Development of Iodinated Indocyanine Green Analogs as a Strategy for Targeted Therapy of Liver Cancer.

Liver cancer is one of the leading causes of cancer-related deaths, with a significant increase in incidence worldwide. Novel therapies are needed to address this unmet clinical need. Indocyanine green (ICG) is a broadly used fluorescence-guided surgery (FGS) agent for liver tumor resection and has significant potential for conversion to a targeted therapy. Here, we report the design, synthesis, and investigation of a series of iodinated ICG analogs (I-ICG), which can be used to develop ICG-based targeted radiopharmaceutical therapy. We applied a CRISPR-based screen to identify the solute carrier transporter, OATP1B3, as a likely mechanism for ICG uptake. Our lead I-ICG compound specifically localizes to tumors in mice bearing liver cancer xenografts. This study introduces the chemistry needed to incorporate iodine onto the ICG scaffold and defines the impact of these modifications on key properties, including targeting liver cancer in vitro and in vivo.

Upregulation of Somatostatin Receptor Type 2 Improves 177Lu-DOTATATE Therapy in Receptor-Deficient Pancreatic Neuroendocrine Tumor Model.

Pancreatic neuroendocrine tumors (PNET) express high levels of somatostatin receptor type 2 (SSTR2), a unique target for both tumor imaging and therapy. This surface expression is lost in metastatic high-grade PNETs, making patients ineligible for SSTR2-targeted 177 Lutetium (Lu)-DOTATATE peptide receptor radionuclide therapy (PRRT), and represents an unmet clinical need. Here, we aimed to restore SSTR2 expression through the reversal of inhibitory epigenetic gene silencing to improve tumor responsiveness to PRRT. We first assessed human SSTR2 promoter methylation and expression levels in 96 patient samples. We then used three NET cell lines (QGP-1, BON-1, GOT-1) with variable SSTR2 expression profiles for functional in vitro studies using histone deacetylase inhibitors (HDACi). Finally, the QGP-1 xenograft mouse model, with low basal SSTR2 expression, was used to assess the therapeutic efficacy of combined HDACi and 177Lu-DOTATATE therapies. We confirm that SSTR expression is decreased and correlates with SSTR2 promoter methylation in patients with high-grade NETs. When exposed to HDACis, SSTR2 surface expression is increased in three NET cell lines in vitro. In an in vivo PNET xenograft model with low basal SSTR2 expression, our studies demonstrate significantly higher tumor uptake of SSTR2-targeted 177Lu-DOTATATE in animals pretreated with HDACis compared with controls. For the first time, we show that this higher tumor uptake results in significant antitumor response when compared with standard PRRT alone. These preclinical results provide a rationale for utilizing HDACi pretreatment to improve targeted radionuclide therapy in patients with SSTR2-negative, metastatic PNETs.

Site-Specifically Conjugated Single-Domain Antibody Successfully Identifies Glypican-3-Expressing Liver Cancer by Immuno-PET.

Primary liver cancer is the third leading cause of cancer-related deaths, and its incidence and mortality are increasing worldwide. Hepatocellular carcinoma (HCC) accounts for 80% of primary liver cancer cases. Glypican-3 (GPC3) is a heparan sulfate proteoglycan that histopathologically defines HCC and represents an attractive tumor-selective marker for radiopharmaceutical imaging and therapy for this disease. Single-domain antibodies are a promising scaffold for imaging because of their favorable pharmacokinetic properties, good tumor penetration, and renal clearance. Although conventional lysine-directed bioconjugation can be used to yield conjugates for radiolabeling full-length antibodies, this stochastic approach risks negatively affecting target binding of the smaller single-domain antibodies. To address this challenge, site-specific approaches have been explored. Here, we used conventional and sortase-based site-specific conjugation methods to engineer GPC3-specific human single-domain antibody (HN3) PET probes. Methods: Bifunctional deferoxamine (DFO) isothiocyanate was used to synthesize native HN3 (nHN3)-DFO. Site-specifically modified HN3 (ssHN3)-DFO was engineered using sortase-mediated conjugation of triglycine-DFO chelator and HN3 containing an LPETG C-terminal tag. Both conjugates were radiolabeled with 89Zr, and their binding affinity in vitro and target engagement of GPC3-positive (GPC3+) tumors in vivo were determined. Results: Both 89Zr-ssHN3 and 89Zr-nHN3 displayed nanomolar affinity for GPC3 in vitro. Biodistribution and PET/CT image analysis in mice bearing isogenic A431 and A431-GPC3+ xenografts, as well as in HepG2 liver cancer xenografts, showed that both conjugates specifically identify GPC3+ tumors. 89Zr-ssHN3 exhibited more favorable biodistribution and pharmacokinetic properties, including higher tumor uptake and lower liver accumulation. Comparative PET/CT studies on mice imaged with both 18F-FDG and 89Zr-ssHN3 showed more consistent tumor accumulation for the single-domain antibody conjugate, further establishing its potential for PET imaging. Conclusion: 89Zr-ssHN3 showed clear advantages in tumor uptake and tumor-to-liver signal ratio over the conventionally modified 89Zr-nHN3 in xenograft models. Our results establish the potential of HN3-based single-domain antibody probes for GPC3-directed PET imaging of liver cancers.

225Ac-MACROPATATE: A Novel α-Particle Peptide Receptor Radionuclide Therapy for Neuroendocrine Tumors.

Neuroendocrine tumors (NETs) express somatostatin receptors (SSTRs) 2 and 5. Modified variants of somatostatin, the cognate ligand for SSTR2 and SSTR5, are used in treatment for metastatic and locoregional disease. Peptide receptor radionuclide therapy with 177Lu-DOTATATE (DOTA-octreotate), a ß-particle-emitting somatostatin derivative, has demonstrated survival benefit in patients with SSTR-positive NETs. Despite excellent results, a subset of patients has tumors that are resistant to treatment, and alternative agents are needed. Targeted α-particle therapy has been shown to kill tumors that are resistant to targeted ß-particle therapy, suggesting that targeted α-particle therapy may offer a promising treatment option for patients with 177Lu-DOTATATE-resistant disease. Although DOTATATE can chelate the clinically relevant α-particle-emitting radionuclide 225Ac, the labeling reaction requires high temperatures, and the resulting radioconjugate has suboptimal stability. Methods: We designed and synthesized MACROPATATE (MACROPA-octreotate), a novel radioconjugate capable of chelating 225Ac at room temperature, and assessed its in vitro and in vivo performance. Results: MACROPATATE demonstrated comparable affinity to DOTATATE (dissociation constant, 21 nM) in U2-OS-SSTR2, a SSTR2-positive transfected cell line. 225Ac-MACROPATATE demonstrated superior serum stability at 37°C over time compared with 225Ac-DOTATATE. Biodistribution studies demonstrated higher tumor uptake of 225Ac-MACROPATATE than of 225Ac-DOTATATE in mice engrafted with subcutaneous H69 NETs. Therapy studies showed that 225Ac-MACROPATATE exhibits significant antitumor and survival benefit compared with saline control in mice engrafted with SSTR-positive tumors. However, the increased accumulation of 225Ac-MACROPATATE in liver and kidneys and subsequent toxicity to these organs decreased its therapeutic index compared with 225Ac-DOTATATE. Conclusion: 225Ac-MACROPATATE and 225Ac-DOTATATE exhibit favorable therapeutic efficacy in animal models. Because of elevated liver and kidney accumulation and lower administered activity for dose-limiting toxicity of 225Ac-MACROPATATE, 225Ac-DOTATATE was deemed the superior agent for targeted α-particle peptide receptor radionuclide therapy.

H2BZmacropa-NCS: A Bifunctional Chelator for Actinium-225 Targeted Alpha Therapy.

Actinium-225 (225Ac) is one of the most promising radionuclides for targeted alpha therapy (TAT). With a half-life of 9.92 days and a decay chain that emits four high-energy α particles, 225Ac is well-suited for TAT when conjugated to macromolecular targeting vectors that exhibit extended in vivo circulation times. The implementation of 225Ac in these targeted constructs, however, requires a suitable chelator that can bind and retain this radionuclide in vivo. Previous work has demonstrated the suitability of a diaza-18-crown-6 macrocyclic chelator H2macropa for this application. Building upon these prior efforts, in this study, two rigid variants of H2macropa, which contain either one (H2BZmacropa) or two (H2BZ2macropa) benzene rings within the macrocyclic core, were synthesized and investigated for their potential use for 225Ac TAT. The coordination chemistry of these ligands with La3+, used as a nonradioactive model for Ac3+, was carried out. Both NMR spectroscopic and X-ray crystallographic studies of the La3+ complexes of these ligands revealed similar structural features to those found for the related complex of H2macropa. Thermodynamic stability constants of the La3+ complexes, however, were found to be 1 and 2 orders of magnitude lower than those of H2macropa for H2BZmacropa and H2BZ2macropa, respectively. The decrease in thermodynamic stability was rationalized via the use of density functional theory calculations. 225Ac radiolabeling and serum stability studies with H2BZmacropa showed that this chelator compares favorably with H2macropa. Based on these promising results, a bifunctional version of this chelator, H2BZmacropa-NCS, was synthesized and conjugated to the antibody codrituzumab (GC33), which targets the liver cancer biomarker glypican-3 (GPC3). The resulting GC33-BZmacropa conjugate and an analogous GC33-macropa conjugate were evaluated for their 225Ac radiolabeling efficiencies, antigen-binding affinities, and in vivo biodistribution in HepG2 liver cancer tumor-bearing mice. Although both conjugates were comparably effective in their radiolabeling efficiencies, [225Ac]Ac-GC33-BZmacropa showed slightly poorer serum stability and biodistribution than [225Ac]Ac-GC33-macropa. Together, these results establish H2BZmacropa-NCS as a new bifunctional chelator for the preparation of 225Ac radiopharmaceuticals.

Towards the stable chelation of radium for biomedical applications with an 18-membered macrocyclic ligand.

Targeted alpha therapy is an emerging strategy for the treatment of disseminated cancer. [223Ra]RaCl2 is the only clinically approved alpha particle-emitting drug, and it is used to treat castrate-resistant prostate cancer bone metastases, to which [223Ra]Ra2+ localizes. To specifically direct [223Ra]Ra2+ to non-osseous disease sites, chelation and conjugation to a cancer-targeting moiety is necessary. Although previous efforts to stably chelate [223Ra]Ra2+ for this purpose have had limited success, here we report a biologically stable radiocomplex with the 18-membered macrocyclic chelator macropa. Quantitative labeling of macropa with [223Ra]Ra2+ was accomplished within 5 min at room temperature with a radiolabeling efficiency of >95%, representing a significant advancement over conventional chelators such as DOTA and EDTA, which were unable to completely complex [223Ra]Ra2+ under these conditions. [223Ra][Ra(macropa)] was highly stable in human serum and exhibited dramatically reduced bone and spleen uptake in mice in comparison to bone-targeted [223Ra]RaCl2, signifying that [223Ra][Ra(macropa)] remains intact in vivo. Upon conjugation of macropa to a single amino acid ß-alanine as well as to the prostate-specific membrane antigen-targeting peptide DUPA, both constructs retained high affinity for 223Ra, complexing >95% of Ra2+ in solution. Furthermore, [223Ra][Ra(macropa-ß-alanine)] was rapidly cleared from mice and showed low 223Ra bone absorption, indicating that this conjugate is stable under biological conditions. Unexpectedly, this stability was lost upon conjugation of macropa to DUPA, which suggests a role of targeting vectors in complex stability in vivo for this system. Nonetheless, our successful demonstration of efficient radiolabeling of the ß-alanine conjugate with 223Ra and its subsequent stability in vivo establishes for the first time the possibility of delivering [223Ra]Ra2+ to metastases outside of the bone using functionalized chelators, marking a significant expansion of the therapeutic utility of this radiometal in the clinic.

Sodium Fluoride-18 and Radium-223 Dichloride Uptake Colocalize in Osteoblastic Mouse Xenograft Tumors.

Background: Patients with osteoblastic bone metastases are candidates for radium-223 (223RaCl2) therapy and may undergo sodium fluoride-18 (18F-NaF) positron emission tomography-computed tomography imaging to identify bone lesions. 18F-NaF has been shown to predict 223RaCl2 uptake, but intratumor distributions of these two agents remain unclear. In this study, the authors evaluate the spatial distribution and relative uptakes of 18F-NaF and 223RaCl2 in Hu09-H3 human osteosarcoma mouse xenograft tumors at macroscopic and microscopic levels to better quantify their correlation. Materials and Methods: 18F-NaF and 223RaCl2 were co-injected into Hu09-H3 xenograft tumor severe combined immunodeficient mice. Tumor content was determined from in vivo biodistributions and visualized by PET, single photon emission computed tomography, and CT imaging. Intratumor distributions were visualized by quantitative autoradiography of tumor tissue sections and compared to histology of the same or adjacent sections. Results: 18F and 223Ra accumulated in proportional amounts in whole Hu09-H3 tumors (r2 = 0.82) and in microcalcified regions within these tumors (r2 = 0.87). Intratumor distributions of 18F and 223Ra were spatially congruent in these microcalcified regions. Conclusions: 18F-NaF and 223RaCl2 uptake are strongly correlated in heterogeneously distributed microcalcified regions of Hu09-H3 xenograft tumors, and thus, tumor accumulation of 18F is predictive of 223Ra accumulation. Hu09-H3 xenograft tumors appear to possess certain histopathological features found in patients with metastatic bone disease and may be useful in clarifying the relationship between administered 223Ra dose and therapeutic effect.

Glypican-3-Targeted Alpha Particle Therapy for Hepatocellular Carcinoma.

Glypican-3 (GPC3) is expressed in 75% of hepatocellular carcinoma (HCC), but not normal liver, making it a promising HCC therapeutic target. GC33 is a full-length humanized monoclonal IgG1 specific to GPC3 that can localize to HCC in vivo. GC33 alone failed to demonstrate therapeutic efficacy when evaluated in patients with HCC; however, we posit that cytotoxic functionalization of the antibody with therapeutic radionuclides, may be warranted. Alpha particles, which are emitted by radioisotopes such as Actinium-225 (Ac-225) exhibit high linear energy transfer and short pathlength that, when targeted to tumors, can effectively kill cancer and limit bystander cytotoxicity. Macropa, an 18-member heterocyclic crown ether, can stably chelate Ac-225 at room temperature. Here, we synthesized and evaluated the efficacy of [225Ac]Ac-Macropa-GC33 in mice engrafted with the GPC3-expressing human liver cancer cell line HepG2. Following a pilot dose-finding study, mice (n = 10 per group) were treated with (1) PBS, (2) mass-equivalent unmodified GC33, (3) 18.5 kBq [225Ac]Ac-Macropa-IgG1 (isotype control), (4) 9.25 kBq [225Ac]Ac-Macropa-GC33, and (5) 18.5 kBq [225Ac]Ac-Macropa-GC33. While significant toxicity was observed in all groups receiving radioconjugates, the 9.25 kBq [225Ac]Ac-Macropa-GC33 group demonstrated a modest survival advantage compared to PBS (p = 0.0012) and 18.5 kBq [225Ac]Ac-IgG1 (p = 0.0412). Hematological analysis demonstrated a marked, rapid reduction in white blood cells in all radioconjugate-treated groups compared to the PBS and unmodified GC33 control groups. Our studies highlight a significant disadvantage of using directly-labeled biomolecules with long blood circulation times for TAT. Strategies to mitigate such treatment toxicity include dose fractionation, pretargeting, and using smaller targeting ligands.

ImmunoPET Predicts Response to Met-targeted Radioligand Therapy in Models of Pancreatic Cancer Resistant to Met Kinase Inhibitors.

Background: Pancreatic ductal adenocarcinoma (PDAC) has limited standard of care therapeutic options. While initially received with enthusiasm, results from targeted therapy with small molecule tyrosine kinases inhibitors (TKIs) have been mixed, in part due to poor patient selection and compensatory changes in signaling networks upon blockade of one or more kinase of tumors. Here, we demonstrate that in PDACs otherwise resistant to rational kinase inhibition, Met-directed immuno-positron emission tomography (immunoPET) can identify targets for cell-signaling independent targeted radioligand therapy (RLT). In this study, we use Met-directed immunoPET and RLT in models of human pancreatic cancer that are resistant to Met- and MEK-selective TKIs, despite over-expression of Met and KRAS-pathway activation. Methods: We assessed cell membrane Met levels in human patient samples and pancreatic ductal adenocarcinoma (PDAC) cell lines (BxPC3, Capan2, Suit2, and MIA PaCa-2) using immunofluorescence, flow cytometry and cell-surface biotinylation assays. To determine whether Met expression levels correlate with sensitivity to Met inhibition by tyrosine kinase inhibitors (TKIs), we performed cell viability studies. A Met-directed imaging agent was engineered by labeling Met-specific onartuzumab with zirconium-89 (Zr-89) and its in vivo performance was evaluated in subcutaneous and orthotopic PDAC xenograft models. To assess whether the immunoPET agent would predict for targeted RLT response, onartuzumab was then labeled with lutetium (Lu-177) as the therapeutic radionuclide to generate our [177Lu]Lu-DTPA-onartuzumab RLT agent. [177Lu]Lu-DTPA-onartuzumab was administered at 9.25MBq (250µCi)/20µg in three fractions separated by three days in mice subcutaneously engrafted with BxPC3 (high cell-membrane Met) or MIA PaCa-2 (low cell-membrane Met). Primary endpoints were tumor response and overall survival. Results: Flow cytometry and cell-surface biotinylation studies showed that cell-membrane Met was significantly more abundant in BxPC3, Capan2, and Suit2 when compared with MIA PaCa-2 pancreatic tumor cells. Crizotinib and cabozantinib, TKIs with known activity against Met and other kinases, decreased PDAC cell line viability in vitro. The TKI with the lowest IC50 for Met, capmatinib, had no activity in PDAC lines. No additive effect was detected on cell viability when Met-inhibition was combined with MEK1/2 inhibition. We observed selective tumor uptake of [89Zr]Zr-DFO-onartuzumab in mice subcutaneously and orthotopically engrafted with PDAC lines containing high cell-surface levels of Met (BxPC3, Capan2, Suit2), but not in mice engrafted with low cell-surface levels of Met (MIA PaCa-2). Significant tumor growth delay and overall survival benefit were observed in both BxPC3 and MIA PaCa-2 engrafted animals treated with RLT when compared to controls, however, the benefit was more pronounced and more durable in the BxPC3 engrafted animals treated with [177Lu]Lu-DTPA-onartuzumab RLT. Conclusions: Our findings demonstrate that while over-expression of Met is not predictive of Met-directed TKI response, immunoPET can detect Met over-expression in vivo and predicts for therapeutic response to Met-selective RLT. This phenomenon can be exploited for other Met-overexpressing tumor types specifically, and to any differentially overexpressed surface molecule more broadly.

Preclinical Evaluation of 203/212Pb-Labeled Low-Molecular-Weight Compounds for Targeted Radiopharmaceutical Therapy of Prostate Cancer.

Targeted radiopharmaceutical therapy (TRT) using α-particle radiation is a promising approach for treating both large and micrometastatic lesions. We developed prostate-specific membrane antigen (PSMA)-targeted low-molecular-weight agents for 212Pb-based TRT of patients with prostate cancer (PC) by evaluating the matching γ-emitting surrogate, 203Pb. Methods: Five rationally designed low-molecular-weight ligands (L1-L5) were synthesized using the lysine-urea-glutamate scaffold, and PSMA inhibition constants were determined. Tissue biodistribution and SPECT/CT imaging of 203Pb-L1-203Pb-L5 were performed on mice bearing PSMA(+) PC3 PIP and PSMA(-) PC3 flu flank xenografts. The absorbed radiation dose of the corresponding 212Pb-labeled analogs was determined using the biodistribution data. Antitumor efficacy of 212Pb-L2 was evaluated in PSMA(+) PC3 PIP and PSMA(-) PC3 flu tumor models and in the PSMA(+) luciferase-expressing micrometastatic model. 212Pb-L2 was also evaluated for dose-escalated, long-term toxicity. Results: All new ligands were obtained in high yield and purity. PSMA inhibitory activities ranged from 0.10 to 17 nM. 203Pb-L1-203Pb-L5 were synthesized in high radiochemical yield and specific activity. Whole-body clearance of 203Pb-L1-203Pb-L5 was fast. The absorbed dose coefficients (mGy/kBq) of the tumor and kidneys were highest for 203Pb-L5 (31.0, 15.2) and lowest for 203Pb-L2 (8.0, 4.2). The tumor-to-kidney absorbed dose ratio was higher for 203Pb-L3 (3.2) and 203Pb-L4 (3.6) than for the other agents, but with lower tumor-to-blood ratios. PSMA(+) tumor lesions were visualized through SPECT/CT as early as 0.5 h after injection. A proof-of-concept therapy study with a single administration of 212Pb-L2 demonstrated dose-dependent inhibition of tumor growth in the PSMA(+) flank tumor model. 212Pb-L2 also demonstrated an increased survival benefit in the micrometastatic model compared with 177Lu-PSMA-617. Long-term toxicity studies in healthy, immunocompetent CD-1 mice revealed kidney as the dose-limiting organ. Conclusion:203Pb-L1-203Pb-L5 demonstrated favorable pharmacokinetics for 212Pb-based TRT. The antitumor efficacy of 212Pb-L2 supports the corresponding 203Pb/212Pb theranostic pair for PSMA-based α-particle TRT in advanced PC.

A Study of 219Rn Outgassing and 211Pb Contamination from 223Ra In dry, Liquid, and Murine Tissue Samples.

INTRODUCTION: A study of Pb contamination caused by the outgassing of Rn from Ra in dry, liquid, and murine tissues samples has been made to help design proper handling procedures for Ra in preclinical biodistribution work. MATERIALS AND METHODS: Pb activity levels were measured from Ra in dry, liquid, and tissue samples using aspiration and autoradiography techniques. RESULTS: Using aspiration techniques on dry samples of Ra, an average Rn outgassing rate of 51% ± 21% was measured with one measurement reaching as high as 81%. 31% ± 4% Pb contamination was measured within a 4.3 cm radius of a dry Ra source placed inside a 10-cm-diameter petri dish where the lip of the petri dish contained the Rn dissemination. Without the containment of the petri dish, Rn can reach as far as 7.8 cm from the source with trace levels spreading further. Using aspiration techniques on liquid samples of Ra, outgassing rates of Rn were 0.9% ± 0.3%. The outgassing levels in harvested organs from a biodistribution were as high as 10.1% ± 0.4% for an intraperitoneally injected mouse and 0.204% ± 0.006% for an intravenously injected mouse. The outgassing of the intravenously injected mouse carcass was less than 0.1%. CONCLUSION: In dry form, the high levels of Rn outgassing from a Ra source necessitate the use of ventilated biohoods when handling or preparing dry Ra from source vials. The very low levels of Rn outgassing from Ra liquid sources reduces exposure to Rn by a factor of 50. Rn exposure from murine organ tissue reaches levels of 10% when handling organs from an intraperitoneal injection and less than 0.2% for an intravenous injection.

Exploration of a F(ab')2 Fragment as the Targeting Agent of α-Radiation Therapy: A Comparison of the Therapeutic Benefit of Intraperitoneal and Intravenous Administered Radioimmunotherapy.

Refinement of treatment regimens enlisting targeted α-radiation therapy (TAT) is an ongoing effort. Among the variables to consider are the target molecule, radionuclide, dosage, and administration route. The panitumumab F(ab')2 fragment targeting epidermal growth factor receptor tolerated modification with the TCMC chelate as well as radiolabeling with 203Pb or 212Pb. Good specific activity was attained when the immunoconjugate was labeled with 212Pb (9.6 ± 1.4 mCi/mg). Targeting of LS-174T tumor xenografts with the 203Pb-panitumumab F(ab')2 demonstrated comparable amounts of uptake to the similarly radiolabeled panitumumab IgG. A dose escalation study was performed to determine an effective working dose for both intraperitoneal (i.p.) and intravenous (i.v.) injections of 212Pb-panitumumab F(ab')2. Therapeutic efficacy, with modest toxicity, was observed with 30 µCi given i.p. Results for the i.v. administration were not as definitive and the experiment was repeated with a higher dose range. From this study, 20 µCi given i.v. was selected as the effective working dose. A subsequent therapy study combined gemcitabine or paclitaxel with i.v. 212Pb-panitumumab F(ab')2, which increased the median survival (MS) of LS-174T tumor-bearing mice to 208 and 239 d, respectively. Meanwhile, the MS of mice treated with i.v. 212Pb-panitumumab F(ab')2 alone was 61 and 11 d for the untreated group of mice. In conclusion, the panitumumab F(ab')2 fragment whether given by i.p. or i.v. injection, is a viable candidate as a delivery vector for TAT of disseminated i.p. disease.

Comparative studies on the therapeutic benefit of targeted α-particle radiation therapy for the treatment of disseminated intraperitoneal disease.

Identification of the appropriate combination of radionuclide, target and targeting vehicle is critical for successful radioimmunotherapy. For the treatment of disseminated peritoneal diseases such as pancreatic or ovarian cancer, α-emitting radionuclides have been proposed for targeted radiation therapy. This laboratory has taken a systematic approach investigating targeted α-radiation therapy, allowing comparisons to now be made between 211At, 227Th, 213Bi and 212Pb. Herein, trastuzumab radiolabeled with 211At and 227Th was evaluated for therapeutic efficacy in the LS-174T i.p. tumor model. A dose escalation study was conducted with each radioimmunoconjugate (RIC). Therapeutic benefit was realized with 211At-trastuzumab with doses of 20, 30 and 40 µCi. At doses >40 µCi, toxicity was observed with greater weight loss and 2-fold higher decrease in the platelet counts. Following a second study comparing the effect of 20, 30 and 40 µCi of 211At-trastuzumab, 30 µCi was selected as the dose for future studies. A parallel study was performed evaluating 0.25, 0.5, 1.0, 2.0 and 5.0 µCi of 227Th-trastuzumab. The 0.5 and 1.0 µCi injected dose resulted in a therapeutic response; a lower degree of weight loss was experienced by the mice in the 0.5 µCi cohort. When the data is normalized for comparing 211At, 227Th, 213Bi and 212Pb, the choice of radionuclide for RIT is perhaps not entirely based on simple therapeutic efficacy, other factors may play a role in choosing the "right" radionuclide.

Targeted α-Particle Radiation Therapy of HER1-Positive Disseminated Intraperitoneal Disease: An Investigation of the Human Anti-EGFR Monoclonal Antibody, Panitumumab.

Identifying molecular targets and an appropriate targeting vehicle, i.e., monoclonal antibodies (mAb) and their various forms, for radioimmunotherapy (RIT) remains an active area of research. Panitumumab, a fully human and less immunogenic mAb that binds to the epidermal growth factor receptor (Erb1; HER1), was evaluated for targeted α-particle radiation therapy using 212Pb, an in vivo α generator. A single dose of 212Pb-panitumumab administered to athymic mice bearing LS-174T intraperitoneal (i.p.) tumor xenografts was found to have greater therapeutic efficacy when directly compared with 212Pb-trastuzumab, which binds to HER2. A dose escalation study determined a maximum effective working dose of 212Pb-panitumumab to be 20µCi with a median survival of 35 days versus 25 days for the untreated controls. Pretreatment of tumor-bearing mice with paclitaxel and gemcitabine 24hours prior to injection of 212Pb-pantiumumab at 10 or 20µCi resulted in the greatest enhanced therapeutic response at the higher dose with median survivals of 106 versus 192 days, respectively. The greatest therapeutic impact, however, was observed in the animals that were treated with topotecan 24hours prior to RIT and then again 24hours after RIT; the best response from this combination was also obtained with the lower 10-µCi dose of 212Pb-panitumumab (median survival >280 days). In summary, 212Pb-panitumumab is an excellent candidate for the treatment of HER1-positive disseminated i.p. disease. Furthermore, the potentiation of the therapeutic impact of 212Pb-pantiumumab by chemotherapeutics confirms and validates the importance of developing a multimodal therapy regimen.

Cell Killing Mechanisms and Impact on Gene Expression by Gemcitabine and 212Pb-Trastuzumab Treatment in a Disseminated i.p. Tumor Model.

In pre-clinical studies, combination therapy with gemcitabine and targeted radioimmunotherapy (RIT) using 212Pb-trastuzumab showed tremendous therapeutic potential in the LS-174T tumor xenograft model of disseminated intraperitoneal disease. To better understand the underlying molecular basis for the observed cell killing efficacy, gene expression profiling was performed after a 24 h exposure to 212Pb-trastuzumab upon gemcitabine (Gem) pre-treatment in this model. DNA damage response genes in tumors were quantified using a real time quantitative PCR array (qRT-PCR array) covering 84 genes. The combination of Gem with α-radiation resulted in the differential expression of apoptotic genes (BRCA1, CIDEA, GADD45α, GADD45γ, IP6K3, PCBP4, RAD21, and p73), cell cycle regulatory genes (BRCA1, CHK1, CHK2, FANCG, GADD45α, GTSE1, PCBP4, MAP2K6, NBN, PCBP4, and SESN1), and damaged DNA binding and repair genes (BRCA1, BTG2, DMC1, ERCC1, EXO1, FANCG, FEN1, MSH2, MSH3, NBN, NTHL1, OGG1, PRKDC, RAD18, RAD21, RAD51B, SEMA4G, p73, UNG, XPC, and XRCC2). Of these genes, the expression of CHK1, GTSE1, EXO1, FANCG, RAD18, UNG and XRCC2 were specific to Gem/212Pb-trastuzumab administration. In addition, the present study demonstrates that increased stressful growth arrest conditions induced by Gem/212Pb-trastuzumab could suppress cell proliferation possibly by up-regulating genes involved in apoptosis such as p73, by down-regulating genes involved in cell cycle check point such as CHK1, and in damaged DNA repair such as RAD51 paralogs. These events may be mediated by genes such as BRCA1/MSH2, a member of BARC (BRCA-associated genome surveillance complex). The data suggest that up-regulation of genes involved in apoptosis, perturbation of checkpoint genes, and a failure to correctly perform HR-mediated DSB repair and mismatch-mediated SSB repair may correlate with the previously observed inability to maintain the G2/M arrest, leading to cell death.

Unexpected Behavior of the Heaviest Halogen Astatine in the Nucleophilic Substitution of Aryliodonium Salts.

Aryliodonium salts have become precursors of choice for the synthesis of (18) F-labeled tracers for nuclear imaging. However, little is known on the reactivity of these compounds with heavy halides, that is, radioiodide and astatide, at the radiotracer scale. In the first comparative study of radiohalogenation of aryliodonium salts with (125) I(-) and (211) At(-) , initial experiments on a model compound highlight the higher reactivity of astatide compared to iodide, which could not be anticipated from the trends previously observed within the halogen series. Kinetic studies indicate a significant difference in activation energy (Ea =23.5 and 17.1 kcal mol(-1) with (125) I(-) and (211) At(-) , respectively). Quantum chemical calculations suggest that astatination occurs via the monomeric form of an iodonium complex whereas iodination occurs via a heterodimeric iodonium intermediate. The good to excellent regioselectivity of halogenation and high yields achieved with diversely substituted aryliodonium salts indicate that this class of compounds is a promising alternative to the stannane chemistry currently used for heavy radiohalogen labeling of tracers in nuclear medicine.

(2S)-2-(3-(1-Carboxy-5-(4-211At-Astatobenzamido)Pentyl)Ureido)-Pentanedioic Acid for PSMA-Targeted α-Particle Radiopharmaceutical Therapy.

Alpha-particle emitters have a high linear energy transfer and short range, offering the potential for treating micrometastases while sparing normal tissues. We developed a urea-based, 211At-labeled small molecule targeting prostate-specific membrane antigen (PSMA) for the treatment of micrometastases due to prostate cancer (PC). METHODS: PSMA-targeted (2S)-2-(3-(1-carboxy-5-(4-211At-astatobenzamido)pentyl)ureido)-pentanedioic acid (211At- 6: ) was synthesized. Cellular uptake and clonogenic survival were tested in PSMA-positive (PSMA+) PC3 PIP and PSMA-negative (PSMA-) PC3 flu human PC cells after 211At- 6: treatment. The antitumor efficacy of 211At- 6: was evaluated in mice bearing PSMA+ PC3 PIP and PSMA- PC3 flu flank xenografts at a 740-kBq dose and in mice bearing PSMA+, luciferase-expressing PC3-ML micrometastases. Biodistribution was determined in mice bearing PSMA+ PC3 PIP and PSMA- PC3 flu flank xenografts. Suborgan distribution was evaluated using α-camera images, and microscale dosimetry was modeled. Long-term toxicity was assessed in mice for 12 mo. RESULTS: 211At- 6: treatment resulted in PSMA-specific cellular uptake and decreased clonogenic survival in PSMA+ PC3 PIP cells and caused significant tumor growth delay in PSMA+ PC3 PIP flank tumors. Significantly improved survival was achieved in the newly developed PSMA+ micrometastatic PC model. Biodistribution showed uptake of 211At- 6: in PSMA+ PC3 PIP tumors and in kidneys. Microscale kidney dosimetry based on α-camera images and a nephron model revealed hot spots in the proximal renal tubules. Long-term toxicity studies confirmed that the dose-limiting toxicity was late radiation nephropathy. CONCLUSION: PSMA-targeted 211At- 6: α-particle radiotherapy yielded significantly improved survival in mice bearing PC micrometastases after systemic administration. 211At- 6: also showed uptake in renal proximal tubules resulting in late nephrotoxicity, highlighting the importance of long-term toxicity studies and microscale dosimetry.

Mechanisms of Cell Killing Response from Low Linear Energy Transfer (LET) Radiation Originating from (177)Lu Radioimmunotherapy Targeting Disseminated Intraperitoneal Tumor Xenografts.

Radiolabeled antibodies (mAbs) provide efficient tools for cancer therapy. The combination of low energy ß(-)-emissions (500 keVmax; 130 keVave) along with a γ-emission for imaging makes (177)Lu (T1/2 = 6.7 day) a suitable radionuclide for radioimmunotherapy (RIT) of tumor burdens possibly too large to treat with α-particle radiation. RIT with (177)Lu-trastuzumab has proven to be effective for treatment of disseminated HER2 positive peritoneal disease in a pre-clinical model. To elucidate mechanisms originating from this RIT therapy at the molecular level, tumor bearing mice (LS-174T intraperitoneal xenografts) were treated with (177)Lu-trastuzumab comparatively to animals treated with a non-specific control, (177)Lu-HuIgG, and then to prior published results obtained using (212)Pb-trastuzumab, an α-particle RIT agent. (177)Lu-trastuzumab induced cell death via DNA double strand breaks (DSB), caspase-3 apoptosis, and interfered with DNA-PK expression, which is associated with the repair of DNA non-homologous end joining damage. This contrasts to prior results, wherein (212)Pb-trastuzumab was found to down-regulate RAD51, which is involved with homologous recombination DNA damage repair. (177)Lu-trastuzumab therapy was associated with significant chromosomal disruption and up-regulation of genes in the apoptotic process. These results suggest an inhibition of the repair mechanism specific to the type of radiation damage being inflicted by either high or low linear energy transfer radiation. Understanding the mechanisms of action of ß(-)- and α-particle RIT comparatively through an in vivo tumor environment offers real information suitable to enhance combination therapy regimens involving α- and ß(-)-particle RIT for the management of intraperitoneal disease.

Bench to Bedside: Stability Studies of GMP Produced Trastuzumab-TCMC in Support of a Clinical Trial.

The first-in-human phase 1 clinical radioimmunotherapy (RIT) trial with 212Pb-1,4,7,10-tetraaza-1,4,7,10-tetra-(2-carbamoylmethyl)-cyclododecane-trastuzumab (212Pb-TCMC-trastuzumab) was completed in October 2014 as a joint effort at the University of Alabama (UAB) and the University of California San Diego Moores Cancer Center. The preliminary reports indicate that after five dose-levels of intraperitoneally administered 212Pb-TCMC-trastuzumab, patients with carcinomatosis experienced minimal agent-related toxicity. This report presents the data accumulated to date on the stability of the clinical grade, produced according to current good manufacturing practices (cGMP), TCMC-trastuzumab conducted in support of that clinical trial. Of the eleven tests performed with the cGMP TCMC-trastuzumab all but one remained within specifications throughout the 5 year testing period. The protein concentration varied by 0.01 mg/mL at 48 months. Two other assays, ion-exchange high performance liquid chromatography (IEX-HPLC) and a competitive radioimmunoassay (RIA) indicated that the cGMP TCMC-trastuzumab integrity may be changing, although the change thus far is within specifications. Subsequent stability testing will confirm if a trend has truly developed. The cGMP TCMC-trastuzumab was also evaluated for tolerance to higher temperatures and the potential of storage at -80 °C. The immunoconjugate proved stable when subjected to the lower temperatures and to multiple freeze-thaw cycles. The size exclusion (SE) HPLC analysis of the 203Pb-TCMC-trastuzumab was the only indicator that cGMP TCMC-trastuzumab may be sensitive to storage at 37 °C for 3 months.

Toxicological Studies of 212Pb Intravenously or Intraperitoneally Injected into Mice for a Phase 1 Trial.

Faced with the novelty of a 212Pb-labeled monoclonal antibody (mAb) for clinical translation, concerns were expressed by the Food and Drug Administration (FDA) regarding 212Pb prematurely released from the mAb-chelate conjugate. The objective of this study was to simulate the worst case scenario of such a failure. Groups of Balb/c mice (n = 9-20) were administered 212Pb by intraperitoneal (0.0925-1.85 MBq) or intravenous (0.0925-1.11 MBq) injection and then euthanized at 7 or 90 days to assess acute or chronic effects. Weights were recorded prior to injection of the 212Pb and at the end of the observation periods. Blood samples were collected for clinical chemistry and blood cell analysis. Thirty tissues were harvested and formalin fixed for histopathological examination. Treatment related effects of the 212Pb were observed in the bone marrow, spleen, kidneys and the liver. Histological alterations in these organs were considered mild to moderate, indicating low grade toxicity, and not considered severe enough to affect function. This data was presented to the FDA and determined to be acceptable. The clinical trial with 212Pb-TCMC-trastuzumab was approved in January 2011 and the trial opened at the University of Alabama at Birmingham (UAB) in July.