Effects of clinically relevant radionuclides on the activation of a type I interferon response by radiopharmaceuticals in syngeneic murine tumor models.

Radiopharmaceutical therapies (RPT) activate a type I interferon (IFN1) response in tumor cells. We hypothesized that the timing and amplitude of this response varies by isotope. We compared equal doses delivered by 90 Y, 177 Lu, and 225 Ac in vitro as unbound radionuclides and in vivo when chelated to NM600, a tumor-selective alkylphosphocholine. Response in murine MOC2 head and neck carcinoma and B78 melanoma was evaluated by qPCR and flow cytometry. Therapeutic response to 225 Ac-NM600+anti-CTLA4+anti-PD-L1 immune checkpoint inhibition (ICI) was evaluated in wild-type and stimulator of interferon genes knockout (STING KO) B78. The timing and magnitude of IFN1 response correlated with radionuclide half-life and linear energy transfer. CD8 + /Treg ratios increased in tumors 7 days after 90 Y- and 177 Lu-NM600 and day 21 after 225 Ac-NM600. 225 Ac-NM600+ICI improved survival in mice with WT but not with STING KO tumors, relative to monotherapies. Immunomodulatory effects of RPT vary with radioisotope and promote STING-dependent enhanced response to ICIs in murine models. Teaser: This study describes the time course and nature of tumor immunomodulation by radiopharmaceuticals with differing physical properties.

Myeloid-derived suppressor cells attenuate the antitumor efficacy of radiopharmaceutical therapy using 90Y-NM600 in combination with androgen deprivation therapy in murine prostate tumors.

RATIONALE: Androgen deprivation therapy (ADT) is pivotal in treating recurrent prostate cancer and is often combined with external beam radiation therapy (EBRT) for localized disease. However, for metastatic castration-resistant prostate cancer, EBRT is typically only used in the palliative setting, because of the inability to radiate all sites of disease. Systemic radiation treatments that preferentially irradiate cancer cells, known as radiopharmaceutical therapy or targeted radionuclide therapy (TRT), have demonstrable benefits for treating metastatic prostate cancer. Here, we explored the use of a novel TRT, 90Y-NM600, specifically in combination with ADT, in murine prostate tumor models. METHODS: 6-week-old male FVB mice were implanted subcutaneously with Myc-CaP tumor cells and given a single intravenous injection of 90Y-NM600, in combination with ADT (degarelix). The combination and sequence of administration were evaluated for effect on tumor growth and infiltrating immune populations were analyzed by flow cytometry. Sera were assessed to determine treatment effects on cytokine profiles. RESULTS: ADT delivered prior to TRT (ADT→TRT) resulted in significantly greater antitumor response and overall survival than if delivered after TRT (TRT→ADT). Studies conducted in immunodeficient NRG mice failed to show a difference in treatment sequence, suggesting an immunological mechanism. Myeloid-derived suppressor cells (MDSCs) significantly accumulated in tumors following TRT→ADT treatment and retained immune suppressive function. However, CD4+ and CD8+ T cells with an activated and memory phenotype were more prevalent in the ADT→TRT group. Depletion of Gr1+MDSCs led to greater antitumor response following either treatment sequence. Chemotaxis assays suggested that tumor cells secreted chemokines that recruited MDSCs, notably CXCL1 and CXCL2. The use of a selective CXCR2 antagonist, reparixin, further improved antitumor responses and overall survival when used in tumor-bearing mice treated with TRT→ADT. CONCLUSION: The combination of ADT and TRT improved antitumor responses in murine models of prostate cancer, however, this was dependent on the order of administration. This was found to be associated with one treatment sequence leading to an increase in infiltrating MDSCs. Combining treatment with a CXCR2 antagonist improved the antitumor effect of this combination, suggesting a possible approach for treating advanced human prostate cancer.

Targeting of Head and Neck Cancer by Radioiodinated CLR1404 in Murine Xenograft Tumor Models with Partial Volume Corrected Theranostic Dosimetry.

Background: Delivery of radiotherapeutic dose to recurrent head and neck cancer (HNC) is primarily limited by locoregional toxicity in conventional radiotherapy. As such, HNC patients stand to benefit from the conformal targeting of primary and remnant disease achievable with radiopharmaceutical therapies. In this study, the authors investigated the tumor targeting capacity of 131I-CLR1404 (iopofosine I-131) in various HNC xenograft mouse models and the impact of partial volume correction (PVC) on theranostic dosimetry based on 124I-CLR1404 (CLR 124) positron emission tomography (PET)/computed tomography (CT) imaging. Methods: Mice bearing flank tumor xenograft models of HNC (six murine cell line and six human patient derived) were intravenously administered 6.5-9.1 MBq of CLR 124 and imaged five times over the course of 6 d using microPET/CT. In vivo tumor uptake of CLR 124 was assessed and PVC for 124I was applied using a novel preclinical phantom. Using subject-specific theranostic dosimetry estimations for iopofosine I-131 based on CLR 124 imaging, a discrete radiation dose escalation study (2, 4, 6, and 8 Gy) was performed to evaluate tumor growth response to iopofosine I-131 relative to a single fraction of external beam radiation therapy (6 Gy). Results: PET imaging demonstrated consistent tumor selective uptake and retention of CLR 124 across all HNC xenograft models. Peak uptake of 4.4% ± 0.8% and 4.2% ± 0.4% was observed in squamous cell carcinoma-22B and UW-13, respectively. PVC application increased uptake measures by 47%-188% and reduced absolute differences between in vivo and ex vivo uptake measurements from 3.3% to 1.0 percent injected activity per gram. Tumor dosimetry averaged over all HNC models was 0.85 ± 0.27 Gy/MBq (1.58 ± 0.46 Gy/MBq with PVC). Therapeutic iopofosine I-131 studies demonstrated a variable, but linear relationship between iopofosine I-131 radiation dose and tumor growth delay (p < 0.05). Conclusions: Iopofosine I-131 demonstrated tumoricidal capacity in preclinical HNC tumor models and the theranostic pairing with CLR 124 presents a promising new treatment approach for personalizing administration of iopofosine I-131.

Radiation to all macroscopic sites of tumor permits greater systemic antitumor response to in situ vaccination.

BACKGROUND: The 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 to all sites of disease would augment systemic antitumor responses to ISV. METHODS: We 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 to 90Y 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. RESULTS: Abscopal 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. CONCLUSIONS: We 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.

Effect of Polar Head Group Modifications on the Tumor Retention of Phospholipid Ether Analogs: Role of the Quaternary Nitrogen.

We have previously described the remarkable capacity of radioiodinated alkyl phospholipids to be sequestered and retained by a variety of tumors in vivo. We have already established the influence of certain structural parameters of iodinated alkyl phospholipids on tumor avidity, such as stereochemistry at the sn-2 carbon of alkylglycerol phosphocholines, meta-or para-position of iodine in the aromatic ring of phenylalkyl phosphocholines, and the length of the alkyl chain in alkyl phospholipids. In order to determine the additional structural requirements for tumor uptake and retention, three new radioiodinated alkylphospholipid analogs, 2-4, were synthesized as potential tumor imaging agents. Polar head groups were modified to determine structure-tumor avidity relationships. The trimethylammonio group in 1 was substituted with a hydrogen atom in 2, an ammonio group in 3 and a tertiary butyl group in 4. All analogs were separately labeled with iodine-125 or iodine-124 and administered to Walker 256 tumor-bearing rats or human PC-3 tumor-bearing SCID mice, respectively. Tumor uptake was assessed by gamma-camera scintigraphy (for [I-125]-labeled compounds) and high-resolution micro-PET scanning (for [I-124]-labeled compounds). It was found that structural modifications in the polar head group of alkyl phospholipids strongly influenced the tumor uptake and tissue distribution of these compounds in tumor-bearing animals. Phosphoethanolamine analog 3 (NM401) displayed a very slight accumulation in tumor as compared with phosphocholine analog 1 (NM346). Analogs 2 (NM400) and 4 (NM402) lacking the positively charged nitrogen atom failed to display any tumor uptake and localized primarily in the liver. This study provided important insights regarding structural requirements for tumor uptake and retention. Replacement of the quaternary nitrogen in the alkyl phospholipid head group with non-polar substituents resulted in loss of tumor avidity.

Next-Generation Cancer Magnetic Resonance Imaging With Tumor-Targeted Alkylphosphocholine Metal Analogs.

OBJECTIVES: In an effort to exploit the elevated need for phospholipids displayed by cancer cells relative to normal cells, we have developed tumor-targeted alkylphosphocholines (APCs) as broad-spectrum cancer imaging and therapy agents. Radioactive APC analogs have exhibited selective uptake and prolonged tumor retention in over 50 cancer types in preclinical models, as well as over 15 cancer types in over a dozen clinical trials. To push the structural limits of this platform, we recently added a chelating moiety capable of binding gadolinium and many other metals for cancer-targeted magnetic resonance imaging (MRI), positron emission tomography imaging, and targeted radionuclide therapy. The aim of this work was to synthesize, characterize, and validate the tumor selectivity of a new broad-spectrum, tumor-targeted, macrocyclic MRI chelate, Gd-NM600, in xenograft and orthotopic tumor models. A secondary aim was to identify and track the in vivo chemical speciation and spatial localization of this new chelate Gd-NM600 in order to assess its Gd deposition properties. MATERIALS AND METHODS: T1 relaxivities of Gd-NM600 were characterized in water and plasma at 1.5 T and 3.0 T. Tumor uptake and subcellular localization studies were performed using transmission electron microscopy. We imaged 8 different preclinical models of human cancer over time and compared the T1-weighted imaging results to that of a commercial macrocyclic Gd chelate, Gd-DOTA. Finally, matrix-assisted laser desorption and ionization-mass spectrometry imaging was used to characterize and map the tissue distribution of the chemical species of Gd-NM600. RESULTS: Gd-NM600 exhibits high T1 relaxivity (approximately 16.4 s-1/mM at 1.5 T), excellent tumor uptake (3.95 %ID/g at 48 hours), prolonged tumor retention (7 days), and MRI conspicuity. Moreover, minimal tumor uptake saturability of Gd-NM600 was observed. Broad-spectrum tumor-specific uptake was demonstrated in 8 different human cancer models. Cancer cell uptake of Gd-NM600 via endosomal internalization and processing was revealed with transmission electron microscopy. Importantly, tissue mass spectrometry imaging successfully interrogated the spatial localization and chemical speciation of Gd compounds and also identified breakdown products of Gd species. CONCLUSIONS: We have introduced a new macrocyclic cancer-targeted Gd chelate that achieves broad-spectrum tumor uptake and prolonged retention. Furthermore, we have demonstrated in vivo stability of Gd-NM600 by ultrahigh resolution MS tissue imaging. A tumor-targeted contrast agent coupled with the enhanced imaging resolution of MRI relative to positron emission tomography may transform oncologic imaging.

Antitumor efficacy of 90Y-NM600 targeted radionuclide therapy and PD-1 blockade is limited by regulatory T cells in murine prostate tumors.

BACKGROUND: 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. METHODS: 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-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. RESULTS: 90Y-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. CONCLUSIONS: Our 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.

Developments in Combining Targeted Radionuclide Therapies and Immunotherapies for Cancer Treatment.

Targeted radionuclide therapy (TRT) and immunotherapy are rapidly growing classes of cancer treatments. Basic, translational, and clinical research are now investigating therapeutic combinations of these agents. In comparison to external beam radiation therapy (EBRT), TRT has the unique advantage of treating all disease sites following intravenous injection and selective tumor uptake and retention-a particularly beneficial property in metastatic disease settings. The therapeutic value of combining radiation therapy with immune checkpoint blockade to treat metastases has been demonstrated in preclinical studies, whereas results of clinical studies have been mixed. Several clinical trials combining TRT and immune checkpoint blockade have been initiated based on preclinical studies combining these with EBRT and/or TRT. Despite the interest in translation of TRT and immunotherapy combinations, many questions remain surrounding the mechanisms of interaction and the optimal approach to clinical implementation of these combinations. This review highlights the mechanisms of interaction between anti-tumor immunity and radiation therapy and the status of basic and translational research and clinical trials investigating combinations of TRT and immunotherapies.

Safety and feasibility of an in situ vaccination and immunomodulatory targeted radionuclide combination immuno-radiotherapy approach in a comparative (companion dog) setting.

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.

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

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

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

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

[124I]CLR1404 PET/CT in High-Grade Primary and Metastatic Brain Tumors.

PURPOSE: There is a continuous search for imaging techniques with high sensitivity and specificity for brain tumors. Positron emission tomography (PET) imaging has shown promise, though many PET agents either have a low tumor specificity or impractical physical half-lives. [124I]CLR1404 is a small molecule alkylphosphocholine analogue that is thought to bind to plasma membrane lipid rafts and has shown high tumor-to-background ratios (TBR) in a previous pilot study in brain tumor patients. This study attempts to define the clinical value of [124I]CLR1404 PET/CT (aka CLR124). PROCEDURES: Adult patients with new or suspected recurrence of high-grade primary or metastatic brain tumors (N = 27) were injected with [124I]CLR1404 followed by PET/CT at 6, 24, and 48 h. Standard uptake values (SUV) and TBR values were calculated for all time points. Uptake of [124I]CLR1404 was qualitatively assessed, compared with magnetic resonance imaging (MRI), and correlated with clinical outcome. Final diagnosis (N = 25) was established based on surgically resected tissue or long-term follow-up. RESULTS: Positive uptake with high TBR was detected in all but one patient with a final diagnosis of primary/recurrent brain tumor (12/13) and in less than half of patients with treatment-related changes (5/12). Concordance between [124I]CLR1404 uptake and contrast enhancement on MRI was seen in < 40 %, with no concordance between T2-hyperintensities and uptake. No significant difference in overall outcome was found between patients with and without [124I]CLR1404 uptake. CONCLUSIONS: The uptake pattern in these patients suggests a very high sensitivity of [124I]CLR1404 PET/CT for diagnosing tumor tissue; however, tumor specificity needs to be further defined. Relative lack of concordance with standard MRI characteristics suggests that [124I]CLR1404 PET/CT provides additional information about brain tumors compared to MRI alone, potentially improving clinical decision-making.

177Lu-NM600 Targeted Radionuclide Therapy Extends Survival in Syngeneic Murine Models of Triple-Negative Breast Cancer.

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.

In Silico Docking of Alkylphosphocholine Analogs to Human Serum Albumin Predicts Partitioning and Pharmacokinetics.

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 suggest pharmacological strategies to alter their PK profiles. Importantly, our methodology may provide a way to design better drugs by better characterizing the PK profile for lead compound optimization.

90Y-NM600 targeted radionuclide therapy induces immunologic memory in syngeneic models of T-cell Non-Hodgkin's Lymphoma.

Finding improved therapeutic strategies against T-cell Non-Hodgkin's Lymphoma (NHL) remains an unmet clinical need. We implemented a theranostic approach employing a tumor-targeting alkylphosphocholine (NM600) radiolabeled with 86Y for positron emission tomography (PET) imaging and 90Y for targeted radionuclide therapy (TRT) of T-cell NHL. PET imaging and biodistribution performed in mouse models of T-cell NHL showed in vivo selective tumor uptake and retention of 86Y-NM600. An initial toxicity assessment examining complete blood counts, blood chemistry, and histopathology of major organs established 90Y-NM600 safety. Mice bearing T-cell NHL tumors treated with 90Y-NM600 experienced tumor growth inhibition, extended survival, and a high degree of cure with immune memory toward tumor reestablishment. 90Y-NM600 treatment was also effective against disseminated tumors, improving survival and cure rates. Finally, we observed a key role for the adaptive immune system in potentiating a durable anti-tumor response to TRT, especially in the presence of microscopic disease.

Preclinical Pharmacokinetics and Dosimetry Studies of 124I/131I-CLR1404 for Treatment of Pediatric Solid Tumors in Murine Xenograft Models.

Cancer is the second leading cause of death for children between the ages of 5 and 14 y. For children diagnosed with metastatic or recurrent solid tumors, for which the utility of external-beam radiotherapy is limited, the prognosis is particularly poor. The availability of tumor-targeting radiopharmaceuticals for molecular radiotherapy (MRT) has demonstrated improved outcomes in these patient populations, but options are nonexistent or limited for most pediatric solid tumors. 18-(p-iodophenyl)octadecylphosphocholine (CLR1404) is a novel antitumor alkyl phospholipid ether analog that broadly targets cancer cells. In this study, we evaluated the in vivo pharmacokinetics of 124I-CLR1404 (CLR 124) and estimated theranostic dosimetry for 131I-CLR1404 (CLR 131) MRT in murine xenograft models of the pediatric solid tumors neuroblastoma, rhabdomyosarcoma, and Ewing sarcoma. Methods: Tumor-bearing mice were imaged with small-animal PET/CT to evaluate the whole-body distribution of CLR 124 and, correcting for differences in radioactive decay, predict that of CLR 131. Image volumes representing CLR 131 provided input for Geant4 Monte Carlo simulations to calculate subject-specific tumor dosimetry for CLR 131 MRT. Pharmacokinetics for CLR 131 were extrapolated to adult and pediatric humans to estimate normal-tissue dosimetry. In neuroblastoma, a direct comparison of CLR 124 with 124I-metaiodobenzylguanidine (124I-MIBG) in an MIBG-avid model was performed. Results: In vivo pharmacokinetics of CLR 124 showed selective uptake and prolonged retention across all pediatric solid tumor models investigated. Subject-specific tumor dosimetry for CLR 131 MRT presents a correlative relationship with tumor-growth delay after CLR 131 MRT. Peak uptake of CLR 124 was, on average, 22% higher than that of 124I-MIBG in an MIBG-avid neuroblastoma model. Conclusion: CLR1404 is a suitable theranostic scaffold for dosimetry and therapy with potentially broad applicability in pediatric oncology. Given the ongoing clinical trials for CLR 131 in adults, these data support the development of pediatric clinical trials and provide detailed dosimetry that may lead to improved MRT treatment planning.

Pretreatment CLR 124 Positron Emission Tomography Accurately Predicts CLR 131 Three-Dimensional Dosimetry in a Triple-Negative Breast Cancer Patient.

INTRODUCTION: CLR1404 is a theranostic molecular agent that can be radiolabeled with 124I (CLR 124) for positron emission tomography (PET) imaging, or 131I (CLR 131) for single-photon emission computed tomography (SPECT) imaging and targeted radionuclide therapy. This pilot study evaluated a pretreatment dosimetry methodology in a triple-negative breast cancer patient who was uniquely enrolled in both a CLR 124 PET imaging clinical trial and a CLR 131 therapeutic dose escalation clinical trial. MATERIALS AND METHODS: Three-dimensional PET/CT images were acquired at 1, 3, 24, 48, and 120 h postinjection of 178 MBq CLR 124. One month later, pretherapy 2D whole-body planar images were acquired at 0.25, 5, 24, 48, and 144 h postinjection of 370 MBq CLR 131. Following the therapeutic administration of 1990 MBq CLR 131, 3D SPECT/CT images were acquired at 74, 147, 334, and 505 h postinjection. The therapeutic CLR 131 voxel-level absorbed dose was estimated from PET (RAPID PET) and SPECT (RAPID SPECT) images using a Geant4-based Monte Carlo dosimetry platform called RAPID (Radiopharmaceutical Assessment Platform for Internal Dosimetry), and region of interest (ROI) mean doses were also estimated using the OLINDA/EXM software based on PET (OLINDA PET), SPECT (OLINDA SPECT), and planar (OLINDA planar) images. RESULTS: The RAPID PET and OLINDA PET tracer-predicted ROI mean doses correlated well (m ≥ 0.631, R2 ≥ 0.694, p ≤ 0.01) with both the RAPID SPECT and OLINDA SPECT therapeutic mean doses. The 2D planar images did not have any significant correlations. The ROI mean doses differed by -4% to -43% between RAPID and OLINDA/EXM, and by -19% to 29% between PET and SPECT. The 3D dose distributions and dose volume histograms calculated with RAPID were similar for the PET/CT and SPECT/CT. CONCLUSIONS: This pilot study demonstrated that CLR 124 pretreatment PET images can be used to predict CLR 131 3D therapeutic dosimetry better than CLR 131 2D planar images. In addition, unlike OLINDA/EXM, Monte Carlo dosimetry methods were capable of accurately predicting dose heterogeneity, which is important for predicting dose-response relationships and clinical outcomes.

Enhanced Radiosensitivity in Solid Tumors using a Tumor-selective Alkyl Phospholipid Ether Analog.

Antitumor alkyl phospholipid (APL) analogs comprise a group of structurally related molecules with remarkable tumor selectivity. Some of these compounds have shown radiosensitizing capabilities. CLR127 is a novel, clinical-grade antitumor APL ether analog, a subtype of synthetic APL broadly targeting cancer cells with limited uptake in normal tissues. The purpose of this study was to investigate the effect of CLR127 to modulate radiation response across several adult and pediatric cancer types in vitro as well as in murine xenograft models of human prostate adenocarcinoma, neuroblastoma, Ewing sarcoma, and rhabdomyosarcoma. In vitro, CLR127 demonstrated selective uptake in cancer cells compared to normal cells. In cancer cells, CLR127 treatment prior to radiation significantly decreased clonogenic survival in vitro, and led to increased radiation-induced double-stranded DNA (dsDNA) breakage compared with radiation alone, which was not observed in normal controls. In animal models, CLR127 effectively increased the antitumor response to fractionated radiotherapy and led to delayed tumor regrowth at potentially clinically achievable doses. In conclusion, our study highlights the ability of CLR127 to increase radiation response in several cancer types. Given almost universal uptake of CLR127 in malignant cells, future research should test whether the observed effects can be extended to other tumor types. Our data provide a strong rationale for clinical testing of CLR127 as a tumor-targeted radiosensitizing agent. Mol Cancer Ther; 17(11); 2320-8. ©2018 AACR.

Targeted Molecular Radiotherapy of Pediatric Solid Tumors Using a Radioiodinated Alkyl-Phospholipid Ether Analog.

External-beam radiotherapy plays a critical role in the treatment of most pediatric solid tumors. Particularly in children, achieving an optimal therapeutic index to avoid damage to normal tissue is extremely important. Consequently, in metastatic disease, the utility of external-beam radiotherapy is limited. Molecular radiotherapy with tumor-targeted radionuclides may overcome some of these challenges, but to date there exists no single cancer-selective agent capable of treating various pediatric malignancies independently of their histopathologic origin. We tested the therapeutic potential of the clinical-grade alkyl-phospholipid ether analog CLR1404, 18-(p-iodophenyl)octadecyl phosphocholine, as a scaffold for tumor-targeted radiotherapy of pediatric malignancies. Methods: Uptake of CLR1404 by pediatric solid tumor cells was tested in vitro by flow cytometry and in vivo by PET/CT imaging and dosimetry. The therapeutic potential of 131I-CLR1404 was evaluated in xenograft models. Results: In vitro, fluorescent CLR1404-BODIPY showed significant selective uptake in a variety of pediatric cancer lines compared with normal controls. In vivo tumor-targeted uptake in mouse xenograft models using 124I-CLR1404 was confirmed by imaging. Single-dose intravenous injection of 131I-CLR1404 significantly delayed tumor growth in all rodent pediatric xenograft models and extended animal survival while demonstrating a favorable side effect profile. Conclusion:131I-CLR1404 has the potential to become a tumor-targeted radiotherapeutic drug with broad applicability in pediatric oncology. Because 131I-CLR1404 has entered clinical trials in adults, our data warrant the development of pediatric clinical trials for this particularly vulnerable patient population.

PET/CT imaging of the diapeutic alkylphosphocholine analog 124I-CLR1404 in high and low-grade brain tumors.

CLR1404 is a cancer-selective alkyl phosphocholine (APC) analog that can be radiolabeled with 124I for PET imaging, 131I for targeted radiotherapy and/or SPECT imaging, or 125I for targeted radiotherapy. Studies have demonstrated avid CLR1404 uptake and prolonged retention in a broad spectrum of preclinical tumor models. The purpose of this pilot trial was to demonstrate avidity of 124I-CLR1404 in human brain tumors and develop a framework to evaluate this uptake for use in larger studies. 12 patients (8 men and 4 women; mean age of 43.9 ± 15.1 y; range 23-66 y) with 13 tumors were enrolled. Eleven patients had suspected tumor recurrence and 1 patient had a new diagnosis of high grade tumor. Patients were injected with 185 MBq ± 10% of 124I-CLR1404 followed by PET/CT imaging at 6-, 24-, and 48-hour. 124I-CLR1404 PET uptake was assessed qualitatively and compared with MRI. After PET image segmentation SUV values and tumor to background ratios were calculated. There was no significant uptake of 124I-CLR1404 in normal brain. In tumors, uptake tended to increase to 48 hours. Positive uptake was detected in 9 of 13 lesions: 5/5 high grade tumors, 1/2 low grade tumors, 1/1 meningioma, and 2/4 patients with treatment related changes. 124I-CLR1404 uptake was not detected in 1/2 low grade tumors, 2/4 lesions from treatment related changes, and 1/1 indeterminate lesion. For 6 malignant tumors, the average tumor to background ratios (TBR) were 9.32 ± 4.33 (range 3.46 to 15.42) at 24 hours and 10.04 ± 3.15 (range 5.17 to 13.17) at 48 hours. For 2 lesions from treatment related change, the average TBR were 5.05 ± 0.4 (range 4.76 to 5.33) at 24 hours and 4.88 ± 1.19 (range 4.04 to 5.72) at 48 hours. PET uptake had areas of both concordance and discordance compared with MRI. 124I-CLR1404 PET demonstrated avid tumor uptake in a variety of brain tumors with high tumor-to-background ratios. There were regions of concordance and discordance compared with MRI, which has potential clinical relevance. Expansion of these studies is required to determine the clinical significance of the 124I-CLR1404 PET findings.