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.

Comparison of planar, PET and well-counter measurements of total tumor radioactivity in a mouse xenograft model.

INTRODUCTION: Quantitative small animal radionuclide imaging studies are often carried out with the intention of estimating the total radioactivity content of various tissues such as the radioactivity content of mouse xenograft tumors exposed to putative diagnostic or therapeutic agents. We show that for at least one specific application, positron projection imaging (PPI) and PET yield comparable estimates of absolute total tumor activity and that both of these estimates are highly correlated with direct well-counting of these same tumors. These findings further suggest that in this particular application, PPI is a far more efficient data acquisition and processing methodology than PET. METHODS: Forty-one athymic mice were implanted with PC3 human prostate cancer cells transfected with prostate-specific membrane antigen (PSMA (+)) and one additional animal (for a total of 42) with a control blank vector (PSMA (-)). All animals were injected with [18F] DCFPyl, a ligand for PSMA, and imaged for total tumor radioactivity with PET and PPI. The tumors were then removed, assayed by well counting for total radioactivity and the values between these methods intercompared. RESULTS: PET, PPI and well-counter estimates of total tumor radioactivity were highly correlated (R2>0.98) with regression line slopes near unity (0.95

Imaging the Met Receptor Tyrosine Kinase (Met) and Assessing Tumor Responses to a Met Tyrosine Kinase Inhibitor in Human Xenograft Mouse Models with a [99mTc] (AH-113018) or Cy 5** (AH-112543) Labeled Peptide.

Developing an imaging agent targeting the hepatocyte growth factor receptor protein (Met) status of cancerous lesions would aid in the diagnosis and monitoring of Met-targeted tyrosine kinase inhibitors (TKIs). A peptide targeting Met labeled with [(99m)Tc] had high affinity in vitro (Kd = 3.3 nM) and detected relative changes in Met in human cancer cell lines. In vivo [(99m)Tc]-Met peptide (AH-113018) was retained in Met-expressing tumors, and high-expressing Met tumors (MKN-45) were easily visualized and quantitated using single-photon emission computed tomography or optical imaging. In further studies, MKN-45 mouse xenografts treated with PHA 665752 (Met TKI) or vehicle were monitored weekly for tumor responses by [(99m)Tc]-Met peptide imaging and measurement of tumor volumes. Tumor uptake of [(99m)Tc]-Met peptide was significantly decreased as early as 1 week after PHA 665752 treatment, corresponding to decreases in tumor volumes. These results were comparable to Cy5**-Met peptide (AH-112543) fluorescence imaging using the same treatment model. [(99m)Tc] or Cy5**-Met peptide tumor uptake was further validated by histologic (necrosis, apoptosis) and immunoassay (total Met, p Met, and plasma shed Met) assessments in imaged and nonimaged cohorts. These data suggest that [(99m)Tc] or Cy5**-Met peptide imaging may have clinical diagnostic, prognostic, and therapeutic monitoring applications.

Preparation and long-term biodistribution studies of a PAMAM dendrimer G5-Gd-BnDOTA conjugate for lymphatic imaging.

AIMS: To demonstrate the use of gadolinium (Gd)-labeled dendrimers as lymphatic imaging agents and establish the long-term biodistribution (90-day) of this type of agent in mice. MATERIALS & METHODS: A G5-Gd-BnDOTA dendrimer was prepared and injected into mice and monkeys for MR lymphangiography, and long-term biodistribution of the conjugate was studied. RESULTS: Administration of G5-Gd-BnDOTA in mice demonstrated a rapid uptake in the deep lymphatic system while injection in monkeys showed enhanced internal iliac nodes, indicating its general utility for lymphatic tracking. Biodistribution studies to 90 days showed that gadolinium conjugate is slowly being eliminated from the liver and other organs. CONCLUSION: The use of G5-Gd-BnDOTA holds great promise for lymphatic imaging, but its slow clearance from the body might hamper its eventual clinical translation.

Simultaneous ECG-gated PET imaging of multiple mice.

INTRODUCTION: We describe and illustrate a method for creating ECG-gated PET images of the heart for each of several mice imaged at the same time. The method is intended to increase "throughput" in PET research studies of cardiac dynamics or to obtain information derived from such studies, e.g. tracer concentration in end-diastolic left ventricular blood. METHODS: An imaging bed with provisions for warming, anesthetic delivery, etc., was fabricated by 3D printing to allow simultaneous PET imaging of two side-by-side mice. After electrode attachment, tracer injection and placement of the animals in the scanner field of view, ECG signals from each animal were continuously analyzed and independent trigger markers generated whenever an R-wave was detected in each signal. PET image data were acquired in "list" mode and these trigger markers were inserted into this list along with the image data. Since each mouse is in a different spatial location in the FOV, sorting of these data using trigger markers first from one animal and then the other yields two independent and correctly formed ECG-gated image sequences that reflect the dynamical properties of the heart during an "average" cardiac cycle. RESULTS: The described method yields two independent ECG-gated image sequences that exhibit the expected properties in each animal, e.g. variation of the ventricular cavity volumes from maximum to minimum and back during the cardiac cycle in the processed animal with little or no variation in these volumes during the cardiac cycle in the unprocessed animal. CONCLUSION: ECG-gated image sequences for each of several animals can be created from a single list mode data collection using the described method. In principle, this method can be extended to more than two mice (or other animals) and to other forms of physiological gating, e.g. respiratory gating, when several subjects are imaged at the same time.

Evaluation of platinum chemotherapy in combination with HER2-targeted α-particle radiation.

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.

In Vitro and In Vivo Pre-Clinical Analysis of a F(ab')(2) Fragment of Panitumumab for Molecular Imaging and Therapy of HER1 Positive Cancers.

BACKGROUND: The objective of this study was to characterize the in vitro and in vivo properties of the F(ab')(2) fragment of panitumumab and to investigate its potential for imaging and radioimmunotherapy. METHODS: The panitumumab F(ab')(2) was generated by enzymatic pepsin digestion. After the integrity and immunoreactivity of the F(ab')(2) was evaluated, the fragment was radiolabeled. In vivo studies included direct quantitation of tumor targeting and normal organ distribution of the radiolabeled panitumumab F(ab')(2) as well as planar γ-scintigraphy and PET imaging. RESULTS: The panitumumab F(ab')(2) was successfully produced by peptic digest. The F(ab')(2) was modified with the CHX-A"-DTPA chelate and efficiently radiolabeled with either (111)In or (86)Y. In vivo tumor targeting was achieved with acceptable uptake of radioactivity in the normal organs. The tumor targeting was validated by both imaging modalities with good visualization of the tumor at 24 h. CONCLUSIONS: The panitumumab F(ab')(2) fragment is a promising candidate for imaging of HER1 positive cancers.

Targeting HER2: a report on the in vitro and in vivo pre-clinical data supporting trastuzumab as a radioimmunoconjugate for clinical trials.

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.

PET imaging of HER1-expressing xenografts in mice with 86Y-CHX-A''-DTPA-cetuximab.

PURPOSE: Cetuximab is a recombinant, human/mouse chimeric IgG(1) monoclonal antibody that binds to the epidermal growth factor receptor (EGFR/HER1). Cetuximab is approved for the treatment of patients with HER1-expressing metastatic colorectal cancer. Limitations in currently reported radiolabeled cetuximab for PET applications prompted the development of (86)Y-CHX-A''-DTPA-cetuximab as an alternative for imaging HER1-expressing cancer. (86)Y-CHX-A''-DTPA-cetuximab can also serve as a surrogate marker for (90)Y therapy. METHODS: Bifunctional chelate, CHX-A''-DTPA was conjugated to cetuximab and radiolabeled with (86)Y. In vitro immunoreactivity was assessed in HER1-expressing A431 cells. In vivo biodistribution, PET imaging and noncompartmental pharmacokinetics were performed in mice bearing HER1-expressing human colorectal (LS-174T and HT29), prostate (PC-3 and DU145), ovarian (SKOV3) and pancreatic (SHAW) tumor xenografts. Receptor blockage was demonstrated by coinjection of either 0.1 or 0.2 mg cetuximab. RESULTS: (86)Y-CHX-A''-DTPA-cetuximab was routinely prepared with a specific activity of 1.5-2 GBq/mg and in vitro cell-binding in the range 65-75%. Biodistribution and PET imaging studies demonstrated high HER1-specific tumor uptake of the radiotracer and clearance from nonspecific organs. In LS-174T tumor-bearing mice injected with (86)Y-CHX-A''-DTPA-cetuximab alone, (86)Y-CHX-A''-DTPA-cetuximab plus 0.1 mg cetuximab or 0.2 mg cetuximab, the tumor uptake values at 3 days were 29.3 +/- 4.2, 10.4 +/- 0.5 and 6.4 +/- 0.3%ID/g, respectively, demonstrating dose-dependent blockage of the target. Tumors were clearly visualized 1 day after injecting 3.8-4.0 MBq (86)Y-CHX-A''-DTPA-cetuximab. Quantitative PET revealed the highest tumor uptake in LS-174T (29.55 +/- 2.67%ID/cm(3)) and the lowest tumor uptake in PC-3 (15.92 +/- 1.55%ID/cm(3)) xenografts at 3 days after injection. Tumor uptake values quantified by PET were closely correlated (r (2) = 0.9, n = 18) with values determined by biodistribution studies. CONCLUSION: This study demonstrated the feasibility of preparation of high specific activity (86)Y-CHX-A''-DTPA-cetuximab and its application for quantitative noninvasive PET imaging of HER1-expressing tumors. (86)Y-CHX-A''-DTPA-cetuximab offers an attractive alternative to previously labeled cetuximab for PET and further investigation for clinical translation is warranted.

Two-step synthesis of galactosylated human serum albumin as a targeted optical imaging agent for peritoneal carcinomatosis.

An optical probe, RG-(gal)(28)GSA, was synthesized to improve the detection of peritoneal implants by targeting the beta-d-galactose receptors highly expressed on the cell surface of a wide variety of cancers arising from the ovary, pancreas, colon, and stomach. Evaluation of RG-(gal)(28)GSA, RG-(gal)(20)GSA, glucose-analogue RG-(glu)(28)GSA, and control RG-HSA demonstrates specificity for the galactose, binding to several human adenocarcinoma cell lines, and cellular internalization. Studies using peritoneally disseminated SHIN3 xenografts in mice also confirmed a preference for galactose with the ability to detect submillimeter size lesions. Preliminary toxicity study for RG-(gal)(28)GSA using Balb/c mice reveal no toxic effects up to 100x of the standard imaging dose of 1 mg/kg administered either intraperitoneally or intravenously. These data indicate that RG-(gal)(28)GSA can selectively target a variety of human adenocarcinomas, can improve intraoperative or endoscopic tumor detection and resection, and may have little or no toxic in vivo effects; hence, it may be clinically translatable.

A new approach in the preparation of dendrimer-based bifunctional diethylenetriaminepentaacetic acid MR contrast agent derivatives.

In this paper, we report a new method to prepare and characterize a contrast agent based on a fourth-generation (G4) polyamidoamine (PAMAM) dendrimer conjugated to the gadolinium complex of the bifunctional diethylenetriamine pentaacetic acid derivative (1B4M-DTPA). The method involves preforming the metal-ligand chelate in alcohol prior to conjugation to the dendrimer. The dendrimer-based agent was purified by a Sephadex G-25 column and characterized by elemental analysis. The analysis and SE-HPLC data gave a chelate to dendrimer ratio of 30:1 suggesting conjugation at approximately every other amine terminal on the dendrimer. Molar relaxivity of the agent measured at pH 7.4 displayed a higher value than that of the analogous G4 dendrimer based agent prepared by the postmetal incorporation method (r(1) = 26.9 vs 13.9 mM(-1) s(-1) at 3 T and 22 degrees C). This is hypothesized to be due to the higher hydrophobicity of this conjugate and the lack of available charged carboxylate groups from noncomplexed free ligands that might coordinate to the metal and thus also reduce water exchange sites. Additionally, the distribution populations of compounds that result from the postmetal incorporation route are eliminated from the current product simplifying characterization as quality control issues pertaining to the production of such agents for clinical use as MR contrast agents. In vivo imaging in mice showed a reasonably fast clearance (t(1/2) = 24 min) suggesting a viable agent for use in clinical application.

A novel side-bridged hybrid phosphonate/acetate pendant cyclam: synthesis, characterization, and 64Cu small animal PET imaging.

Copper-64 (t(1/2)=12.7h; beta(+): 0.653 MeV, 17.4%; beta(-): 0.578 MeV, 39%) is produced in a biomedical cyclotron and has applications in both imaging and therapy. Macrocyclic chelators are widely used as bifunctional chelators to bind copper radionuclides to antibodies and peptides owing to their relatively high kinetic stability. A novel side-bridged cyclam featuring both pendant acetate and phosphonate groups was synthesized using a Kabachnik-Fields approach followed by hydrobromic acid deprotection. The Cu(II) complex of the novel ligand was synthesized, radiolabeling with (64)Cu was demonstrated, and in vitro (serum) stability was performed. In addition, in vivo distribution and clearance of the (64)Cu-labeled complex was visualized by positron emission tomography (PET) imaging. This novel chelate may be useful in (64)Cu-mediated diagnostic positron emission tomography (PET) imaging as well as targeted radiotherapeutic applications.

Cetuximab: preclinical evaluation of a monoclonal antibody targeting EGFR for radioimmunodiagnostic and radioimmunotherapeutic applications.

The monoclonal antibody, cetuximab, binds to epidermal growth-factor receptor and thus provides an opportunity to create both imaging and therapies that target this receptor. The potential of cetuximab as a radioimmunoconjugate, using the acyclic bifunctional chelator, CHX-A"-DTPA, was investigated. The pharmacokinetic behavior in the blood was determined in mice with and without tumors. Tumor targeting and scintigraphic imaging were evaluated in mice bearing xenografts of LS-174T (colorectal), SHAW (pancreatic), SKOV3 (ovarian), DU145 (prostate), and HT-29 (colorectal). Excellent tumor targeting was observed in each of the models with peak tumor uptakes of 59.8 +/- 18.1, 22.5 +/- 4.7, 33.3 +/- 5.7, 18.2 +/- 7.8, and 41.7 +/- 10.8 injected dose per gram (%ID/g) at 48-72 hours, respectively. In contrast, the highest tumor %ID/g obtained in mice bearing melanoma (A375) xenografts was 6.3 +/- 1.1 at 72 hours. The biodistribution of (111)In-cetuximab was also evaluated in nontumor-bearing mice. The highest %ID/g was observed in the liver (9.3 +/- 1.3 at 24 hours) and the salivary glands (8.1 +/- 2.8 at 72 hours). Scintigraphy showed excellent tumor targeting at 24 hours. Blood pool was evident, as expected, but cleared over time. At 168 hours, the tumor was clearly discernible with negligible background.

Multimodality therapy: potentiation of high linear energy transfer radiation with paclitaxel for the treatment of disseminated peritoneal disease.

PURPOSE: Studies herein explore paclitaxel enhancement of the therapeutic efficacy of alpha-particle-targeted radiation therapy. EXPERIMENTAL DESIGN: Athymic mice bearing 3 day i.p. LS-174T xenografts were treated with 300 or 600 microg paclitaxel at 24 h before, concurrently, or 24 h after [213Bi] or [212Pb]trastuzumab. RESULTS: Paclitaxel (300 or 600 microg) followed 24 h later with [213Bi]trastuzumab (500 microCi) provided no therapeutic enhancement. Paclitaxel (300 microg) administered concurrently with [213Bi]trastuzumab or [213Bi]HuIgG resulted in median survival of 93 and 37 days, respectively; no difference was observed with 600 microg paclitaxel. Mice receiving just [213Bi]trastuzumab or [213Bi]HuIgG or left untreated had a median survival of 31, 21, and 15 days, respectively, 23 days for just either paclitaxel dose alone. Paclitaxel (300 or 600 microg) given 24 h after [213Bi]trastuzumab increased median survival to 100 and 135 days, respectively. The greatest improvement in median survival (198 days) was obtained with two weekly doses of paclitaxel (600 microg) followed by [213Bi]trastuzumab. Studies were also conducted investigating paclitaxel administered 24 h before, concurrently, or 24 h after [212Pb]trastuzumab (10 microCi). The 300 microg paclitaxel 24 h before radioimmunotherapy (RIT) failed to provide benefit, whereas 600 microg extended the median survival from 44 to 171 days. CONCLUSIONS: These results suggest that regimens combining chemotherapeutics and high linear energy transfer (LET) RIT may have tremendous potential in the management and treatment of cancer patients. Dose dependency and administration order appear to be critical factors requiring careful investigation.

Synthesis of a cross-bridged cyclam derivative for peptide conjugation and 64Cu radiolabeling.

The increased use of copper radioisotopes in radiopharmaceutical applications has created a need for bifunctional chelators (BFCs) that form stable radiocopper complexes and allow covalent attachment to biological molecules. Previous studies have established that 4,11-bis-(carbo- tert-butoxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (H 2CB-TE2A), a member of the ethylene "cross-bridged" cyclam (CB-cyclam) class of bicyclic tetraaza macrocycles, forms highly kinetically stable complexes with Cu(II) and is less susceptible to in vivo transchelation than its nonbridged analogue, 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA). Herein, we report a convenient synthesis of a novel cross-bridged BFC that is structurally analogous to CB-TE2A in that it possesses two coordinating acetate arms, but in addition possesses a third orthogonally protected arm for conjugation to peptides and other targeting agents. Application of this strategy to cross-bridged chelators may also enable the development of even further improved agents for (64)Cu-mediated diagnostic positron emission tomography (PET) imaging as well as for targeted radiotherapeutic applications.

Synthesis, characterization, and biological evaluation of integrin alphavbeta3-targeted PAMAM dendrimers.

Ligand size and valency strongly influence the receptor uptake and clearance of tumor angiogenesis imaging agents. The structures of successful imaging agents exhibit a high degree of variability, encompassing small monovalent arginine-glycine-aspartic acid (RGD)-containing peptides, multivalent RGD-oligomers, and a monoclonal antibody against integrin alpha-v-beta-3 (alpha-v-beta-3). We have pursued a nanoscale approach to imaging of angiogenesis using rationally designed polyamidoamine (PAMAM) dendrimers covalently adorned with RGD-cyclopeptides. An orthogonal oxime-ligation strategy was applied to chemoselectively effect conjugation of the PAMAM dendrimers with RGD-cyclopeptides for targeting alpha vbeta 3. Fluorescent dyes for optical imaging and chelates for gadolinium-based magnetic resonance (MR) imaging were subsequently appended to create robust multimodal macromolecular imaging agents. Fluorescence microscopy revealed selective binding of the resulting RGD peptide-bearing dendrimer with empty chelates to alpha-v-beta-3-expressing cells, but somewhat reduced selectivity was observed following Gd(III) complexation. The expected incomplete saturation of chelates with Gd(III) ions permitted radiometal complexation, and an in vivo tissue distribution of the resulting agent in M21 melanoma tumor-bearing mice showed mostly renal and reticuloendothelial accumulation, with the tumor:blood ratio peaking (3.30+/-0.03) at 2 h postinjection.

A dual CT-MR dendrimer contrast agent as a surrogate marker for convection-enhanced delivery of intracerebral macromolecular therapeutic agents.

The feasibility of using Gd dendrimer-based macromolecules (Gd-G8 dendrimer) as a dual CT and MR contrast agent for monitoring convection-enhanced delivery of therapy in the brain is evaluated both in vitro and in vivo with optimal dosing established. In vitro CT attenuation values of the Gd-based agents ( approximately 6.0 HU mM(-1)) were approximately 1.6 times greater than iodine-based agents and the attenuation of the Gd-DTPA was comparable to Gd-G8 dendrimer. Visible enhancement was observed on both CT and MR using Gd-G8 dendrimer over a range of 23-78 mM; however, a concentration of at least 47 mM in Gd was required for adequate delineation of the injection site on both CT and MR. MR offers greater sensitivity than CT in estimating the volume of distribution (V(d)) and effectively quantified the agent's concentration and diffusion using T(1) mapping at much lower concentrations of Gd (<10 mM in [Gd]).

A novel bifunctional maleimido CHX-A'' chelator for conjugation to thiol-containing biomolecules.

A novel bifunctional maleimido CHX-A'' DTPA chelator 5 was developed and conjugated to the monoclonal antibody trastuzumab (Herceptin) and subsequently radiolabeled with (111)In. The resulting (111)In labeled immunoconjugate 2 was demonstrated to bind to SKOV-3 ovarian cancer cells comparably to an isothiocyanato CHX-A'' DTPA modified native trastuzumab, 1. Through efficient thiol-maleimide chemistry, antibodies, peptides or other targeting vectors can now be modified with an established radioactive metal chelating agent CHX-A'' DTPA for imaging and/or therapies of cancer.

Pretargeted radioimmunotherapy of mesothelin-expressing cancer using a tetravalent single-chain Fv-streptavidin fusion protein.

UNLABELLED: Mesothelin is a glycoprotein that is overexpressed in several human tumors, including mesotheliomas and ovarian cancers, and has been identified as a potential target for therapy. We evaluated the biodistribution and tumor-targeting ability of an antimesothelin tetravalent single-chain Fv-streptavidin fusion protein (SS1scFvSA) in mice. METHODS: SS1scFvSA was labeled with 125I or 111In for evaluation of internalization in vitro and for optimization of its biodistribution. The A431-K5 mesothelin transfected cell line was used as the target. We used a 3-step pretargeting approach consisting of injections of (i) SS1scFvSA, followed 20 h later by (ii) a synthetic clearing agent, and (iii) 4 h later, radiolabeled (111In, 88Y/90Y, or 177Lu) 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA)-biotin. To optimize the tumor uptake, the effect of the specific activity of 111In-DOTA-biotin was evaluated. RESULTS: Approximately 60% of SS1sc FvSA internalized within 6 h. The optimal dose of SS1scFvSA for pretargeting was 600 microg. Decreasing the specific activity of DOTA-biotin by administering 0.1-5 microg of DOTA-biotin resulted in tumor uptake decreasing from 31.8 to 5.5 %ID/g (percentage injected dose per gram) at 2 h. Pretargeted therapy of A431-K5 tumor with 90Y doses of 11.1-32.4 MBq resulted in a dose-dependent tumor response. With 32.4 MBq, 86% of mice survived tumor free for 110 d. All nontreated mice died, with a median survival of 16 d. CONCLUSION: SS1scFvSA localized in the mesothelin-expressing tumor, resulting in a high accumulation of radiolabeled DOTA-biotin. The specific activity of DOTA-biotin had a significant effect on its tumor uptake. Therapeutic tumor doses were obtained without dose-limiting toxicity.

Semi-automated 86Y purification using a three-column system.

The separation of (86)Y from (86)Sr was optimized by a semi-automated purification system involving the passage of the target sample through three sequential columns. The target material was dissolved in 4 N HNO(3) and loaded onto a Sr-selective (Sr-Spec) column to retain the (86)Sr. The yttrium was eluted with 4 N HNO(3) onto the second Y-selective (RE-Spec) column with quantitative retention. The RE-Spec column was eluted with a stepwise decreasing concentration of HNO(3) to wash out potential metallic impurities to a waste container. The eluate was then pumped onto an Aminex A5 column with 0.1 N HCl and finally with 3 N HCl to collect the radioyttrium in 0.6-0.8 mL with a >80% recovery. This method enabled us to decontaminate Sr by 250,000 times and label 30 micro g of DOTA-Biotin with a >95% yield.