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Background: Patients with osteoblastic bone metastases are candidates for radium-223 ((223)RaCl(2)) therapy and may undergo sodium fluoride-18 ((18)F-NaF) positron emission tomography-computed tomography imaging to identify bone lesions. (18)F-NaF has been shown to predict (223)RaCl(2) uptake, but intratumor distributions of these two agents remain unclear. In this study, the authors evaluate the spatial distribution and relative uptakes of (18)F-NaF and (223)RaCl(2) in Hu09-H3 human osteosarcoma mouse xenograft tumors at macroscopic and microscopic levels to better quantify their correlation. Materials and Methods: (18)F-NaF and (223)RaCl(2) 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: (18)F and (223)Ra accumulated in proportional amounts in whole Hu09-H3 tumors (r(2) = 0.82) and in microcalcified regions within these tumors (r(2) = 0.87). Intratumor distributions of (18)F and (223)Ra were spatially congruent in these microcalcified regions. Conclusions: (18)F-NaF and (223)RaCl(2) uptake are strongly correlated in heterogeneously distributed microcalcified regions of Hu09-H3 xenograft tumors, and thus, tumor accumulation of (18)F is predictive of (223)Ra 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 (223)Ra dose and therapeutic effect.

Actinium-225 ((225)Ac) 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, (225)Ac is well-suited for TAT when conjugated to macromolecular targeting vectors that exhibit extended in vivo circulation times. The implementation of (225)Ac 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 H(2)macropa for this application. Building upon these prior efforts, in this study, two rigid variants of H(2)macropa, which contain either one (H(2)BZmacropa) or two (H(2)BZ(2)macropa) benzene rings within the macrocyclic core, were synthesized and investigated for their potential use for (225)Ac TAT. The coordination chemistry of these ligands with La(3+), used as a non-radioactive model for Ac(3+), was carried out. Both NMR spectroscopic and X-ray crystallographic studies of the La(3+) complexes of these ligands revealed similar structural features as those found for the related complex of H(2)macropa. Thermodynamic stability constants of the La(3+) complexes, however, were found to be one and two orders of magnitude lower than those of H(2)macropa for H(2)BZmacropa and H(2)BZ(2)macropa, respectively. The decrease in thermodynamic stability was rationalized via the use of density functional theory calculations. (225)Ac radiolabeling and serum stability studies with H(2)BZmacropa showed that this chelator compares favorably with H(2)macropa. Based on these promising results, a bifunctional version of this chelator, H(2)BZmacropa-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 were evaluated for their (225)Ac radiolabeling efficiencies, antigen-binding affinity, and in vivo biodistribution in HepG2 liver cancer tumor-bearing mice. Although both conjugates were comparably effective in their radiolabeling efficiencies, [(225)Ac]Ac-GC33-BZmacropa showed slightly poorer serum stability and biodistribution than [(225)Ac]Ac-GC33-macropa. Together, these results establish H(2)BZmacropa-NCS as a new bifunctional chelator for the preparation of (225)Ac radiopharmaceuticals.

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.

INTRODUCTION: A study of (211)Pb contamination caused by the outgassing of (219)Rn from (223)Ra in dry, liquid, and murine tissues samples has been made to help design proper handling procedures for (223)Ra in preclinical biodistribution work. MATERIALS AND METHODS: (211)Pb activity levels were measured from (223)Ra in dry, liquid, and tissue samples using aspiration and autoradiography techniques. RESULTS: Using aspiration techniques on dry samples of (223)Ra, an average (219)Rn outgassing rate of 51% ± 21% was measured with one measurement reaching as high as 81%. 31% ± 4% (211)Pb contamination was measured within a 4.3 cm radius of a dry (223)Ra source placed inside a 10-cm-diameter petri dish where the lip of the petri dish contained the (219)Rn dissemination. Without the containment of the petri dish, (219)Rn can reach as far as 7.8 cm from the source with trace levels spreading further. Using aspiration techniques on liquid samples of (223)Ra, outgassing rates of (219)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 (219)Rn outgassing from a (223)Ra source necessitate the use of ventilated biohoods when handling or preparing dry (223)Ra from source vials. The very low levels of (219)Rn outgassing from (223)Ra liquid sources reduces exposure to (219)Rn by a factor of 50. (219)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.

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 β-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-β-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 β-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., The therapeutic alpha-emitter 223Ra can be stably complexed in vivo, creating opportunities for the development of targeted radiopharmaceutical agents with this radionuclide.

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&ndash, Macropa&ndash, 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&ndash, IgG1 (isotype control), (4) 9.25 kBq [225Ac]Ac&ndash, GC33, and (5) 18.5 kBq [225Ac]Ac&ndash, GC33. While significant toxicity was observed in all groups receiving radioconjugates, the 9.25 kBq [225Ac]Ac&ndash, GC33 group demonstrated a modest survival advantage compared to PBS (p = 0.0012) and 18.5 kBq [225Ac]Ac&ndash, 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.

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 212 Pb, an in vivo α generator. A single dose of 212 Pb-panitumumab administered to athymic mice bearing LS-174T intraperitoneal (i.p.) tumor xenografts was found to have greater therapeutic efficacy when directly compared with 212 Pb-trastuzumab, which binds to HER2. A dose escalation study determined a maximum effective working dose of 212 Pb-panitumumab to be 20 μCi with a median survival of 35 days versus 25 days for the untreated controls. Pretreatment of tumor-bearing mice with paclitaxel and gemcitabine 24 hours prior to injection of 212 Pb-pantiumumab at 10 or 20 μCi 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 24 hours prior to RIT and then again 24 hours after RIT; the best response from this combination was also obtained with the lower 10-μCi dose of 212 Pb-panitumumab (median survival >280 days). In summary, 212 Pb-panitumumab is an excellent candidate for the treatment of HER1-positive disseminated i.p. disease. Furthermore, the potentiation of the therapeutic impact of 212 Pb-pantiumumab by chemotherapeutics confirms and validates the importance of developing a multimodal therapy regimen.

BackgroundHepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. While conventional imaging approaches like ultrasound, CT, and MRI play critical roles in the diagnosis and surveillance of HCC, improved methods for detection and assessment of treatment response are needed. One promising approach is the use of radiolabeled antibodies for positron emission tomography (immunoPET) imaging. Glypican-3 (GPC3) is a proteoglycan that is highly expressed in the majority of HCC tumors. GPC3-specific antibodies are used to diagnose HCC histopathologically, and have been proposed as a treatment of HCC. Here, we design, synthesize and demonstrate that our humanized immunoPET agent, [89Zr]Zr-DFO-TAB-H14, can stoichiometrically bind to models of human liver cancer with varied GPC3 expression. Methods: The GPC3-specific monoclonal humanized IgG1, TAB-H14, was used as a scaffold for engineering our immunoPET agent. Fluorescent and deferroxamine (DFO) chelate conjugates of TAB-H14 were characterized using mass spectrometry. Binding affinity of TAB-H14 and conjugates for GPC3 was determined in cell-free biolayer interferometry, and cell-based radioimmunoassays. GPC3-expression was assessed by flow cytometry and immunofluorescence using commercially available anti-GPC3 antibodies and TAB-H14 in GPC3−(A431) and GPC3+cell lines including an engineered line (A431-GPC3+, G1) and liver cancer lines (HepG2, Hep3B, and Huh7). DFO-TAB-H14, was radiolabeled with Zr-89. Mice were subcutaneously engrafted with the aforementioned cell lines and in vivo target engagement of the immunoPET agent [89Zr]Zr-DFO-TAB-H14 was determined using PET/CT, quantitative biodistribution, and autoradiography. Results: TAB-H14 demonstrated subnanomolar to nanomolar affinity for human GPC3. Fluorescently tagged TAB-H14 was able to bind to GPC3 on cell membranes of GPC3-expressing lines by flow cytometry. These results were confirmed by immunofluorescence staining of A431, G1 HepG2, Hep3B, and Huh7 tumor sections. ImmunoPET imaging with [89Zr]Zr-DFO-TAB-H14 showed stoichiometric tumor uptake corresponding to the cell surface expression levels. Autoradiography and immunostaining confirmed in vivo findings. Conclusion: We systematically demonstrate that the humanized immnoPET agent [89Zr]Zr-DFO-TAB-H14 specifically and stoichiometrically binds to GPC3 in several models of human liver cancer, serving as a promising in vivo GPC3 sensor. This agent may provide utility in HCC diagnosis and surveillance, and the selection of candidates for GPC3-directed therapies.

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 (203)Pb or (212)Pb. Good specific activity was attained when the immunoconjugate was labeled with (212)Pb (9.6 ± 1.4 mCi/mg). Targeting of LS-174T tumor xenografts with the (203)Pb-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 (212)Pb-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. (212)Pb-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. (212)Pb-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.

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.

There is growing interest in small and rigid peptidomimetic αvβ3 integrin antagonists that are readily synthesized and characterized and amenable to physiological conditions. Peptidomimetic 4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino)ethyloxy]benzoyl-2-[N-(3-amino-neopenta-1-carbamyl)]-aminoethylsulfonyl-amino-β-alanine (IAC) was successfully conjugated to DOTA, complexed with Gd(III) and radiolabeled with 153Gd. Radioassay results demonstrated specificity of the labeled conjugate by blocking ~95% binding with the addition of a 50-fold molar excess of cold IAC to the reaction solution. Relaxometry was used to support the hypothesis that the specificity of the Gd-peptidomimetic targeting αvβ3 integrin would increase the contrast and therefore enhance the sensitivity of an MRI scan of αvβ3 integrin positive tissues. Magnetic resonance imaging of cell pellets (M21 human melanoma) was also performed, and the images clearly show that cells reacted with Gd(III)-DOTA-IAC display a brighter image than cells without the Gd(III)-DOTA-IAC contrast agent. In addition, Gd(III)-DOTA-IAC and IAC, with IC50 of 300 nM and 230 nM, respectively, are 2.1 and 2.7 times more potent than c(RGDfK) whose IC50 is 625 nM. This promising preliminary data fuels further investigation of DOTA-IAC conjugates for targeting tumor associated angiogenesis and αvβ3 integrin positive tumors using magnetic resonance imaging.

(86)Y (half-life = 14.74 h, 33% β(+)) is within an emerging class of positron-emitting isotopes with relatively long physical half-lives that enables extended imaging of biologic processes. We report the synthesis and evaluation of 3 low-molecular-weight compounds labeled with (86)Y for imaging the prostate-specific membrane antigen (PSMA) using PET. Impetus for the study derives from the need to perform dosimetry estimates for the corresponding (90)Y-labeled radiotherapeutics.Multistep syntheses were used in preparing (86)Y- 4: - 6: PSMA inhibition constants were evaluated by competitive binding assay. In vivo characterization using tumor-bearing male mice was performed by PET/CT for (86)Y- 4: - 6: and by biodistribution studies of (86)Y- 4: and (86)Y- 6: out to 24 h after injection. Quantitative whole-body PET scans were recorded to measure the kinetics for 14 organs in a male baboon using (86)Y- 6 RESULTS: Compounds (86)Y- 4: - 6: were obtained in high radiochemical yield and purity, with specific radioactivities of more than 83.92 GBq/μmol. PET imaging and biodistribution studies using PSMA-positive PC-3 PIP and PSMA-negative PC-3 flu tumor-bearing mice revealed that (86)Y- 4-6: had high site-specific uptake in PSMA-positive PC-3 PIP tumor starting at 20 min after injection and remained high at 24 h. Compound (86)Y- 6: demonstrated the highest tumor uptake and retention, with 32.17 ± 7.99 and 15.79 ± 6.44 percentage injected dose per gram (%ID/g) at 5 and 24 h, respectively. Low activity concentrations were associated with blood and normal organs, except for the kidneys, a PSMA-expressing tissue. PET imaging in baboons reveals that all organs have a 2-phase (rapid and slow) clearance, with the highest uptake (8 %ID/g) in the kidneys at 25 min. The individual absolute uptake kinetics were used to calculate radiation doses using the OLINDA/EXM software. The highest mean absorbed dose was received by the renal cortex, with 1.9 mGy per MBq of (86)Y- 6:Compound (86)Y- 6: is a promising candidate for quantitative PET imaging of PSMA-expressing tumors. Dosimetry calculations indicate promise for future (90)Y or other radiometals that could use a similar chelator/scaffold combination for radiopharmaceutical therapy based on the structure of 6.

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.

Indocyanine green (IC-Green), the only FDA approved near-infrared (NIR) fluorophore for clinical use, is attractive to researchers for the development of targeted optical imaging agents by modification of its structure and conjugation to monoclonal antibodies (mAbs) or their fragments. IC-Green derivative, ICG-sulfo-OSu (ICG-sOSu), is frequently used for antibody conjugation. However, ICG-sOSu is amphiphilic and readily facilitates aggregation of mAbs that is not easily separable from the desired immunoconjugates. Complications originating from this behavior are frequently overlooked by researchers. This study examined detailed chemical and biological characteristics of an ICG-sOSu-labeled mAb, panitumumab, and provided a clinically applicable strategy to deliver a pure conjugation product. Size-exclusion high-performance liquid chromatography (SE-HPLC) analysis of conjugation reactions, performed at molar reaction ratios of ICG-sOSu: mAb of 5, 10, or 20, resulted in isolable desired ICG-sOSu-panitumumab conjugation product in 72%, 53%, and 19% yields, respectively, with the remainder consisting of high molecular weight aggregates (>150 kDa) 14%, 30%, and 51%, respectively. The HPLC-purified ICG-sOSu-panitumumab products were analyzed by native and SDS polyacrylamide gel electrophoresis (PAGE) followed by optical imaging. Results indicated that the interaction between ICG-sOSu and panitumumab was due to both covalent and noncovalent binding of the ICG-sOSu to the protein. Noncovalently bound dye in the ICG-sOSu-panitumumab conjugate products was removed by extraction with ethyl acetate to further purify the HPLC-isolated conjugation products. With conserved immunoreactivity, excellent target-specific uptake of the doubly purified bioconjugates was observed with minimal liver retention in athymic nude mice bearing HER1-expressing tumor xenografts. In summary, the preparation of well-defined bioconjugate products labeled with commercial ICG-sOSu dye is not a simple process and control of the conjugation reaction ratio and conditions is crucial. Furthermore, absolute purification and characterization of the products is necessitated prior to in vivo optical imaging. Use of validated and characterized dye conjugate products should facilitate the development of clinically viable and reproducible IC-Green derivative and other NIR dye mAb conjugates for optical imaging applications.

Introduction Alpha particles possess an exquisite degree of cytotoxicity when employed for targeted α-particle therapy (TAT) or radioimmunotherapy (RIT). 212 Pb, which acts as an in vivo generator of the α-emitting nuclide 212 Bi has shown great promise in pre-clinical studies when used to label the HER2 binding antibody, trastuzumab. Currently, the first RIT clinical trial employing 212 Pb radiolabeled trastuzumab is in progress. This report provides detailed current protocol operations and steps that were generated for use in the clinical trial as well as the relevant pre-clinical experimentation, and describes in detail the labeling of proteins or peptides with 212 Pb as provided via a 224 Ra based generator system. Methods 212 Pb was eluted from the 224 Ra/ 212 Pb generator using hydrochloric acid (2M). The generator eluate was evaporated and digested with nitric acid (8M) followed by extraction of the 212 Pb with dilute nitric acid (0.1M). The dilute nitric acid solution of 212 Pb was used to label the immunoconjugate Trastuzumab-TCMC (2-(4-isothiocyanatobenzyl-1,4,7,10-tetraaza-1,4,7,10,tetra-(2-carbamonylmethyl)-cyclododecane) at pH5.5. Results Elution of 212 Pb from the generator was efficient yielding>90% of available 212 Pb. Trastuzumab-TCMC was efficiently labeled with a radiochemical yield of 94%±4% (n=7) by ITLC and an isolated yield of 73%±3% (n=7). Conclusions The results show the feasibility of generating radioimmunoconjugates and peptide conjugates for use as in vivo α generator systems in the clinic. The technology holds promise in applications involving the treatment of minimal disease such as micrometastases and residual tumor after surgical debulking, hematological cancers, infections, and compartmental cancers, such as ovarian cancer.

Despite the great potential of targeted α-radioimmunotherapy (RIT) as demonstrated by pre-clinical and clinical trials, limited progress has been made on the improvement of chelation chemistry for (212)Bi and (213)Bi. A new bifunctional ligand 3p-C-NETA was evaluated for targeted α RIT using (212)Bi and (213)Bi.Radiolabeling of 3p-C-NETA with (205/6)Bi, a surrogate of (212)Bi and (213)Bi, was evaluated at pH5.5 and room temperature. In vitro stability of the (205/6)Bi-3p-C-NETA-trastuzumab conjugate was evaluated using human serum (pH7, 37 °C). Immunoreactivity and specific activity of the (205/6)Bi-3p-C-NETA-trastuzumab conjugate were measured. An in vivo biodistribution study was performed to evaluate the in vivo stability and tumor targeting properties of the (205/6)Bi-3p-C-NETA-trastuzumab conjugate in athymic mice bearing subcutaneous LS174T tumor xenografts.The 3p-C-NETA-trastuzumab conjugate was extremely rapid in complexing with (205/6)Bi, and the corresponding (205/6)Bi-3p-C-NETA-trastuzumab was stable in human serum. (205/6)Bi-3p-C-NETA-trastuzumab was prepared with a high specific activity and retained immunoreactivity. (205/6)Bi-3p-C-NETA-trastuzumab conjugate displayed excellent in vivo stability and targeting as evidenced by low normal organ and high tumor uptake.The results of the in vitro and in vivo studies indicate that 3p-C-NETA is a promising chelator for RIT applications using (212)Bi and (213)Bi. Further detailed in vivo evaluations of 3p-C-NETA for targeted α RIT are warranted.

The studies described herein assess the potential of combining platinum-based chemotherapy with high-linear energy transfer (LET) α-particle-targeted radiation therapy using trastuzumab as the delivery vehicle. An initial study explored the combination of cisplatin with (213)Bi-trastuzumab in the LS-174T i.p. xenograft model. This initial study determined the administration sequence of cisplatin and (213)Bi-trastuzumab. Cisplatin coinjected with (213)Bi-trastuzumab increased the median survival (MS) to 90 days versus 65 days for (213)Bi-trastuzumab alone. Toxicity was observed with a weight loss of 17.6% in some of the combined treatment groups. Carboplatin proved to be better tolerated. Maximal therapeutic benefit, that is, a 5.1-fold increase in MS, was obtained in the group injected with (213)Bi-trastuzumab, followed by carboplatin 24 hours later. This was further improved by administration of multiple weekly doses of carboplatin. The MS achieved with administration of 3 doses of carboplatin was 180 days versus 60 days with (213)Bi-trastuzumab alone. The combination of carboplatin with (212)Pb radioimmunotherapy was also evaluated. The therapeutic efficacy of (212)Pb-trastuzumab (58-day MS) increased when the mice were pretreated with carboplatin 24 hours prior (157-day MS). These results again demonstrate the necessity of empirically determining the administration sequence when combining therapeutic modalities.

Background: Paclitaxel has recently been reported by this laboratory to potentiate the high-LET radiation therapeutic 212Pb-TCMC-trastuzumab, which targets HER2. To elucidate mechanisms associated with this therapy, targeted α-particle radiation therapeutic 212Pb-TCMC-trastuzumab together with paclitaxel was investigated for the treatment of disseminated peritoneal cancers. Methods: Mice bearing human colon cancer LS-174T intraperitoneal xenografts were pre-treated with paclitaxel, followed by treatment with 212Pb-TCMC-trastuzumab and compared with groups treated with paclitaxel alone, 212Pb-TCMC-HuIgG, 212Pb-TCMC-trastuzumab and 212Pb-TCMC-HuIgG after paclitaxel pre-treatment. Results: 212Pb-TCMC-trastuzumab with paclitaxel given 24 h earlier induced increased mitotic catastrophe and apoptosis. The combined modality of paclitaxel and 212Pb-TCMC-trastuzumab markedly reduced DNA content in the S-phase of the cell cycle with a concomitant increase observed in the G2/M-phase. This treatment regimen also diminished phosphorylation of histone H3, accompanied by an increase in multi-micronuclei, or mitotic catastrophe in nuclear profiles and positively stained γH2AX foci. The data suggests, possible effects on the mitotic spindle checkpoint by the paclitaxel and 212Pb-TCMC-trastuzumab treatment. Consistent with this hypothesis, 212Pb-TCMC-trastuzumab treatment in response to paclitaxel reduced expression and phosphorylation of BubR1, which is likely attributable to disruption of a functional Aurora B, leading to impairment of the mitotic spindle checkpoint. In addition, the reduction of BubR1 expression may be mediated by the association of a repressive transcription factor, E2F4, on the promoter region of BubR1 gene. Conclusion: These findings suggest that the sensitisation to therapy of 212Pb-TCMC-trastuzumab by paclitaxel may be associated with perturbation of the mitotic spindle checkpoint, leading to increased mitotic catastrophe and cell death.

Purpose To elucidate the mechanism of the therapeutic efficacy of targeted α-particle radiation therapy using 212 Pb-TCMC-trastuzumab together with gemcitabine for treatment of disseminated peritoneal cancers. Methods and Materials Mice bearing human colon cancer LS-174T intraperitoneal xenografts were pretreated with gemcitabine, followed by 212 Pb-TCMC-trastuzumab and compared with controls. Results Treatment with 212 Pb-TCMC-trastuzumab increased the apoptotic rate in the S-phase-arrested tumors induced by gemcitabine at earlier time points (6 to 24 hours). 212 Pb-TCMC-trastuzumab after gemcitabine pretreatment abrogated G2/M arrest at the same time points, which may be associated with the inhibition of Chk1 phosphorylation and, in turn, cell cycle perturbation, resulting in apoptosis. 212 Pb-TCMC-trastuzumab treatment after gemcitabine pretreatment caused depression of DNA synthesis, DNA double-strand breaks, accumulation of unrepaired DNA, and down-regulation of Rad51 protein, indicating that DNA damage repair was blocked. In addition, modification in the chromatin structure of p21 may be associated with transcriptionally repressed chromatin states, indicating that the open structure was delayed at earlier time points. Conclusion These findings suggest that the cell-killing efficacy of 212 Pb-TCMC-trastuzumab after gemcitabine pretreatment may be associated with abrogation of the G2/M checkpoint, inhibition of DNA damage repair, and chromatin remodeling.

An α-particle, a 4He nucleus, is exquisitely cytotoxic and indifferent to many limitations associated with conventional chemo- and radiotherapy. The exquisite cytotoxicity of α-radiation, the result of its high mean energy deposition [high linear energy transfer (LET)] and limited range in tissue, provides for a highly controlled therapeutic modality that can be targeted to selected malignant cells [targeted α-therapy (TAT)] with minimal normal tissue effects. A burgeoning interest in the development of TAT is buoyed by the increasing number of ongoing clinical trials worldwide. The short path length renders α-emitters suitable for treatment and management of minimal disease such as micrometastases or residual tumor after surgical debulking, hematologic cancers, infections, and compartmental cancers such as ovarian cancer or neoplastic meningitis. Yet, despite decades of study of high LET radiation, the mechanistic pathways of the effects of this modality remain not well defined. The modality is effectively presumed to follow a simple therapeutic mechanism centered on catastrophic double-strand DNA breaks without full examination of the actual molecular pathways and targets that are activated that directly affect cell survival or death. This Molecular Pathways article provides an overview of the mechanisms and pathways that are involved in the response to and repair of TAT-induced DNA damage as currently understood. Finally, this article highlights the current state of clinical translation of TAT as well as other high-LET radionuclide radiation therapy using α-emitters such as 225Ac, 211At, 213Bi, 212Pb, and 223Ra. Clin Cancer Res; 19(3); 530–7. ©2012 AACR.

Radiolabeled antibodies (mAbs) provide efficient tools for cancer therapy. The combination of low energy β(-)-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 β(-)- and α-particle RIT comparatively through an in vivo tumor environment offers real information suitable to enhance combination therapy regimens involving α- and β(-)-particle RIT for the management of intraperitoneal disease.

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.

Methodology for site-specific modification and chelate conjugation of a cyclic RGD (cRGD) peptide for the preparation of a radiotracer molecular imaging agent suitable for detecting α(v)β(3) integrin is described. The method involves functionalizing the peptide with an aldehyde moiety and conjugation to a 1,4,7,10-tetraazacyclododecane-N,N',N″,N‴-tetraacetic acid (DOTA) derivative that possesses an aldehyde reactive aminooxy group. The binding assay of the (111)In-labeled peptide conjugate with α(v)β(3) integrin showed 60% bound when four equivalents of the integrin was added, a reasonable binding affinity for a mono-valent modified RGD peptide.

Integrins are a family of transmembrane glycoproteins with associated α and β subunits forming 25 unique heterodimers that facilitate adhesion and migration of cells on the extracellular matrix proteins found in intercellular spaces and basement membranes.1 One of these integrins, αvβ3 integrin, interacts with vitronectin, fibronectin, fibrinogen, thrombospondin, collagen, laminin and von Willebrand factor. This integrin is over-expressed in tumor induced angiogenic vessels and in various human tumors, but is found at low levels on epithelial and endothelial cells. It is therefore a widely recognized target for the development of molecular probes for imaging angiogenesis and cancer therapy. Towards this end, the tumor imaging capability of several RGD peptides that act as αvβ3 integrin antagonists has been demonstrated by several research groups. Additionally, several of these peptides have been shown to inhibit tumor angiogenesis and interrupt metastasis in many models.2–4 There is growing interest in peptidomimetic αvβ3 integrin antagonists composed of a stable core scaffold with basic and acidic groups that mimic the guanidine and carboxylate pharmacophore of RGD peptides. Peptidomimetics tend to have higher activity, specificity and longer duration of action compared to the peptides. One such peptidomimetic αvβ3 integrin antagonist, 4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino)ethyloxy]benzoyl-2-aminoethylsulfonyl-amino-β-alanine (IA) was synthesized by Hood et al.5 Subsequent, modification of IA to the corresponding carbamate derivatives by the Danthi group resulted in 4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino)ethyloxy]benzoyl-2-[N-(3-amino-neopenta-1-carbamyl)]-aminoethylsulfonyl-amino-β-alanine (IAC), with a binding affinity 20 times greater than that of IA.6 A SPECT (single photon emission computed tomography) imaging study with 111In-DOTA-Bz-SCN- IAC was also performed and tumor was clearly visualized at 4 h p.i.7111In-DOTA-Bz-SCN- IAC was prepared using the bifunctional chelate DOTA-Bz-SCN which differs from the DOTA-GA described in this study. Clinically, SPECT and PET (positron emission tomography) play significant roles allowing noninvasive imaging of internal physiological and biochemical function and pathologies in vivo. While PET is more expensive, it has significant advantages over SPECT with respect to its ability to better quantify images. Of metallic radionuclides currently being investigated for PET applications, gallium-68 (68Ga) has grown in popularity.8, 9 The popularity of 68Ga stems from the ease of on site production from a long lived generator system (68Ge/68Ga) rather than a cyclotron, and automation for incorporation into radiolabeled compounds.10 The 67.7 min half-life of 68Ga is an appropriate match to the biological half-lives of peptides. Gallium(III) typically binds with chelates possessing multiple anionic oxygen donors preferring a coordination number of six in an octahedral geometry. Fitting these preferences, the macrocyclic ligand, 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), is well established as forming very stable complexes with a wide variety of metals, Ga(III) being one of them.11 Several NOTA derivatives have been reported (Figure 1) for use in the radiolabeling of proteins and peptides. Recently, Knetsch et al reported a 68Ga-labeled NODA-GA-conjugated RGD peptide ([68Ga]NODAGA-RGD) that showed better tumor to blood ratio in vivo than the corresponding [68Ga]DOTA-RGD derivative.12 Figure 1 Structures of NOTA derivatives. 7-(5-Maleimido-1-ethoxycarbonylphenyl)-1,4,7-triazacyclononane-1,4-diylacetic acid (1), 2-(4-Aminobutyl)-1,4,7-triazacyclononane-1,4,7-triyltriacetic acid (2), nNOTA (3), p-SCN-Bn-NOTA (4), NETA (5), and NODA-GA(tBu)3 ( ... While interest in 68Ga for PET imaging is currently significant, there are Pb(II) isotopes that have also been of interest for biomedical applications. Specifically, 203Pb and 212Pb are radiometals possessing favorable properties for use in nuclear medicine for potential diagnostic and therapeutic applications, respectively.13–21203Lead (t1/2 = 51.9 h) emits a γ-ray (279 keV) that is ideal for single photon emission computed tomography (SPECT) imaging and is suitable for pharmacokinetic and pharmacodynamic tracer studies. In addition, 203Pb can serve as one half of a potential matched-pair of radioisotopes when combined with 212Pb for therapeutic applications.212 Lead (t1/2 = 10.6 h) has been studied as an `in vivo generator' of 212Bi (t1/2 = 60 min) to overcome the short half-life of that daughter isotope. The macrocyclic polyaminocarboxylate chelate DOTA, (1,4,7,10-tetraazacyclododecane-N,N',N”,N”'-tetraacetic acid labeled with 212Pb provides a complex that is adequately stable in vivo to sequester the radionuclide.17 The Macke group in Switzerland have synthesized a DOTA derivative analogous to NOTA-GA, , 1-(1-carboxy-3-carbotertbutoxymethyl)-1,4,7,10-tetraazacyclododecane (DOTA-GA(tBu)4).22 The DOTA-GA(tBu)4 affords four intact carboxylic acid functional groups with a free carboxylate side chain ready for conjugation to the N-terminus of peptides which makes it useful for biomedical applications. In the present study, the objective was to move beyond the use of RGD peptides as delivery vectors to the various integrin targets and explore the utility of IAC for such applications. 68Gallium labeling was investigated for PET applications using NODA-GA and 67Ga as a surrogate for 68Ga, and 203Pb for SPECT imaging using DOTA-GA. To this end, IAC was successfully conjugated to NODAGA (Scheme 1) and DOTA-GA (Scheme 2) and the conjugates were radiolabeled with 111In or 67Ga for the NODA-GA conjugate and 203Pb for the DOTA-GA conjugate. In brief, NODA-GA(tBu)3 or DOTAGA(tBu)4, N-hydroxysuccinimide, and EDC were dissolved in dichloromethane and the reaction mixture was stirred for 24 h. The mixture was extracted with saturated NaCl solution, 5% NaHCO3, and saturated NaCl again. The organic layer was dried over MgSO4, filtered, and dried under vacuum resulting in the formation of yellowish oils 2 or 5. The IAC and 2 or 5 were combined in anhydrous DMF and diisopropylethylamine was added to the mixture which was then stirred overnight at room temperature. Reverse-phase HPLC purification followed by TFA deprotection yielded 1 or 4, respectively.23, 24 To evaluate the radiolabeling efficiency of the NODA-GA and DOTA-GA conjugates,111In, 67Ga and 203Pb were employed to demonstrate facile formation of complexes with these radionuclides. The NODA-GA conjugate 1 was efficiently radiolabeled (> 90 %) with 111In and 67Ga within 30 min (Fig. 2A and 2B, respectively). The radiolabeling of the DOTA-GA conjugate 4 with 203Pb was equally efficient (Fig. 2C). Non-radioactive Ga(III)-1 and Pb(II)-4 were also synthesized in order to characterize the radiolabeled 67Ga and 203Pb complexes.25, 26 Figures 3 and ​and44 demonstrate HPLC profiles of the mixture containing both Ga(III)-1 and 67Ga-1; and Pb(II)-4 and 203Pb-4, respectively. Figure 2 Radio-HPLC profiles of 111In-1, A; 67Ga-1, B; and 203Pb-4, C and Figure 3 HPLC profiles of Ga(III)-1 (top) and 67Ga-1 (bottom) Figure 4 HPLC profiles of Pb(II)-4 (top) and 203Pb-4 (bottom). Scheme 1 Synthesis of compound 1. Scheme 2 Synthesis of compound 4. A radioimmunoassay was performed to assess the binding ability of the radiolabeled NODA-GA and DOTA-GA conjugates with αvβ3 integrin. The 111In-labeled 1 (2 × 106 cpm, 0.47 μM), 67Ga-labeled 1 (5 × 105 cpm, 0.45 μM) or 203Pb-labeled 4 (3 × 105 cpm, 0.5 μM) was incubated with 0, 0.5, 1.0 and 2.0 μM of purified human αvβ3 integrin (MW 237,000) in a total volume of 25 μL PBS for 3 h at 37 °C. For non-specific binding, excess IAC (20 μM) was added to the reaction mixture to block binding. The reaction mixture was then separated on a 10 mL Sephadex G50 column using PBS as eluent. Fractions (0.5 mL) were collected and subsequently counted in a γ-counter. As indicated in Table 1, the labeled conjugates bound the integrin to varying degrees. The binding of 111In-1 was greatest followed by 67Ga-1 and then 203Pb-4. In addition, binding was blocked ~95% by the addition of a 10 to 20-fold molar excess of the cold IAC to the reaction solution indicating specific binding of the labeled conjugates. Furthermore, it is worth noting that the reactivity of the 111In-1 with αvβ3 integrin (88 %) is higher than that reported for 111In-DOTA-IAC (72 %).7 Table 1 Binding of 111In-1, 67Ga-1 and 203Pb-4 to purified αvβ3 integrin. In conclusion, the peptidomimetic αvβ3 integrin antagonist (IAC) was conjugated to NODA-GA and DOTA-GA and successfully radiolabeled with 111In, 67Ga and 203Pb. This promising preliminary data is fueling further investigation of NODA-GA-IAC and DOTA-GA-IAC conjugates for targeting tumor associated angiogenesis and αvβ3 integrin positive tumors using PET and SPECT imaging. Other potential applications include the use of radionuclides such as 90Y, 177Lu and 212Pb for radiotherapy.

Studies from this laboratory have demonstrated the potential of targeting HER2 for therapeutic and imaging applications with medically relevant radionuclides. To expand the repertoire of trastuzumab as a radioimmunoconjugate (RIC) vector, use of (177)Lu was investigated. The combination of a 6.7 d half-life, lower energy β(-)-emissions (500 keV max; 130 keV ave), and an imagable γ-emission make (177)Lu an attractive candidate for radioimmunotherapy (RIT) regimens for treatment of larger tumor burdens not possible with α-zparticle radiation. Radiolabeling trastuzumab-CHX-A"-DTPA with (177)Lu was efficient with a specific binding of 60.8 ± 6.8% with HER2 positive SKOV-3 cells. Direct quantitation of tumor targeting and normal tissue uptake was performed with athymic mice bearing subcutaneous and intraperitoneal LS-174T xenografts; a peak tumor %ID/g of 24.70 ± 10.29 (96 h) and 31.70 ± 16.20 (72 h), respectively, was obtained. Normal tissue uptake of the RIC was minimal. Tumor targeting was also demonstrated by γ-scintigraphy. A therapy study administering escalating doses of (177)Lu-trastuzumab to mice bearing three day LS-174T i.p. xenografts established the effective therapeutic dose of i.p. administered (177)Lu-trastuzumab at 375 μCi with a median survival of 124.5 d while a median survival of 10 d was noted for the control (untreated) group. In conclusion, trastuzumab radiolabeled with (177)Lu has potential for treatment of disseminated, HER2 positive, peritoneal disease.

The potential of the HER2-targeting antibody trastuzumab as a radioimmunoconjugate useful for both imaging and therapy was investigated. Conjugation of trastuzumab with the acyclic bifunctional chelator CHX-A"-DTPA yielded a chelate:protein ratio of 3.4 ± 0.3; the immunoreactivity of the antibody unaffected. Radiolabeling was efficient, routinely yielding a product with high specific activity. Tumor targeting was evaluated in mice bearing subcutaneous (s.c.) xenografts of colorectal, pancreatic, ovarian, and prostate carcinomas. High uptake of the radioimmunoconjugate, injected intravenously (i.v.), was observed in each of the models, and the highest tumor %ID/g (51.18 ± 13.58) was obtained with the ovarian (SKOV-3) tumor xenograft. Specificity was demonstrated by the absence of uptake of 111In-trastuzumab by melanoma (A375) s.c. xenografts and 111In-HuIgG by s.c. LS-174T xenografts. Minimal uptake of i.v. injected 111In-trastuzumab in normal organs was confirmed in non-tumor-bearing mice. The in vivo behavior of 111In-trastuzumab in mice bearing intraperitoneal (i.p.) LS-174T tumors resulted in a tumor %ID/g of 130.85 ± 273.34 at 24 h. Visualization of tumor, s.c. and i.p. xenografts, was achieved by γ-scintigraphy and PET imaging. Blood pool was evident as expected, but cleared over time. The blood pharmacokinetics of i.v. and i.p. injected 111In-trastuzumab was determined in mice with and without tumors. The data from these in vitro and in vivo studies supported advancement of radiolabeled trastuzumab into two clinical studies, a Phase 0 imaging study in the Molecular Imaging Program of the National Cancer Institute and a Phase 1 radioimmunotherapy study at the University of Alabama.

Bevacizumab is a humanized monoclonal antibody that binds to tumor-secreted vascular endothelial growth factor (VEGF)-A and inhibits tumor angiogenesis. In 2004, the antibody was approved by the US Food and Drug Administration (FDA) for the treatment of metastatic colorectal carcinoma in combination with chemotherapy. This report describes the preclinical evaluation of a radioimmunoconjugate, (86)Y-CHX-A″-DTPA-bevacizumab, for potential use in Positron Emission Tomography (PET) imaging of VEGF-A tumor angiogenesis and as a surrogate marker for (90)Y-based radioimmunotherapy. Bevacizumab was conjugated to CHX-A″-DTPA and radiolabeled with (86)Y. In vivo biodistribution and PET imaging studies were performed on mice bearing VEGF-A-secreting human colorectal (LS-174T), human ovarian (SKOV-3) and VEGF-A-negative human mesothelioma (MSTO-211H) xenografts. Biodistribution and PET imaging studies demonstrated highly specific tumor uptake of the radioimmunoconjugate. In mice bearing VEGF-A-secreting LS-174T, SKOV-3 and VEGF-A-negative MSTO-211H tumors, the tumor uptake at 3 days postinjection was 13.6 ± 1.5, 17.4 ± 1.7 and 6.8 ± 0.7 % ID/g, respectively. The corresponding tumor uptake in mice coinjected with 0.05 mg cold bevacizumab were 5.8 ± 1.3, 8.9 ± 1.9 and 7.4 ± 1.0 % ID/g, respectively at the same time point, demonstrating specific blockage of the target in VEGF-A-secreting tumors. The LS-174T and SKOV3 tumors were clearly visualized by PET imaging after injecting 1.8-2.0 MBq (86)Y-CHX-A″-DTPA-bevacizumab. Organ uptake quantified by PET closely correlated (r(2) = 0.87, p = 0.64, n = 18) to values determined by biodistribution studies. This preclinical study demonstrates the potential of the radioimmunoconjugate, (86)Y-CHX-A″-DTPA-bevacizumab, for noninvasive assessment of the VEGF-A tumor angiogenesis status and as a surrogate marker for (90)Y-CHX-A″-DTPA-bevacizumab radioimmunotherapy.

The studies described here are the first to evaluate the in vitro and in vivo properties of (111)In-CHX-A''-panitumumab for radioimmunotherapy (alpha- and beta(-)-emitters) and radioimmunoimaging (single photon emission computed tomography and positron emission tomography).Twenty-seven human carcinoma cell lines were analysed for expression of epidermal growth factor receptors by flow cytometry. Panitumumab was conjugated with CHX-A''-DTPA (diethylenetriamine-pentaacetic acid) and radiolabelled with (111)In. Immunoreactivity of the CHX-A''-DTPA-panitumumab and (111)In-CHX-A''-DTPA-panitumumab was evaluated by radioimmunoassays. Tumour targeting was determined in vivo by direct quantitation of tumour and normal tissues and by gamma-scintigraphy.For 26 of 27 human tumour cell lines, 95% of the cells expressed epidermal growth factor receptors over a range of intensity. Immunoreactivity of panitumumab was retained after modification with CHX-A''-DTPA. Radiolabelling of the immunoconjugate with (111)In was efficient with a specific activity of 19.5 +/- 8.9 mCi.mg(-1) obtained. Immunoreactivity and specificity of binding of the (111)In-panitumumab was shown with A431 cells. Tumour targeting by (111)In-panitumumab was demonstrated in athymic mice bearing A431, HT-29, LS-174T, SHAW or SKOV-3 s.c. xenografts with little uptake observed in normal tissues. The (111)In-panitumumab was also evaluated in non-tumour-bearing mice. Pharmacokinetic studies compared the plasma retention time of the (111)In-panitumumab in both non-tumour-bearing and A431 tumour-bearing mice. Tumour targeting was also visualized by gamma-scintigraphy.Panitumumab can be efficiently radiolabelled with (111)In with high labelling yields. Based on the efficiency in tumour targeting and low normal tissue uptake, panitumumab may be an effective targeting component for radioimmunodiagnostic and radioimmunotherapeutic applications.

Two novel imaging agents trastuzumab-Cy5.5-CHX-A''1 and cetuximab-Cy7-CHX-A''2, bearing both a chelating moiety (CHX-A'') for sequestering metallic radionuclides ((86)Y or (111)In) and the near infrared dye Cy5.5/Cy7, were prepared by a novel modular synthetic strategy as examples of dual-labeled, antibody-based imaging probe library. Fluorescent microscopy illustrated that 1 and 2 strongly bind to HER2-expressing cancer cells (e.g., NIH3T3-HER2(+), SKOV-3) and to EGFR-expressing cancer cells (e.g., A431), respectively, thereby demonstrating that the functionality of the targeting moiety is conserved. Hence, the described novel synthesis strategy can be applied to engineer other tumor-targeted monoclonal antibody based probes for multimodality imaging.

An efficient and short synthetic route to a novel decadentate ligand 7-[2-(bis-carboxymethyl-amino)-ethyl]-4,10-bis-carboxymethyl-1,4,7,10-tetraaza-cyclododec-1-yl-acetic acid (DEPA) with both macrocyclic and acyclic binding moieties is reported. A reproducible and scalable synthetic method to a precursor molecule of DEPA, 1,4,7-tris(tert-butoxycarbonylmethyl)tetraazacyclododecane was developed. DEPA was evaluated as a chelator of (177)Lu, (212)Bi, and (213)Bi for potential use in an antibody-targeted cancer therapy, radioimmunotherapy (RIT) using Arsenazo III based spectroscopic complexation kinetics, in vitro serum stability, and in vivo biodistribution studies.

IL-2 is well described as a cytokine with two markedly distinct functionalities: as a necessary signal during CD4+ and CD8+ T cell activation/expansion and as an essential cytokine for the maintenance of CD4+CD25+FoxP3+ T cells (regulatory T (TREG) cells) during homeostasis. In this study we demonstrate for the first time that, compared with the use of IL-2 alone, a complex of IL-2 and anti-IL-2 Ab (IL-2 complex) enhances the effectiveness of a viral vaccine in a mouse model with known Ag specificity. IL-2 complex led to an increase in the number of Ag-specific effector/memory CD8+ T cells, cytokine production, and CTL lysis following Ag-specific restimulation in a vaccination setting. Our results further demonstrate that this effect is temporary and declines over the course of a few days after the IL-2 complex treatment cycle. Moreover, in contrast to the use of IL-2 alone, IL-2 complex greatly increased the ratio of effector/memory CD8+ T cells to TREG cells. This phenomenon can thus potentially be used in the enhancement of immune responses to vaccination.

The use of monoclonal antibodies (mAbs) as targeting vectors to selectively deliver a lethal dose of radiation to cancer cells through either a β- or α-emitting radionuclide, radioimmunotherapy (RIT), has been extensively investigated for cancer therapies.1-3 The targeted nature of such therapies as well as imaging with γ- or β+-emitters (SPECT / PET), radioimmunoimaging (RII), offers the promise of greater efficacy and less toxicity.4 This field has attracted greater attention particularly after the recent Food and Drug Administration approval of two radionuclide-bearing monoclonal antibody therapies (90Y-ibritumomab and 131I-tositumomab) for the treatment of lymphohematopoietic malignancies.5, 6 The development of suitable bifunctional chelating agents for modification of proteins for RII and RIT continues to receive great attention. To achieve useful radiolabeled mAbs in the clinical setting, chelating agents must form thermodynamically and kinetically stable complexes to prevent loss of radionuclides in vivo, while retaining mAb immunoreactivity.7, 8 A variety of DTPA and DOTA derivatives bearing isothiocyanate reactive groups are well documented for this purpose and some have been carried forward into antibody-targeted radiation therapy clinical trials (Figure 1).4 The acyclic bifunctional ligand, CHX-A” DTPA (N-[(R)-2-amino-3-(p-aminophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-N,N,N’,N”,N”-pentaacetic acid) reported in 1997, has been found to be excellent at sequestering radionuclides such as 86/90Y, 212/213Bi, 111In, and 177Lu with great in vivo stability. 9-12 In particular, radiolabeling of CHX-A” DTPA modified mAb with the therapeutic α-emitter 212Bi was found to be statistically comparable to protein conjugates formed with a bifunctional DOTA derivative (2-(p-isothiocyanatobenzyl)-l,4,7,10-tetraazacyclododecane tetraacetic acid) in stability, without the detraction of the slow formation kinetics inherent to the DOTA macrocycle.11 Figure 1 Structures of CHX-A” DTPA, 1B4M-DTPA, c-DOTA and PA-DOTA. In this laboratory, the isothiocyanate form of CHX-A” DTPA reacts with primary e-amine groups of lysine or terminal α-amine residues of proteins and has routinely been used for conjugation to mAb (Figure 2). This conjugation method can be problematic in controlling the precise number and exact position of chelates that have been attached. This can lead to concerns regarding potential interference with antigen binding, particularly so if there are lysine residues in the antigen binding region. Figure 2 A schematic presentation of CHX-A” DTPA conjugated trastuzumab 1, conjugation through an isothiocyanato CHX-A” DTPA chelator, and 2 conjugation through the maleimido CHX-A” DTPA chelator, 5, to prepare 2. An alternate conjugation strategy is available via the selectivity of the Michael addition between a thiol and a maleimide.13 The maleimide group reacts efficiently and specifically with thiol-terminal biomolecules and has been widely used to form stable protein conjugates through thioether linkages. This conjugation method is widely considered to give greater control over the position of the chelating agent on a protein when the thiol group(s), e.g., engineered cysteine residues, can be introduced at the genetic level. One the other hand, thiol groups are less likely than charged groups (e.g. amino groups) currently used for conjugation to be found in at mAb’s binding sites, thereby offering better chemistry to retain immunoreactivity of mAbs. A small number of chelating agents that exploit this strategy have been reported,14-17 but few have seen extensive use for in vivo studies of radiolabeled antibodies or peptides for either imaging or therapy. In part, this may be due to a lack of familiarity with this chemistry and/or more problematically, the lack of appropriate derivatives of previously established chelating agents. Herein, the synthesis of a novel maleimido CHX-A” DTPA chelator, 5, is described as well as its successful conjugation to the monoclonal antibody trastuzumab. This novel malemide CHX-A” DTPA derivative thus offers an alternative route to modify antibodies, peptides, and other targeting vectors with an established radiometal chelate. This conjugation strategy might be particularly useful considering the availability of engineered Fab or Fab’ antibody fragments containing free sulfhydryl groups. For the synthesis of 5, we used the tert-butyl ester protected p-amino functionalized CHX-A” DTPA, 3, which was recently developed in this laboratory (Figure 3).18, 19 This aniline derivative was reacted with N-e-maleimidocaproic acid (EMCA) using standard peptide coupling conditions to provide maleimide 4 (63%).20 The tert-butyl esters in 4 were quantitatively cleaved by TFA as monitored by TLC to generate 5.21 Thereafter, the liberated five carboxyl groups, along with the three tertiary amines, became available for sequestering radioactive metals, such as 111In, 86/90Y, 212/213Bi or 177Lu. The maleimide moiety in 5 provides a highly reactive group towards thiol groups either extant or introduced into proteins or peptides. To conjugate 5 to trastuzumab, the mAb was first thiolated with 15 equivalents of Traut’s agent using standard procedures with minor modifications.22 Routinely, ∼ 2 - 4 thiol groups per trastuzumab were introduced as quantified by Ellman’s reagent. Thiolated trastuzumab was then reacted with 10 equivalents of 5 to produce conjugate 2.23 Unreacted thiolated conjugate was capped with iodoacetamide to minimize cross-linking of the mAb conjugate product and to promote a longer shelf life of the immunoconjugate. Finally, the reaction mixture was dialyzed into PBS buffer at 4 °C to remove all small molecules from the protein solution. The final average ratio of CHX-A” DTPA moieties per trastuzumab achieved by this process was calculated to be ∼ 2 based on an Arsenazo (III) assay.24 The immunoconjugate 2 was radiolabeled efficiently (> 95%) with the SPECT radionuclide 111In within 30 min at room temperature.25 Figure 4 shows the size-exclusion HPLC profile of the isothiocyanate and maleimide-functionalized trastuzumab as compared to their respective 111In labeled analogs as well as to the unmodified mAb. The profiles of the maleimide-modified analogs were similar to those of the previously reported isothiocyanate-modified analogs, both radiolabeled and unlabeled, as well as to unmodified trastuzumab. Immunoreactivity of the resulting 111In labeled immunoconjugate 2 was demonstrated by binding to SKOV-3, a human ovarian carcinoma cell line. There was comparable binding between 111In labeled immunoconjugate 2 (44.9%) and the 111In-labeled isothiocyanato CHX-A” modified trastuzumab, 1 (38.6%).19,26 Figure 3 Synthesis of the bifunctional maleimido CHX-A” DTPA chelator, 5. Figure 4 SE-HPLC chromatograph of (a) 1; (b) 2; (c) 111In-1; (d) 111In-2; (e) trastuzumab. (a), (b), and (e) were monitored by UV at 280 nm, while (c) and (d) were recorded by an in-line HPLC radiodetector. In conclusion, a novel bifunctional maleimido DTPA derivative 5 was designed, synthesized and characterized for conjugation to thiol containing biomolecules such as antibodies, peptides or other targeting vectors, which was based on a previously established chelating agent, CHX-A”. Successful conjugation of 5 to the monoclonal antibody trastuzumab (Herceptin) was achieved by efficient thiol-maleimide chemistry. One should note that the direct use of 4 would also be possible for solution or solid phase conjugation to peptides and other vectors. Subsequent cleavage of the esters would provide a conjugate product suitable for radiolabeling. Therefore, this newly developed maleimido CHX-A” DTPA allows a new route to providing an established radioactive metal chelating agent on biomolecules for imaging and/or therapies of cancers.

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.

99mTc-labeled bombesin analogues have shown promise for noninvasive detection of many tumors that express bombesin (BN)/gastrin-releasing peptide (GRP) receptors. 99mTc-labeled peptides, however, have a tendency to accumulate in the liver and intestines due to hepatobiliary clearance as a result of the lipophilicity of the 99mTc chelates. This makes the imaging of lesions in the abdominal area difficult. In this study, we have synthesized a new high affinity 99mTc-labeled BN analogue, [DTPA1, Lys3(99mTc-Pm-DADT), Tyr4]BN, having a built-in pharmacokinetic modifier, DTPA, and labeled with 99mTc using a hydrophilic diaminedithiol chelator (Pm-DADT) to effect low hepatobiliary clearance. In vitro binding studies using human prostate cancer PC-3 cell membranes showed that the inhibition constant (Ki) for [DTPA1, Lys3(99Tc-Pm-DADT), Tyr4]BN was 4.1 +/- 1.4 nM. Biodistribution studies of [DTPA1, Lys3(99mTc-Pm-DADT), Tyr4]BN in normal mice showed very low accumulation of radioactivity in the liver and intestines (1.32 +/- 0.13 and 4.58 +/- 0.50% ID, 4 h postinjection, respectively). There was significant uptake (7.71 +/- 1.37% ID/g, 1 h postinjection) in the pancreas which expresses BN/GRP receptors. The uptake in the pancreas could be blocked by BN, partially blocked by neuromedin B, but not affected by somatostatin, indicating that the in vivo binding was BN/GRP receptor specific. Scintigraphic images showed specific, high contrast delineation of prostate cancer PC-3 xenografts in SCID mice. Thus, the new peptide has a great potential for imaging BN/GRP receptor-positive cancers located even in the abdomen.

Previously, we showed that labeled bitistatin analogues possessed excellent characteristics for imaging both deep-vein thrombosis and pulmonary embolism. We hypothesized that the N-terminal amino acid sequence of bitistatin, which is different from other disintegrins, likely interacts with the binding site of platelets to confer desirable properties to bitistatin for imaging. In this study, we present the design, synthesis, and initial biological testing of a short-chain analogue of the native 83-amino-acid bitistatin sequence. Our initial molecular modeling of the binding loop of bitistatin showed that the minimal sequence that represented the binding region was a cyclic 10 amino acid sequence cyclo[Cys-Arg-Ile-Ala-Arg-Gly-Asp-Trp-Asn-Cys(S)]. Systematic modeling of a truncated N-terminal sequence of bitistatin fused with the optimized binding region having a thioether sequence through a Gaba spacer ultimately yielded the 24-amino acid peptide, cyclo-[CH(2)CO-Arg-Ile-Ala-Arg-Gly-Asp-Trp-Asn-Cys(S-)]-Gaba-Gly-Asn-Glu-Ile-Leu-Glu-Gln-Gly-Glu-Asp-Ser-Asp-Ser-Lys-OH, 1. The peptide was then coupled to the hydrazino-nicotinic acid bifunctional chelating agent and the purified adduct labeled with (99m)Tc using tricine as a coligand. Binding of the unlabeled and labeled peptide to stimulated human platelets was assayed in vitro. The (99m)Tc labeling yield was > 90%. The in vitro binding assays showed that the IC(50) for inhibition of platelet aggregation was 3694 nM, while the Kd of the (99m)Tc labeled peptide was 185 nM, indicating moderate affinity for the receptor. The (99m)Tc-labeled peptide was able to identify sites of experimental thrombi and emboli in a canine model. The results suggest initial success in attempting to mimic the behavior of bitistatin for imaging thrombi and emboli.

The peripheral benzodiazepine receptor (PBR) has been used as a sensitive marker to visualize and measure glial cell activation associated with various forms of brain injury and inflammation. Previous studies have shown that increased PBR levels following brain injury are specific to areas expressing activated glial cells. However, the contribution of glial cell types responsible for the increases in PBR levels following brain injury is not well defined. In the present study, we used a murine model of cuprizone-induced demyelination to broaden the application of PBR as a marker of brain injury and to validate the relationship between PBR levels and glial cell types. C57BL/6J mice were maintained on a cuprizone-containing or control diet and sacrificed at specific time points after initiation of treatment. Quantitative autoradiography of the PBR-selective ligand [(3)H]-(R)-PK11195 and [(125)I]-(R)-PK11195 showed that increased PBR levels were associated with the degree of demyelination assessed by Black-Gold histochemistry and activation of glial cells assessed by glial fibrillary acidic protein (GFAP) immunohistochemistry for astrocytes and CD11b (Mac-1) for microglia. Our findings indicate that brain PBR levels increased as a function of dose and duration of cuprizone treatment and it was detectable prior to observable demyelination. Increased PBR levels were associated with the degree of demyelination and temporal activation of glial cell types in different anatomical regions. In the corpus striatum, we found a close anatomical correlation between microglial activation and increased PBR levels in demyelinating fibre tracts. In the deep cerebellar nuclei, the temporal increases in PBR paralleled demyelination and microglia and astrocyte activation. On the other hand, in the corpus callosum there was an apparent temporal shift in the increase in PBR levels by different glial cell types from an early and predominantly microglial contribution to a late microglial and astrocytic response. High-resolution emulsion autoradiography of [(3)H]-(R)-PK11195 binding to PBR coupled with GFAP or Mac-1 immunohistochemistry showed that demyelination-induced increases in PBR levels were co-localized to both microglia and astrocytes. These findings support the notion that PBR is a sensitive and specific marker for the in vitro and in vivo visualization and quantification of neuropathological changes in the brain.

The low level of oxygenation within tumors is a major cause of radiation treatment failures. We theorized that anaerobic bacteria that can selectively destroy the hypoxic regions of tumors would enhance the effects of radiation. To test this hypothesis, we used spores of Clostridium novyi-NT to treat transplanted tumors in mice. The bacteria were found to markedly improve the efficacy of radiotherapy in several of the mouse models tested. Enhancement was noted with external beam radiation derived from a Cs-137 source, systemic radioimmunotherapy with an I-131-conjugated monoclonal antibody, and a previously undescribed form of experimental brachytherapy using plaques loaded with I-125 seeds. C. novyi-NT spores added little toxicity to the radiotherapeutic regimens, and the combination resulted in long-term remissions in a significant fraction of animals.

Although the epidermal growth factor receptor (EGFR), also known as HER1, has been studied for over a decade, it continues to be a molecule of great interest and focus of investigators for development of targeted therapies. The marketed monoclonal antibody cetuximab binds to HER1, and thus might serve as the basis for creation of imaging or therapies that target this receptor. The potential of cetuximab as a vehicle for the delivery of α-particle radiation was investigated in an intraperitoneal tumor mouse model. The effective working dose of 10 μCi of (212)Pb-cetuximab was determined from a dose (10-50 μCi) escalation study. Toxicity, as indicated by the lack of animal weight loss, was not evident at the 10 μCi dose of (212)Pb-cetuximab. A subsequent study demonstrated (212)Pb-cetuximab had a therapeutic efficacy similar to that of (212)Pb-trastuzumab (p = 0.588). Gemcitabine given 24 h prior to (212)Pb-cetuximab increased the median survival from 174 d to 283 d, but carboplatin suppressed the effectiveness of (212)Pb-cetuximab. Notably, concurrent treatment of tumor-bearing mice with (212)Pb-labeled cetuximab and trastuzumab provided therapeutic benefit that was greater than either antibody alone. In conclusion, cetuximab proved to be an effective vehicle for targeting HER1-expressing tumors with α-radiation for the treatment of disseminated intraperitoneal disease. These studies provide further evidence that the multimodality therapy regimens may have greater efficacy and benefit in the treatment of cancer patients.

A new bifunctional ligand C-DEPA was designed and synthesized as a component for antibody-targeted radiation therapy (radioimmunotherapy, RIT) of cancer. C-DEPA was conjugated to a tumor targeting antibody, trastuzumab, and the corresponding C-DEPA-trastuzumab conjugate was evaluated for radiolabeling kinetics with (205/6)Bi. C-DEPA-trastuzumab conjugate rapidly bound (205/6)Bi, and (205/6)Bi-C-DEPA-trastuzumab conjugate was stable in human serum for 72 h. The in vitro radiolabeling kinetics and serum stability data suggest that C-DEPA is a potential chelate for preclinical RIT applications using (212)Bi and (213)Bi.

Potent antagonists of bombesin-like peptides have shown great potential for applications in cancer therapy. A 99mTc-labeled agent capable of identifying patients who could benefit from these emerging therapies would have a great impact on patient management. This study involves the synthesis and initial evaluation of technetium diaminedithiolate analogues derived from the potent bombesin analogue Pyr-Gln-Lys-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2 (Lys3-bombesin). We coupled two diaminedithiol (DADT) bifunctional chelating agents (BCAs 1 and 2) to the Lys3 residue at the N-terminal region that is not required for binding to the receptor. 99mTc labeling was performed by ligand exchange on addition of [99mTc]glucoheptonate to a solution of the adduct at room temperature. Two products were obtained from each adduct on analysis by HPLC. The major to minor product ratios of the 99mTc-labeled analogues were 3:1 for products from BCA 1 and 9:1 for the products from BCA 2. Macroscopic amounts of the 99Tc analogues were similarly prepared using [99Tc]glucoheptonate. In this case, the major to minor ratios were 2:1 for the products from both BCAs. For initial evaluation of the binding of the Tc-labeled peptides to bombesin receptors, the 99Tc analogues were used in vitro in competitive binding assays in rat brain cortex membranes against [125I-Tyr4]bombesin. Results of the in vitro assays showed that the inhibition constants (Ki) of the major and minor products were 3.5+/-0.7 and 3.9+/-1.5 nM, respectively, for the products from BCA 1; and 7.4+/-2.0 and 5.2+/-1.5 nM for the products derived from BCA 2, respectively. The high affinity exhibited by these technetium analogues is an indication of their potential for use in non-invasive in vivo biochemical characterization of cancers that possess receptors for bombesin.

Nalpha-for-Nle-Leu-Phe-Nle-Tyr-Lys, a chemotactic peptide that binds with high affinity to the chemoattractant receptor on granulocytes and monocytes, was labeled with 99mTc using the diaminedithiol (DADT) chelating system to coordinate the Tc. 99mTc labeling of the DADT-coupled peptide was accomplished in 84% overall yield (room temperature for 10 min) using [99mTc]glucoheptonate as the donor of prereduced Tc. HPLC analysis showed two major 99mTc-labeled peptide peaks, 99mTc-DADT-Pep-I and 99mTc-DADT-Pep-II, were obtained in a ratio of 1:0.85. Using an iodoacetamide-derivatized gel to remove unlabeled peptide from the 99mTc labeling mixtures, essentially no-carrier-added (nca) high-specific activity 99mTc-labeled chemotactic peptides were obtained. The 99Tc analogues of the peptides were synthesized (72% yield) in a similar fashion and correlated with 99mTc complexes I and II by HPLC. In vitro competitive receptor binding assays of the isolated 99Tc analogues were performed against the tritiated chemotactic peptide [3H]N-for-Met-Leu-Phe ([3H]fMLF) using isolated granulocytes. The 99Tc-derivatized peptides showed similar binding affinities to the chemoattractant receptor as the unlabeled Nalpha-for-Nle-Leu-Phe-Nle-Tyr-Lys. The nca 99mTc-labeled peptides gave high contrast images of experimental inflammation in rabbits without causing neutropenia. Thus, it is feasible to attach the Tc-DADT chelate to low-molecular weight receptor binding chemotactic peptides and retain substantial binding to the receptor. Chemotactic peptides labeled with 99mTc via the DADT ligand system have the potential for imaging focal sites of inflammation without toxic effects, an important consideration in the successful utilization of chemotactic peptide agonists.

To better understand the molecular basis of the enhanced cell killing effected by the combined modality of paclitaxel and 212Pb-trastuzumab (Pac/212Pb-trastuzumab), gene expression in LS-174T i.p. xenografts was investigated 24 h after treatment. Employing a real time quantitative PCR array (qRT-PCR array), 84 DNA damage response genes were quantified. Differentially expressed genes following therapy with Pac/212Pb-trastuzumab included those involved in apoptosis (BRCA1, CIDEA, GADD45α, GADD45γ, GML, IP6K3, PCBP4, PPP1R15A, RAD21, and p73), cell cycle (BRCA1, CHK1, CHK2, GADD45α, GML, GTSE1, NBN, PCBP4, PPP1R15A, RAD9A, and SESN1), and damaged DNA repair (ATRX, BTG2, EXO1, FEN1, IGHMBP2, OGG1, MSH2, MUTYH, NBN, PRKDC, RAD21, and p73). This report demonstrates that the increased stressful growth arrest conditions induced by the Pac/212Pb-trastuzumab treatment suppresses cell proliferation through the regulation of genes which are involved in apoptosis and damaged DNA repair including single and double strand DNA breaks. Furthermore, the study demonstrates that 212Pb-trastuzumab potentiation of cell killing efficacy results from the perturbation of genes related to the mitotic spindle checkpoint and BASC (BRCA1-associated genome surveillance complex), suggesting cross-talk between DNA damage repair and the spindle damage response.

Solid phase chemistry with UltraLink Iodoacetyl gel was investigated as a method for removing excess ligand present in preparations of diaminedithiol (DADT) 99mTc radiopharmaceuticals. The process was optimized and tested under 99mTc labeling conditions with 2,2,4,9,9-pentamethyl-4,7-diaza-1,10-decanedithiol (N-methyl-DADT), 1, and 4-(1-(2'-methoxyphenyl) piperazinylbutyl)-2,2,9,9-tetramethyl-4,7-diaza-1,10 -decanedithiol (DADT-4C-MPP), 2, a serotonin receptor-binding ligand. The results illustrated that this gel removed > or = 99.9% excess ligand used in labeling reactions, and was comparable to the level of purification usually obtained by the more labor intensive method of HPLC.