were euthanized when >25% weight loss was observed. Similar experiments using 90Y-NM600 and BEMPEG were performed in additional syngeneic mouse cancer models including SCC7 (HNSCC), 4T1 (breast), LLC (lung), and Panc02 (pancreatic). In the immunologically “cold” MOC2 HNSCC model, 62.5% of mice treated with 90Y-NM600 (TRT), BEMPEG, and anti-CTLA4; and 44.4% of mice treated with 90Y-NM600, BEMPEG, and anti-PDL1 experienced complete tumor response with no observable primary or metastatic disease at day 60. In mice treated with 90Y-NM600 and BEMPEG, tumor regression followed by escape was seen without ICI. By day 60, no mice receiving single or dual treatment combinations exhibited a complete tumor response. Comprehensive whole blood analysis did not show any major hematologic toxicities. This treatment was explored in mice bearing SCC7, 4T1, Panc02, and LLC tumors, and similar trends were seen. Combination of 90Y-NM600, BEMPEG, and ICI displays robust anti-tumor activity that prevents metastatic disease progression and prolongs survival in spontaneously metastatic, immunologically “cold” tumor models. Clinical studies are warranted to test the safety and efficacy of this promising combined modality treatment regimen. Citation Format: Elizabeth G. Sumiec, Amber M. Bates, Reinier Hernandez, Joseph J. Grudzinski, Ian R. Marsh, Sarah E. Emma, Erin J. Nystuen, Justin C. Jagodinsky, Alexander A. Pieper, Gustavo A. Sosa, Bryan P. Bednarz, Ravi B. Patel, Jamey Weichert, Zachary S. Morris. In vivo synergy of 90Y-NM600 and Bempegaldesleukin improves anti-tumor efficacy of immune checkpoint inhibitors in syngeneic murine cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 679.

The purpose of this study was to evaluate mechanisms of cooperative therapeutic effects of bempegaldesleukin (BEMPEG; NKTR-214) and 90Y-NM600 in head and neck squamous cell carcinoma (HNSCC). BEMPEG is a first in class, CD122-preferential interleukin-2 (IL2) pathway agonist tailored to stimulate antitumor immunity through promoting activation and proliferation of CD8+ T and NK cells. We have observed that targeted radionuclide therapy (TRT) delivered at low doses to sites of metastatic cancer can enhance immune susceptibility in immunologically “cold” tumors such as the MOC2 syngeneic mouse HNSCC model. NM600 is an alkylphosphocholine analog that is selectively sequestered and retained by most human and murine cancer cells. We hypothesized that combining BEMPEG and 90Y-NM600 would cooperate to increase immunosusceptibility and immune cell tumor infiltration and create an overall more favorable immune microenvironment in MOC2 HNSCC tumors. MOC2 tumors were engrafted in the flank of C57BL/6 female mice. Once tumors reached ~100 mm³, mice were randomized into one of eight groups for a survival study utilizing varying combinations of BEMPEG (16 µg, days 6, 15, 24 IV), 90Y-NM600 (100 µCi, day 1 IV), and anti-CTLA4 (200 µg, days 4, 7, 10 IP). In vivo dosimetry was performed prior to day 1 using the Monte Carlo based RAPID platform. Serial 86Y-NM600 PET/CT imaging indicated the dose delivered to the tumor was ~8 Gy. Cohorts of mice in a parallel study were treated with PBS (control), BEMPEG, 90Y-NM600, or BEMPEG and 90Y-NM600. Flow cytometry, qPCR analysis, multiplex cytokine analyses were used to evaluate tumors collected at day 14. In this immunologically “cold” murine HNSCC model, a complete tumor response was observed in 62.5% of mice treated with the triple combination therapy of BEMPEG, 90Y-NM600, and anti-CTLA4. Compared to single therapy groups, mice treated with BEMPEG and 90Y-NM600 had increased CD8+ T cell tumor infiltrate, and 90Y-NM600 induced increased expression of the IL2βγ receptor, CD122, on the surface of CD8 T cells. Tumors from mice treated with BEMPEG and 90Y-NM600 had increased expression of genes associated with tumor cell immune susceptibility (Pdl1, Mhc1, Fas), a type 1 interferon response (Mx1, Trex1, Oas2, Oas3), tumor immune cell recruitment (MIP1α , Tnfα, Mac1, Csf1, Cxcl11) activation of cytotoxic T lymphocytes (Ifny, Cxcl10), and production of immune stimulatory cytokines (IFNy, IL3, IL4, IL5, IL17, RANTES, TNFα, MIP1β, MIP1α, CXCL10, CXCL9). These results suggest a synergistic interaction between BEMPEG and 90Y-NM600 that improves the immune microenvironment in this difficult to treat murine model of HNSCC, which may thereby enhance the response to immune checkpoint blockade. Further studies are warranted to examine the therapeutic implications of this combination in patients. Citation Format: Sarah E. Emma, Amber M. Bates, Reinier Hernandez, Joseph J. Grudzinski, Ian R. Marsh, Justin C. Jagodinsky, Bryan P. Bednarz, Alexander A. Pieper, Elizabeth G. Sumiec, Erin J. Nystuen, Gustavo A. Sosa, Ravi B. Patel, Jamey Weichert, Zachary S. Morris. Mechanisms of cooperative response to bempegaldesleukin (BEMPEG) and 90Y-NM600 targeted radionuclide therapy in the treatment of a syngeneic murine model of head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 508.

Overexpression of tissue factor (TF) has been associated with increased tumor growth, tumor angiogenesis, and metastatic potential in many malignancies, including pancreatic cancer. Additionally, high TF expression was shown to strongly correlate with poor prognoses and decreased survival in pancreatic cancer patients. Herein, we exploited the potential targeting of TF for positron emission tomography (PET) imaging of pancreatic cancer. The TF-targeted tracer was developed through radiolabeling of the anti-human TF monoclonal antibody (ALT-836) with 89Zr. The tracer was characterized by fluorescence microscopy and flow cytometry assays in BXPC-3 and PANC-1 cells, two pancreatic cancer cell lines with high and low TF expression levels, respectively. Non-invasive PET scans were acquired in tumor-bearing mice injected with 89Zr-Df-ALT-836. Additionally, ex vivo biodistribution, blocking, and histological studies were performed to establish the affinity and specificity of 89Zr-Df-ALT-836 for TF in vivo. 89Zr-labeling of Df-ALT-836 was achieved in high yield and good specific activity. FACS and microscopy studies revealed no detectable difference in TF-binding affinity between ALT-836 and Df-ALT-836 in vitro. Longitudinal PET scans unveiled a lasting and prominent 89Zr-Df-ALT-836 uptake in BXPC-3 tumors (peak at 31.5 ± 6.0 %ID/g at 48 h post-injection; n=3), which was significantly abrogated (2.3 ± 0.5 %ID/g at 48 h post-injection; n=3) when mice were pre-injected with a blocking dose (50 mg/kg) of unlabeled ALT-836. Ex vivo biodistribution data confirmed the accuracy of the PET results, and histological analysis correlated high tumor uptake with in situ TF expression. Taken together, these results attest to the excellent affinity and TF-specificity of 89Zr-Df-ALT-836 in vivo. With elevated, persistent, and specific accumulation in TF-positive BXPC-3 tumors, PET imaging using 89Zr-Df-ALT-836 promises to open new avenues for improving future diagnosis, stratification, and treatment response assessment in pancreatic cancer patients.

Nivolumab is a human monoclonal antibody specific for programmed cell death-1 (PD-1), a negative regulator of T-cell activation and response. Acting as an immune checkpoint inhibitor, nivolumab binds to PD-1 expressed on the surface of many immune cells and prevents ligation by its natural ligands. Nivolumab is only effective in a subset of patients, and there is limited evidence supporting its use for diagnostic, monitoring, or stratification purposes. 89Zr-Df-nivolumab was synthesized to map the biodistribution of PD-1-expressing tumor infiltrating T-cells in vivo using a humanized murine model of lung cancer. The tracer was developed by radiolabeling the antibody with the positron emitter zirconium-89 (89Zr). Imaging results were validated by ex vivo biodistribution studies, and PD-1 expression was validated by immunohistochemistry. Data obtained from PET imaging were used to determine human dosimetry estimations. The tracer showed elevated binding to stimulated PD-1 expressing T-cells in vitro and in vivo. PET imaging of 89Zr-Df-nivolumab allowed for clear delineation of subcutaneous tumors through targeting of localized activated T-cells expressing PD-1 in the tumors and salivary glands of humanized A549 tumor-bearing mice. In addition to tumor uptake, salivary and lacrimal gland infiltration of T-cells was noticeably visible and confirmed via histological analysis. These data support our claim that PD-1-targeted agents allow for tumor imaging in vivo, which may assist in the design and development of new immunotherapies. In the future, noninvasive imaging of immunotherapy biomarkers may assist in disease diagnostics, disease monitoring, and patient stratification.

Increased angiogenesis is a marker of aggressiveness in many cancers. Targeted radionuclide therapy of these cancers with angiogenesis-targeting agents may curtail this increased blood vessel formation and slow the growth of tumors, both primary and metastatic. CD105, or endoglin, has a primary role in angiogenesis in a number of cancers, making this a widely applicable target for targeted radioimmunotherapy. The anti-CD105 antibody, TRC105 (TRACON Pharmaceuticals), was conjugated with DTPA for radiolabeling with 177Lu (t 1/2 6.65 days). Balb/c mice were implanted with 4T1 mammary carcinoma cells, and five study groups were used: 177Lu only, TRC105 only, 177Lu-DTPA-IgG (a nonspecific antibody), 177Lu-DTPA-TRC105 low-dose, and 177Lu-DTPA-TRC105 high-dose. Toxicity of the agent was monitored by body weight measurements and analysis of blood markers. Biodistribution studies of 177Lu-DTPA-TRC105 were also performed at 1 and 7 days after injection. Ex vivo histology studies of various tissues were conducted at 1, 7, and 30 days after injection of high-dose 177Lu-DTPA-TRC105. Biodistribution studies indicated steady uptake of 177Lu-DTPA-TRC105 in 4T1 tumors between 1 and 7 days after injection (14.3 ± 2.3%ID/g and 11.6 ± 6.1%ID/g, respectively; n = 3) and gradual clearance from other organs. Significant inhibition of tumor growth was observed in the high-dose group, with a corresponding significant increase in survival (p

Manganese has long been employed as a T1-shortening agent in magnetic resonance imaging (MRI) applications, but these techniques are limited by the biotoxicity of bulk-manganese. Positron emission tomography (PET) offers superior contrast sensitivity compared with MRI, and recent preclinical PET studies employing 52gMn (t1/2: 5.6 d, β+: 29%) show promise for a variety of applications including cell tracking, neural tract tracing, immunoPET, and functional β-cell mass quantification. The half-life and confounding gamma emissions of 52gMn are prohibitive to clinical translation, but the short-lived 51Mn (t1/2: 46 min, β+: 97%) represents a viable alternative. This work develops methods to produce 51Mn on low-energy medical cyclotrons, characterizes the in vivo behavior of 51MnCl2 in mice, and performs preliminary human dosimetry predictions. 51Mn was produced by proton irradiation of electrodeposited isotopically-enriched 54Fe targets. Radiochemically isolated 51MnCl2 was intravenously administered to ICR mice which were scanned by dynamic and static PET, followed by ex vivo gamma counting. Rapid blood clearance was observed with stable uptake in the pancreas, kidneys, liver, heart, and salivary gland. Dosimetry calculations predict that 370 MBq of 51Mn in an adult human male would yield an effective dose equivalent of approximately 13.5 mSv, roughly equivalent to a clinical [18F]-FDG procedure.

Item does not contain fulltext Pretargeted radioimmunotherapy (PRIT) with the beta-emitting radionuclide 177Lu is an attractive approach to treat carcinoembryonic antigen (CEA)-expressing tumors. The therapeutic efficacy of PRIT might be improved using alpha-emitting radionuclides such as 213Bi. Herein, we report and compare the tumor-targeting properties and therapeutic efficacy of 213Bi and 177Lu for PRIT of CEA-expressing xenografts, using the bispecific monoclonal antibody TF2 (anti-CEA x anti-histamine-succinyl-glycine [HSG]) and the di-HSG-DOTA peptide IMP288. Methods: The in vitro binding characteristics of 213Bi-IMP288 were compared with those of 177Lu-IMP288. Tumor targeting of 213Bi-IMP288 and 177Lu-IMP288 was studied in mice bearing subcutaneous LS174T tumors that were pretargeted with TF2. Finally, the effect of 213Bi-IMP288 (6, 12, or 17 MBq) and 177Lu-IMP288 (60 MBq) on tumor growth and survival was assessed. Toxicity was determined by monitoring body weight, analyzing blood samples for hematologic and renal toxicity (hemoglobin, leukocytes, platelets, creatinine), and immunohistochemical analysis of the kidneys. Results: The in vitro binding characteristics of 213Bi-IMP288 (dissociation constant, 0.45 +/- 0.20 nM) to TF2-pretargeted LS174T cells were similar to those of 177Lu-IMP288 (dissociation constant, 0.53 +/- 0.12 nM). In vivo accumulation of 213Bi-IMP288 in LS174T tumors was observed as early as 15 min after injection (9.2 +/- 2.0 percentage injected dose [%ID]/g). 213Bi-IMP288 cleared rapidly from the circulation; at 30 min after injection, the blood levels were 0.44 +/- 0.28 %ID/g. Uptake in normal tissues was low, except for the kidneys, where uptake was 1.8 +/- 1.1 %ID/g at 30 min after injection. The biodistribution of 213Bi-IMP288 was comparable to that of 177Lu-IMP288. Mice treated with a single dose of 213Bi-IMP288 or 177Lu-IMP288 showed significant inhibition of tumor growth. Median survival for the groups treated with phosphate-buffered saline, 6 MBq 213Bi-IMP288, 12 MBq 213Bi-IMP288, and 60 MBq 177Lu-IMP288 was 22, 31, 45, and 42 d, respectively. Mice receiving 17 MBq 213Bi-IMP288 showed significant weight loss, resulting in a median survival of only 24 d. No changes in hemoglobin, platelets, or leukocytes were observed in the treatment groups. However, immunohistochemical analysis of the kidneys of mice treated with 17 or 12 MBq 213Bi-IMP288 showed signs of tubular damage, indicating nephrotoxicity. Conclusion: To our knowledge, this study shows for the first time that PRIT with TF2 and 213Bi-IMP288 is feasible and at least as effective as 177Lu-IMP288. However, at higher doses, kidney toxicity was observed. Future studies are warranted to determine the optimal dosing schedule to improve therapeutic efficacy while reducing renal toxicity.