Vaccinia virus and peptide-receptor radiotherapy synergize to improve treatment of peritoneal carcinomatosis.

Tumor-specific overexpression of receptors enables a variety of targeted cancer therapies, exemplified by peptide-receptor radiotherapy (PRRT) for somatostatin receptor (SSTR)-positive neuroendocrine tumors. While effective, PRRT is restricted to tumors with SSTR overexpression. To overcome this limitation, we propose using oncolytic vaccinia virus (vvDD)-mediated receptor gene transfer to permit molecular imaging and PRRT in tumors without endogenous SSTR overexpression, a strategy termed radiovirotherapy. We hypothesized that vvDD-SSTR combined with a radiolabeled somatostatin analog could be deployed as radiovirotherapy in a colorectal cancer peritoneal carcinomatosis model, producing tumor-specific radiopeptide accumulation. Following vvDD-SSTR and 177Lu-DOTATOC treatment, viral replication and cytotoxicity, as well as biodistribution, tumor uptake, and survival, were evaluated. Radiovirotherapy did not alter virus replication or biodistribution, but synergistically improved vvDD-SSTR-induced cell killing in a receptor-dependent manner and significantly increased the tumor-specific accumulation and tumor-to-blood ratio of 177Lu-DOTATOC, making tumors imageable by microSPECT/CT and causing no significant toxicity. 177Lu-DOTATOC significantly improved survival over virus alone when combined with vvDD-SSTR but not control virus. We have therefore demonstrated that vvDD-SSTR can convert receptor-negative tumors into receptor-positive tumors and facilitate molecular imaging and PRRT using radiolabeled somatostatin analogs. Radiovirotherapy represents a promising treatment strategy with potential applications in a wide range of cancers.

Correlation of Somatostatin Receptor-2 Expression with Gallium-68-DOTA-TATE Uptake in Neuroblastoma Xenograft Models.

Peptide-receptor imaging and therapy with radiolabeled somatostatin analogs such as 68Ga-DOTA-TATE and 177Lu-DOTA-TATE have become an effective treatment option for SSTR-positive neuroendocrine tumors. The purpose of this study was to evaluate the correlation of somatostatin receptor-2 (SSTR2) expression with 68Ga-DOTA-TATE uptake and 177Lu-DOTA-TATE therapy in neuroblastoma (NB) xenograft models. We demonstrated variable SSTR2 expression profiles in eight NB cell lines. From micro-PET imaging and autoradiography, a higher uptake of 68Ga-DOTA-TATE was observed in SSTR2 high-expressing NB xenografts (CHLA-15) compared to SSTR2 low-expressing NB xenografts (SK-N-BE(2)). Combined autoradiography-immunohistochemistry revealed histological colocalization of SSTR2 and 68Ga-DOTA-TATE uptake in CHLA-15 tumors. With a low dose of 177Lu-DOTA-TATE (20 MBq/animal), tumor growth inhibition was achieved in the CHLA-15 high SSTR2 expressing xenograft model. Although, in vitro, NB cells showed variable expression levels of norepinephrine transporter (NET), a molecular target for 131I-MIBG therapy, low 123I-MIBG uptake was observed in all selected NB xenografts. In conclusion, SSTR2 expression levels are associated with 68Ga-DOTA-TATE uptake and antitumor efficacy of 177Lu-DOTA-TATE. 68Ga-DOTA-TATE PET is superior to 123I-MIBG SPECT imaging in detecting NB tumors in our model. Radiolabeled DOTA-TATE can be used as an agent for NB tumor imaging to potentially discriminate tumors eligible for 177Lu-DOTA-TATE therapy.

64Cu-Labeled Trastuzumab Fab-PEG24-EGF Radioimmunoconjugates Bispecific for HER2 and EGFR: Pharmacokinetics, Biodistribution, and Tumor Imaging by PET in Comparison to Monospecific Agents.

Heterodimerization of EGFR with HER2 coexpressed in breast cancer (BC) promotes tumor growth, and increased EGFR expression is associated with trastuzumab resistance. Our aim was to construct 64Cu-labeled bispecific radioimmunoconjugates (bsRIC) composed of trastuzumab Fab, which binds HER2 linked through a polyethylene glycol (PEG24) spacer to EGF, and to compare their pharmacokinetic, biodistribution, and tumor imaging characteristics by positron-emission tomography (PET). bsRICs were generated by linking maleimide modified trastuzumab Fab with thiolated EGF through a thioether bond. HER2 and EGFR binding were assessed in vitro in MDA-MB-231 (EGFRmod/HER2low), MDA-MB-468 (EGFRhigh/HER2neg), MDA-MB-231-H2N (EGFRmod/HER2mod), and SKOV3 (EGFRlow/HER2high) cells by competition and saturation cell binding assays to estimate the dissociation constant (Kd). The elimination of the 64Cu-NOTA-trastuzumab Fab-PEG24-EGF bsRICs from the blood of Balb/c mice was compared to monospecific 64Cu-NOTA-trastuzumab Fab and 64Cu-NOTA-EGF. MicroPET/CT imaging was performed in NOD/SCID mice bearing subcutaneous MDA-MB-468, MDA-MB-231/H2N, or SKOV3 human BC xenografts at 24 and 48 h postinjection (p.i.) of bsRICs. Tumor and normal tissue uptake were quantified by biodistribution studies and compared to monospecific agents. The binding of bsRICs to MDA-MB-231 cells was decreased to 24.5 ± 5.2% by excess EGF, while the binding of bsRICs to SKOV3 cells was decreased to 38.6 ± 5.4% by excess trastuzumab Fab, demonstrating specific binding to both EGFR and HER2. 64Cu-labeled bsRICs incorporating the PEG24 spacer were eliminated more slowly from the blood than 64Cu-bsRICs without the PEG spacer and were cleared much more slowly than 64Cu-NOTA-Fab or 64Cu-NOTA-EGF. All three tumor xenografts were visualized by microPET/CT at 24 and 48 h p.i. of bsRICs. Biodistribution studies at 48 h p.i. in NOD/SCID mice with MDA-MB-231/H2N tumors demonstrated significantly greater tumor uptake of 64Cu-NOTA-Fab-PEG24-EGF (4.9 ± 0.4%ID/g) than 64Cu-NOTA-Fab (1.9 ± 0.3%ID/g; P < 0.0001) and 64Cu-NOTA-EGF (0.7 ± 0.2%ID/g; P < 0.0001). Furthermore, preadministration of an excess of trastuzumab Fab or trastuzumab Fab-PEG24-EGF significantly decreased the tumor uptake of 64Cu-NOTA-Fab-PEG24-EGF in SK-OV-3 and MDA-MB-468 xenografts by 4.4-fold (P = 0.0012) and 1.8-fold (P = 0.0031), respectively. 64Cu-labeled bsRICs bound HER2 or EGFR and were taken up specifically in vivo in tumor xenografts expressing one or both receptors. The PEG24 linker prolonged the blood residence time contributing to the higher tumor uptake of the bsRICs than monospecific agents.

The Necrosis-Avid Small Molecule HQ4-DTPA as a Multimodal Imaging Agent for Monitoring Radiation Therapy-Induced Tumor Cell Death.

PURPOSE: Most effective antitumor therapies induce tumor cell death. Non-invasive, rapid and accurate quantitative imaging of cell death is essential for monitoring early response to antitumor therapies. To facilitate this, we previously developed a biocompatible necrosis-avid near-infrared fluorescence (NIRF) imaging probe, HQ4, which was radiolabeled with 111Indium-chloride (111In-Cl3) via the chelate diethylene triamine pentaacetic acid (DTPA), to enable clinical translation. The aim of the present study was to evaluate the application of HQ4-DTPA for monitoring tumor cell death induced by radiation therapy. Apart from its NIRF and radioactive properties, HQ4-DTPA was also tested as a photoacoustic imaging probe to evaluate its performance as a multimodal contrast agent for superficial and deep tissue imaging. MATERIALS AND METHODS: Radiation-induced tumor cell death was examined in a xenograft mouse model of human breast cancer (MCF-7). Tumors were irradiated with three fractions of 9 Gy each. HQ4-DTPA was injected intravenously after the last irradiation, NIRF and photoacoustic imaging of the tumors were performed at 12, 20, and 40 h after injection. Changes in probe accumulation in the tumors were measured in vivo, and ex vivo histological analysis of excised tumors was performed at experimental endpoints. In addition, biodistribution of radiolabeled [111In]DTPA-HQ4 was assessed using hybrid single-photon emission computed tomography-computed tomography (SPECT-CT) at the same time points. RESULTS: In vivo NIRF imaging demonstrated a significant difference in probe accumulation between control and irradiated tumors at all time points after injection. A similar trend was observed using in vivo photoacoustic imaging, which was validated by ex vivo tissue fluorescence and photoacoustic imaging. Serial quantitative radioactivity measurements of probe biodistribution further demonstrated increased probe accumulation in irradiated tumors. CONCLUSION: HQ4-DTPA has high specificity for dead cells in vivo, potentiating its use as a contrast agent for determining the relative level of tumor cell death following radiation therapy using NIRF, photoacoustic imaging and SPECT in vivo. Initial preclinical results are promising and indicate the need for further evaluation in larger cohorts. If successful, such studies may help develop a new multimodal method for non-invasive and dynamic deep tissue imaging of treatment-induced cell death to quantitatively assess therapeutic response in patients.

Kit for the preparation of 111In-labeled pertuzumab injection for imaging response of HER2-positive breast cancer to trastuzumab (Herceptin).

We previously reported that 111In-labeled pertuzumab imaged trastuzumab (Herceptin)-mediated changes in HER2 expression preclinically in breast cancer tumors. To advance 111In-labeled pertuzumab to a Phase I/II clinical trial, a kit was designed for preparing this agent in a form suitable for human administration. Unit-dose kits containing pertuzumab modified with 2-(4-isothiocyanatobenzyl)-diethylenetriaminepentaacetic acid (BzDTPA) were prepared that labeled to high efficiency (>90%) with 111In and met specifications for pharmaceutical quality. The kits were stable for 4 months and the final radiopharmaceutical was stable for 24h. Imaging studies demonstrated high and specific uptake in HER2-positive tumors in mice using this clinical kit formulation.

Phase I trial to evaluate the tumor and normal tissue uptake, radiation dosimetry and safety of (111)In-DTPA-human epidermal growth factor in patients with metastatic EGFR-positive breast cancer.

The safety, pharmacokinetics, biodistribution and radiation dosimetry of (111)In-DTPA-hEGF, an Auger electron-emitting radiopharmaceutical, were evaluated in a first-in-human trial. Dose escalation was performed in patients with EGFR-positive metastatic breast cancer who had received ≥2 prior courses of systemic treatment. (111)In-DTPA-hEGF (0.25 mg) was administered once intravenously (i.v.). Blood was collected for biochemistry/hematology testing and pharmacokinetic and immunogenicity analyses at selected times post injection (p.i.). Whole body planar images were acquired at 1, 4-6, 24 and 72 h p.i. and SPECT images at 24 and/or 72 h p.i. Macrodosimetry (MIRD) for the whole body and organs was estimated using OLINDA. Correlative radiological imaging was obtained at baseline, 1 and 3 months and then 6 monthly. Toxicity was scored using Common Terminology Criteria for Adverse Events (CTCAE)v2.0. Sixteen patients, median age 47 yr (range, 35-59), received (111)In-DTPA-hEGF as follows: 357-434 MBq (7), 754-805 MBq (3), 1,241-1,527 MBq (3) and 2,030-2,290 MBq (3). Fifteen were evaluable for toxicity. The commonest adverse events (AE) were flushing, chills, nausea, and vomiting occurring during or immediately p.i. One patient experienced Grade 3 thrombocytopenia (attributed to bone marrow infiltration by cancer). There were no other Grade 3 or 4 AEs. Maximum tolerated dose was not reached. Clear accumulation of radiopharmaceutical in at least one known site of disease was observed in 47% of patients. (111)In-DTPA-hEGF was cleared biexponentially from the blood with α-phase T½ of 0.16 ± 0.03 h and ß-phase T½ of 9.41 ± 1.93 h. (111)In-DTPA-hEGF was not immunogenic. The mean radiation dose estimates in mGy/MBq for whole body, liver, kidneys, spleen and thyroid were 0.08, 0.86, 0.74, 0.37 and 0.30, respectively. No objective antitumor responses were observed at the doses studied. In summary, administered amounts of up to 2,290 MBq (0.25 mg) of (111)In-DTPA-hEGF were well tolerated as a single i.v. injection.

Phase I trial of intraoperative detection of tumor margins in patients with HER2-positive carcinoma of the breast following administration of 111In-DTPA-trastuzumab Fab fragments.

INTRODUCTION: Our aim was to conduct a Phase I clinical trial to determine the feasibility of intraoperative detection of tumor margins in HER2 positive breast carcinoma using a hand-held γ-probe following administration of (111)In-DTPA-trastuzumab Fab fragments. Accurate delineation of tumor margins is important for preventing local recurrence. METHODS: Six patients with HER2-positive in situ or invasive ductal carcinoma were administered 74MBq (0.5mg) of (111)In-DTPA-trastuzumab Fab fragments and counts in the tumor, surgical cavity wall and en face margins were measured intraoperatively at 72h post-injection using the Navigator or C-Trak γ-probes. Margins were evaluated histologically. Quantitative whole body planar imaging was performed to estimate radiation absorbed doses using OLINDA/EXM software. SPECT imaging of the thorax was performed to evaluate tumor uptake. The pharmacokinetics of elimination from the blood and plasma were determined over 72h. RESULTS: There were no acute adverse reactions from (111)In-DTPA-trastuzumab Fab fragments and no changes in hematological or biochemical indices were found over a 3month period. (111)In-DTPA-trastuzumab Fab fragments exhibited a biphasic elimination from the blood and plasma with t1/2α=11.9h and 7.5h, respectively, and t1/2ß=26.6 and 20.7h, respectively. The radiopharmaceutical accumulated in the liver, spleen and kidneys. SPECT imaging did not reveal tumor in any patient. The mean effective dose was 0.146mSv/MBq (10.8mSv for 74MBq). Counts in excised tumors were low but were higher than in margins. Margins in two patients harboured tumor but this was not correlated with counts obtained using the γ-probes. Surgical cavity counts were high and likely due to detection of γ-photons outside the surgical field. CONCLUSION: We conclude that it was not feasible, at least at the administered amount of radioactivity used in this study, to reliably detect the margins of disease in patients with in situ or invasive ductal carcinoma intraoperatively using a hand-held γ-probe and (111)In-DTPA-trastuzumab Fab fragments due to low uptake in the tumor and involved margins.

MicroSPECT/CT imaging of co-expressed HER2 and EGFR on subcutaneous human tumor xenografts in athymic mice using ¹¹¹In-labeled bispecific radioimmunoconjugates.

Epidermal growth factor receptors (EGFR) form heterodimers with HER2 in breast cancer, and increased EGFR expression has been found in HER2-positive tumors resistant to trastuzumab (Herceptin). Our objective was to synthesize bispecific radioimmunoconjugates (bsRICs) that recognize HER2 and EGFR and evaluate their ability to image tumors in athymic mice that express one or both receptors by microSPECT/CT. Bispecific radioimmunoconjugates were constructed by conjugating maleimide-derivatized trastuzumab Fab fragments that bind HER2 to a thiolated form of EGF with an intervening 24 mer polyethylene glycol (PEG24) spacer. Bispecific radioimmunoconjugates were derivatized with diethylenetriaminepentaacetic acid for labeling with (111)In. The ability of (111)In-bsRICs to bind HER2 or EGFR was determined in competition assays using cells expressing one or both receptors. Tumor and normal tissue uptake were examined in CD1 athymic mice bearing subcutaneous tumor xenografts that expressed HER2, EGFR, or both receptors, with or without pre-administration of Fab or EGF to determine specificity. HER2 and EGFR binding and displacement of binding by competitors were found for (111)In-bsICs. The highest uptake of (111)In-bsRICs [7.3 ± 3.5 %ID/g] in 231-H2N human breast cancer xenografts (HER2+/EGFR+) occurred at 48 h post-injection. Pre-administration of trastuzumab Fab decreased uptake in SK-OV-3 (HER2+/EGFR-) human ovarian cancer xenografts from 7.1 ± 1.2 to 2.4 ± 1.5 %ID/g. Pre-administration of excess EGF decreased uptake in MDA-MB-231 (HER2-/EGFR+) human breast cancer xenografts from 5.9 ± 0.5 to 2.0 ± 0.1 %ID/g. All tumors were imaged by microSPECT/CT. We conclude that (111)In-bsRICs composed of trastuzumab Fab and EGF exhibited specific binding in vitro to tumor cells displaying HER2 or EGFR, and were taken up specifically in vivo in tumors expressing one or both receptors, permitting tumor visualization by microSPECT/CT. These agents may ultimately be useful for imaging heterodimerized HER2-EGFR complexes since their bivalent properties permit more avid binding to these complexes.

The effect of metal-chelating polymers (MCPs) for 111In complexed via the streptavidin-biotin system to trastuzumab Fab fragments on tumor and normal tissue distribution in mice.

PURPOSE: To study the effects of backbone composition and charge of biotin-functionalized metal-chelating polymers (Bi-MCPs) for (111)In complexed to streptavidin (SAv)-trastuzumab Fab fragments on tumor and normal tissue localization. METHODS: Bi-MCPs were synthesized with a polyacrylamide [Bi-PAm(DTPA)(40)], polyaspartamide [Bi-PAsp(DTPA)(33)] or polyglutamide [Bi-PGlu(DTPA)(28)] backbone and harboured diethylenetriaminepentaacetic acid (DTPA) chelators for (111)In. Bi-PAm(DTPA)(40) had a net negative charge; Bi-PAsp(DTPA)(33) and Bi-PGlu(DTPA)(28) were zwitterionic with a net neutral charge. Binding to HER2+ SKOV-3 human ovarian carcinoma cells was determined. Tissue uptake was studied in Balb/c mice by MicroSPECT/CT imaging and biodistribution studies. Tumor and normal tissue uptake of (111)In-labeled Bi-PAsp(DTPA)(33) or Bi-PGlu(DTPA)(28) complexed to SAv-Fab was evaluated 48 h post-injection in athymic mice with subcutaneous SKOV-3 xenografts. RESULTS: SAv-Fab complexed to MCPs bound specifically to SKOV-3 cells; but specific binding was decreased 2-fold. Liver uptake was 5-13 fold higher for Bi-PAm(DTPA)(40) than Bi-PAsp(DTPA)(33) and Bi-PGlu(DTPA)(28) but was reduced by decreasing negative charges by saturation with indium. (111)In-Bi-PAsp(DTPA)(33) complexed to SAv-Fab accumulated in SKOV-3 tumors; low tumor uptake was found for (111)In-Bi-PGlu(DTPA)(28)-SAv-Fab. CONCLUSIONS: Zwitterionic MCPs composed of polyaspartamide with a net neutral charge are most desirable for constructing radioimmunoconjugates.

Small-animal SPECT/CT of HER2 and HER3 expression in tumor xenografts in athymic mice using trastuzumab Fab-heregulin bispecific radioimmunoconjugates.

UNLABELLED: Heterodimerization of human epidermal growth factor receptor 2 (HER2) with HER3 initiates aberrant downstream growth-signaling pathways in tumors. Our objective was to construct bispecific radioimmunoconjugates (bsRICs) that recognize HER2 and HER3 and evaluate their ability to image tumors in athymic mice that express one or both receptors using small-animal SPECT/CT. METHODS: bsRICs were constructed by reacting the maleimide-derivatized trastuzumab Fab fragments that bind HER2 with a thiolated form of the HER3-binding peptide of heregulin-ß1 (HRG) with or without a 12- or 24 mer polyethylene glycol (PEG) spacer. bsRICs were derivatized with diethylenetriaminepentaacetic acid for labeling with (111)In. The ability of (111)In-bsRICs to bind HER2 or HER3 was determined in competition assays with unlabeled Fab or HRG on cells expressing one or both receptors. Tumor and normal-tissue uptake were examined in CD1 athymic mice bearing subcutaneous tumor xenografts that expressed HER2, HER3, or both receptors, with or without the preadministration of unlabeled Fab or HRG to determine the specificity of uptake. RESULTS: Conjugation of Fab to HRG was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis-Western blot and size-exclusion high-performance liquid chromatography. Improved HER2 and HER3 binding and greater displacement of binding by competitors was found for (111)In-bsRICs that incorporated a PEG spacer, with the PEG(24) spacer being optimal. The highest uptake of (111)In-bsRICs (7.8% ± 2.1% injected dose per gram [%ID/g]) in BT-474 human breast cancer xenografts (HER2-positive/HER3-positive) occurred at 48 h after injection. The preadministration of trastuzumab Fab decreased uptake in SK-OV-3 (HER2-positive/HER3-negative) human ovarian cancer xenografts from 7.0 ± 1.2 to 2.6 ± 1.5 %ID/g (P < 0.001). The preadministration of an excess of HRG decreased uptake in MDA-MB-468 (HER2-negative/HER3-positive) human breast cancer xenografts from 4.4 ± 0.9 to 2.6 ± 0.5 %ID/g (P < 0.05). All tumors were imaged by small-animal SPECT/CT. CONCLUSION: (111)In-bsRICs composed of trastuzumab Fab and HRG exhibited specific binding in vitro to tumor cells displaying HER2 or HER3 and were taken up specifically in vivo in tumors expressing one or both receptors, permitting tumor visualization by small-animal SPECT/CT. These agents could be useful for imaging heterodimerized HER2 and HER3 receptors because their bivalent properties may result in preferential binding to the heterodimerized forms. The approach may also be extended to constructing bsRICs for visualizing other peptide growth factor receptors.

Role of antibody-mediated tumor targeting and route of administration in nanoparticle tumor accumulation in vivo.

In this study, we have looked at enhancing tumor uptake and intracellular delivery of gold nanoparticles (AuNPs) while reducing the systemic exposure by systematic evaluation of the impact of targeting and route of administration on organ distribution. High-resolution microSPECT/CT imaging was used to track the in vivo fate of (111)In-labeled nontargeted and human epidermal growth factor receptor-2 (HER-2) targeted AuNPs following intravenous (i.v.) or intratumoral (i.t.) injection. For i.v. injection, the effects of GdCl3 (for deactivation of macrophages) and nonspecific (anti-CD20) antibody rituximab (for blocking of Fc mediated liver and spleen uptake) were studied. It was found that HER-2 targeting via attachment of trastuzumab paradoxically decreased tumor uptake as a result of faster elimination of the targeted AuNPs from the blood while improving internalization in HER-2-positive tumor cells as compared to nontargeted AuNPs. I.T. injections with HER-2 targeted AuNPs resulted in high tumor retention with low systemic exposure and represents an attractive delivery strategy. Our results provide a strategy for optimizing tumor delivery and quantifying organ distribution of this widely studied class of nanomaterial.

Influence of formulation variables on the biodistribution of multifunctional block copolymer micelles.

The physico-chemical characteristics and composition of block copolymer micelles (BCMs) may influence the pharmacokinetics and consequently, the desired delivery characteristics. In this study the influence of formulation variables such as size, density of targeting ligand [i.e. epidermal growth factor (hEGF)] and the bifunctional chelator (BFC) used for labelling the BCMs with (111)In, on the pharmacokinetics and biodistribution in mice were evaluated. BCMs were prepared from Me-PEG(x)-b-PCL(y) (x=2.5 k, y=1.2 k for 15 nm BCMs and x=5 k, y=5 k for 60 nm BCMs) with (targeted, 1 or 5 mol% hEGF) or without (non-targeted) hEGF-PEG(x)-b-PCL(y). To investigate the effect of the BFC on the pharmacokinetics, the BCMs were labelled with (111)In using p-SCN-Bn-DOTA (Bn-DOTA-PEG(x)-b-PCL(y)), H(2)N-DOTA (DOTA-PEG(x)-b-PCL(y)), DTPA anhydride (DTPA-PEG(x)-b-PCL(y)) or p-SCN-Bn-DTPA (Bn-DTPA-PEG(x)-b-PCL(y)). The resulting 15 nm or 60 nm non-targeted or targeted (1 or 5 mol% hEGF) were injected via a tail vein to mice bearing MDA-MB-468 human breast cancer xenograft that overexpress EGFR, followed by pharmacokinetics and biodistribution studies. Pharmacokinetic parameters were determined by fitting the blood concentration vs time data using a two compartment model with i.v. bolus input. Pharmacokinetic parameters were found to depend on BCM size, the BFC used as well as the density of hEGF on the surface of the BCMs. BCMs labelled with p-SCN-Bn-DTPA ((111)In-Bn-BCMs) showed improved pharmacokinetics (i.e. extended circulation lifetime) and tumor uptake compared to those labelled with DOTA-PEG(x)-b-PCL(y), p-SCN-Bn-DOTA or DTPA dianhydride. Formulations with a high density of hEGF (5 mol% hEGF) had short circulation half-lives. BCMs labelled with (111)In via p-SCN-Bn-DTPA showed highest accumulation in the liver and spleen and slower whole body elimination. Smaller sized BCMs were rapidly cleared from the circulation. Increasing the density of hEGF on the surface did not improve tumor uptake due to faster clearance from the circulation. To achieve improved pharmacokinetics and in turn effective exploitation of the EPR effect, p-SCN-Bn-DTPA emerged as the optimal BFC for radiolabelling BCMs while a lower density of hEGF gave more favourable organ distribution.

Comparisons of [18F]-1-deoxy-1-fluoro-scyllo-inositol with [18F]-FDG for PET imaging of inflammation, breast and brain cancer xenografts in athymic mice.

INTRODUCTION: The aim of the study was to evaluate the uptake of [(18)F]-1-deoxy-1-fluoro-scyllo-inositol ([(18)F]-scyllo-inositol) in human breast cancer (BC) and glioma xenografts, as well as in inflammatory tissue, in immunocompromised mice. Studies of [(18)F]-2-fluoro-2-deoxy-d-glucose ([(18)F]-FDG) under the same conditions were also performed. METHODS: Radiosynthesis of [(18)F]-scyllo-inositol was automated using a commercial synthesis module. Tumour, inflammation and normal tissue uptakes were evaluated by biodistribution studies and positron emission tomography (PET) imaging using [(18)F]-scyllo-inositol and [(18)F]-FDG in mice bearing subcutaneous MDA-MB-231, MCF-7 and MDA-MB-361 human BC xenografts, intracranial U-87 MG glioma xenografts and turpentine-induced inflammation. RESULTS: The radiosynthesis of [(18)F]-scyllo-inositol was automated with good radiochemical yields (24.6%±3.3%, uncorrected for decay, 65±2 min, n=5) and high specific activities (≥195 GBq/µmol at end of synthesis). Uptake of [(18)F]-scyllo-inositol was greatest in MDA-MB-231 BC tumours and was comparable to that of [(18)F]-FDG (4.6±0.5 vs. 5.5±2.1 %ID/g, respectively; P=.40), but was marginally lower in MDA-MB-361 and MCF-7 xenografts. Uptake of [(18)F]-scyllo-inositol in inflammation was lower than [(18)F]-FDG. While uptake of [(18)F]-scyllo-inositol in intracranial U-87 MG xenografts was significantly lower than [(18)F]-FDG, the tumour-to-brain ratio was significantly higher (10.6±2.5 vs. 2.1±0.6; P=.001). CONCLUSIONS: Consistent with biodistribution studies, uptake of [(18)F]-scyllo-inositol was successfully visualized by PET imaging in human BC and glioma xenografts, with lower accumulation in inflammatory tissue than [(18)F]-FDG. The tumour-to-brain ratio of [(18)F]-scyllo-inositol was also significantly higher than that of [(18)F]-FDG for visualizing intracranial glioma xenografts in NOD SCID mice, giving a better contrast.

A kit to prepare (111)In-DTPA-trastuzumab (Herceptin) Fab fragments injection under GMP conditions for imaging or radioimmunoguided surgery of HER2-positive breast cancer.

INTRODUCTION: The human epidermal growth factor receptor-2 (HER2) gene is amplified in 25% of invasive breast cancers, and receptor overexpression has been noted in up to 60% of early stages of the disease [ductal carcinoma in situ (DCIS)]. Preclinical studies have revealed high tumor/blood ratios (>27:1) for (111)In-labeled Fab fragments of the HER2 monoclonal antibody, trastuzumab (Herceptin) ((111)In-DTPA-trastuzumab Fab) at 72 h pi in athymic mice bearing subcutaneous human breast cancer xenografts. Our aim in this study was to formulate a kit for preparation of (111)In-DTPA-trastuzumab Fab injection under good manufacturing practice (GMP) conditions suitable for human administration in a Phase I clinical trial of imaging and radioimmunoguided surgery (RIGS) of HER2-positive breast cancer. METHODS: Fab fragments were produced by digestion of trastuzumab IgG (Herceptin) with immobilized papain for 20 h at 37°C. Fab fragments were purified by ultrafiltration, then reacted with a 10-fold molar excess of diethylenetriaminepentaacetic acid (DTPA) dianhydride. DTPA-Fab fragments were purified, then sterilized by filtration into unit dose glass vials (kits). Kits were tested against specifications for volume (0.9-1.1 ml), protein concentration (0.45-0.55 mg/ml), pH (5.5-6.5), DTPA substitution (0.5-4.0 mol DTPA/mol Fab), appearance (clear, colorless and particle free), labeling efficiency (≥ 85%), and sterility and apyrogenicity (USP XXXII). Immunoreactivity of (111)In-DTPA-trastuzumab Fab towards HER2 was measured by saturation radioligand binding assays using SKBR-3 human breast cancer cells (specifications: K(a) = 0.6-9.6 × 10(7) L/mol; B(max) = 0.6-10.4 × 10(6) sites/cell). (111)In-DTPA-trastuzumab Fab injection was prepared by adding 80-100 MBq of (111)InCl(3) to a single kit vial and incubating for 30 min at room temperature. (111)In-DTPA-trastuzumab Fab was assayed for the amount of radioactivity and tested for pH, radiochemical purity (RCP), appearance and sterility. RESULTS: Pure and homogeneous Fab fragments were produced. Eleven lots of kits met established quality specifications. The labeling efficiency with (111)In was 90.6 ± 2.2%. (111)In-DTPA-trastuzumab Fab bound specifically to HER2 on SKBR-3 cells (K(a) = 4.8 ± 2.5 × 10(7) L/mol and B(max) = 1.6 ± 0.8 × 10(6) sites/cell). Thirteen lots of (111)In-DTPA-trastuzumab injection met all established specifications. Kits were stable for 90 days and (111)In-DTPA-trastuzumab Fab injection was stable for 24 h stored at 4 °C. CONCLUSIONS: A kit was formulated under GMP conditions for the preparation of (111)In-DTPA-trastuzumab Fab injection suitable for human administration. The kits were approved by Health Canada.

A comparison of 111In- or 64Cu-DOTA-trastuzumab Fab fragments for imaging subcutaneous HER2-positive tumor xenografts in athymic mice using microSPECT/CT or microPET/CT.

BACKGROUND: Our objective was to compare 111In- or 64Cu-DOTA-trastuzumab Fab fragments for imaging small or large s.c. tumor xenografts in athymic mice that display a wide range of human epidermal growth factor receptor-2 (HER2) expression using microSPECT/CT or microPET/CT. METHODS: Trastuzumab Fab were labeled with 111In or 64Cu by conjugation to 1,4,7,10-tetraazacyclododecane N, N', N'', N'''-tetraacetic acid (DOTA). The purity of 111In- and 64Cu-DOTA-trastuzumab Fab was measured by SDS-PAGE and HPLC. HER2 binding affinity was determined in saturation radioligand binding assays using SKBR-3 cells (1.3 × 106 HER2/cell). MicroSPECT/CT and microPET/CT were performed in athymic mice bearing s.c. BT-20 and MDA-MB-231 xenografts with low (0.5 to 1.6 × 105 receptors/cell), MDA-MB-361 tumors with intermediate (5.1 × 105 receptors/cell) or SKOV-3 xenografts with high HER2 expression (1.2 × 106 receptors/cell) at 24 h p.i. of 70 MBq (10 µg) of 111In-DOTA-trastuzumab Fab or 22 MBq (10 µg) of 64Cu-DOTA-trastuzumab Fab or irrelevant 111In- or 64Cu-DOTA-rituximab Fab. Tumor and normal tissue uptake were quantified in biodistribution studies. RESULTS: 111In- and 64Cu-DOTA-trastuzumab were > 98% radiochemically pure and bound HER2 with high affinity (Kd = 20.4 ± 2.5 nM and 40.8 ± 3.5 nM, respectively). MDA-MB-361 and SKOV-3 tumors were most clearly imaged using 111In- and 64Cu-DOTA-trastuzumab Fab. Significantly higher tumor/blood (T/B) ratios were found for 111In-DOTA-trastuzumab Fab than 111In-DOTA-rituximab Fab for BT-20, MDA-MB-231 and MDA-MB-361 xenografts, and there was a direct association between T/B ratios and HER2 expression. In contrast, tumor uptake of 64Cu-DOTA-trastuzumab Fab was significantly higher than 64Cu-DOTA-rituximab Fab in MDA-MB-361 tumors but no direct association with HER2 expression was found. Both 111In- and 64Cu-DOTA-trastuzumab Fab imaged small (5 to 10 mm) or larger (10 to 15 mm) MDA-MB-361 tumors. Higher blood, liver, and spleen radioactivity were observed for 64Cu-DOTA-trastuzumab Fab than 111In-DOTA-trastuzumab Fab. CONCLUSIONS: We conclude that 111In-DOTA-trastuzumab Fab was more specific than 64Cu-DOTA-trastuzumab Fab for imaging HER2-positive tumors, especially those with low receptor density. This was due to higher levels of circulating radioactivity for 64Cu-DOTA-trastuzumab Fab which disrupted the relationship between HER2 density and T/B ratios. Use of alternative chelators that more stably bind 64Cu may improve the association between T/B ratios and HER2 density for 64Cu-labeled trastuzumab Fab.

18F-FDG small-animal PET/CT differentiates trastuzumab-responsive from unresponsive human breast cancer xenografts in athymic mice.

UNLABELLED: Breast cancers (BCs) with high human epidermal growth factor receptor type 2 (HER2) expression are most likely to respond to trastuzumab; however, the mechanisms of action of trastuzumab are complex and there are no established biomarkers to accurately monitor treatment outcome in individual patients. Therefore, our aim was to determine, in human BC xenografts in athymic mice treated with trastuzumab, whether there were any changes in (18)F-FDG uptake that were associated with response to the drug and that could have utility in monitoring response in patients. METHODS: Baseline tumor uptake of (18)F-FDG was measured in mice with MDA-MB-361 HER2-overexpressing xenografts and MDA-MB-231 xenografts with low HER2 expression by small-animal PET imaging on day 0. Mice were treated with phosphate-buffered saline (PBS) or trastuzumab (4 mg/kg), and small-animal PET was repeated 2 d after treatment. Maintenance doses of trastuzumab (2 mg/kg) or PBS were administered on days 7 and 14, and mice were imaged again on days 9 and 16. Tumor uptake was measured as percentage injected dose per gram (%ID/g) by volume-of-interest analysis on days 0 (baseline), 2, 9, and 16, followed by biodistribution studies on day 16. Tumor growth was measured, and a tumor growth index was calculated. RESULTS: The treatment of mice with trastuzumab, compared with control mice treated with PBS, resulted in a significant decrease in tumor uptake of (18)F-FDG in HER2-overexpressing MDA-MB-361 xenografts after 16 d of treatment (2.6 +/- 0.8 %ID/g vs. 4.6 +/- 1.8 %ID/g, respectively; P < 0.03) but not after 2 or 9 d of treatment (P = 0.28-0.32). In contrast, there was no significant change in the tumor uptake of MDA-MB-231 xenografts with low HER2 expression during the entire course of therapy (4.4 +/- 1.7 %ID/g vs. 3.6 +/- 1.1 %ID/g, respectively; P = 0.31). Trastuzumab treatment, compared with PBS treatment of controls, resulted in significant growth inhibition of MDA-MB-361 xenografts as early as 10 d from the initiation of treatment (tumor growth index, 0.7 +/- 0.2 vs. 1.7 +/- 0.3, respectively; P < 0.0005), whereas no tumor growth inhibition was observed for MDA-MB-231 xenografts (5.3 +/- 2.7 and 5.2 +/- 3.0; P = 0.95). CONCLUSION: Changes in the tumor uptake of (18)F-FDG after therapy accurately identified responding and nonresponding human BC xenografts in athymic mice treated with trastuzumab; however, diminished glucose utilization did not precede changes in tumor volume.

Micro-SPECT/CT with 111In-DTPA-pertuzumab sensitively detects trastuzumab-mediated HER2 downregulation and tumor response in athymic mice bearing MDA-MB-361 human breast cancer xenografts.

UNLABELLED: Pertuzumab is a HER2 dimerization inhibitor that binds to an epitope unique from that of trastuzumab. Our objective was to determine whether SPECT with (111)In-diethylenetriaminepentaacetic acid-pertuzumab ((111)In-DTPA-pertuzumab) could sensitively detect an early molecular response to trastuzumab manifested by HER2 downregulation and a later tumor response revealed by a decreased number of HER2-positive viable tumor cells. METHODS: Changes in HER2 density in SKBr-3 and MDA-MB-361 BC cells exposed to trastuzumab (14 microg/mL) in vitro were measured by saturation binding assays using (111)In-DTPA-pertuzumab and by confocal immunofluorescence microscopy and flow cytometry with fluorescein isothiocyanate-labeled HER2/neu antibodies. Imaging of HER2 downregulation was studied in vivo in athymic mice with subcutaneous MDA-MB-361 tumors treated for 3 d with trastuzumab (4 mg/kg) or nonspecific human IgG (hIgG) or phosphate-buffered saline (PBS). Imaging of tumor response to trastuzumab was studied in mice bearing subcutaneous MDA-MB-361 xenografts treated with trastuzumab (4 mg/kg), followed by weekly doses of nonspecific hIgG or rituximab or PBS (2 mg/kg). Mice were imaged on a micro-SPECT/CT system at 72 h after injection of (111)In-DTPA-pertuzumab. Tumor and normal-tissue biodistribution was determined. RESULTS: (111)In-DTPA-pertuzumab saturation binding to SKBr-3 and MDA-MB-361 cells was significantly decreased at 72 h after exposure in vitro to trastuzumab (14 microg/mL), compared with untreated controls (62% +/- 2%, P < 0.0001; 32% +/- 9%, P < 0.0002, respectively). After 3 d of trastuzumab, in vivo tumor uptake of (111)In-DTPA-pertuzumab decreased 2-fold in trastuzumab- versus PBS-treated mice (13.5 +/- 2.6 percentage injected dose per gram [%ID/g] vs. 28.5 +/- 9.1 %ID/g, respectively; P < 0.05). There was also a 2-fold decreased tumor uptake in trastuzumab- versus PBS-treated mice by image volume-of-interest analysis (P = 0.05), suggesting trastuzumab-mediated HER2 downregulation. After 3 wk of trastuzumab, tumor uptake of (111)In-DTPA-pertuzumab decreased 4.5-fold, compared with PBS-treated mice (7.6 +/- 0.4 vs. 34.6 +/- 9.9 %ID/g, respectively; P < 0.001); this decrease was associated with an almost-completed eradication of HER2-positive tumor cells determined immunohistochemically. CONCLUSION: (111)In-DTPA-pertuzumab sensitively imaged HER2 downregulation after 3 d of treatment with trastuzumab and detected a reduction in viable HER2-positive tumor cells after 3 wk of therapy in MDA-MB-361 human breast cancer xenografts.

Associations between the uptake of 111In-DTPA-trastuzumab, HER2 density and response to trastuzumab (Herceptin) in athymic mice bearing subcutaneous human tumour xenografts.

PURPOSE: The purpose of the study was to investigate the associations between uptake of (111)In-DTPA-trastuzumab, tumour HER2 density and response to trastuzumab (Herceptin) of human breast cancer (BC) xenografts in athymic mice. MATERIALS AND METHODS: The tumour uptake of (111)In-DTPA-trastuzumab in athymic mice bearing BC xenografts with increasing HER2 density (0 to 3+) was evaluated. Specific uptake ratios were established in biodistribution (SUR) and imaging studies (ROI-SUR) using (111)In-labeled mouse IgG ((111)In-DTPA-mIgG). Further corrections were made for circulating radioactivity using tumour-to-blood ratios defined as a localization index (LI) and region-of-interest localization index (ROI-LI), respectively. Mice were treated with trastuzumab (Herceptin). A tumour growth inhibition index (TGI) was calculated and relative TGIs calculated by dividing the TGI of control by that of trastuzumab-treated mice. RESULTS: Strong, nonlinear associations with HER2 density were obtained if the uptake of (111)In-DTPA-trastuzumab was corrected for nonspecific IgG localization (i.e., SUR; r (2) = 0.99) and circulating radioactivity (i.e., LI; r (2) = 0.87), but without these corrections, the association between HER2 density and tumour uptake was poor (r (2) = 0.22). There was a strong association between ROI-SUR and ROI-LI values and HER2 expression (r (2) = 0.90 and r (2) = 0.95, respectively. All tumours were imaged. Relative TGI values were associated with increasing uncorrected tumour uptake of (111)In-DTPA-trastuzumab but not always with HER2 density (i.e., MCF-HER2-18 cells with trastuzumab-resistance). CONCLUSION: HER2 expression (0 to 3+) can be differentiated using (111)In-DTPA-trastuzumab, but requires correction of tumour uptake for nonspecific IgG localization and circulating radioactivity. The uncorrected uptake of (111)In-DTPA-trastuzumab was associated with tumour response to trastuzumab.

Relationship between induction of phosphorylated H2AX and survival in breast cancer cells exposed to 111In-DTPA-hEGF.

UNLABELLED: The Auger electron-emitting radiopharmaceutical 111In-diethylenetriaminepentaacetic acid human epidermal growth factor (111In-DTPA-hEGF) binds the epidermal growth factor receptor (EGFR), is internalized, and translocates to the nucleus. The purpose of this study was to investigate the relationship between EGFR expression, DNA damage, and cytotoxicity in cells exposed to 111In-DTPA-hEGF. METHODS: Breast cancer cell lines with a range of EGFR expression levels were exposed to 111In-DTPA-hEGF or gamma-radiation. The cell lines (followed by number of EGFR per cell in parentheses) were MDA-MB-468 (1.3 x 10(6)), MDA-MB-231 (1.3 x 10(5)), and MCF-7 (1.5 x 10(4)). The proportion of radioactivity partitioning into the nucleus was measured by cell fractionation. DNA double-strand breaks were evaluated using the gamma-H2AX assay. Clonogenic survival assays were used to measure cytotoxicity. RESULTS: All data are presented as mean +/- SD. The amount of 111In-DTPA-hEGF that translocated to the nucleus (in mBq/nucleus) in MDA-MB-468, MDA-MB-231, and MCF-7 cells incubated with 111In-DTPA-hEGF (5.2 MBq/mL, 43 nM) for 20 h was 131 +/- 6, 8.1 +/- 0.1, and 1.1 +/- 0.9, respectively. The number of gamma-H2AX foci per nucleus was 35 +/- 15, 19 +/- 10, and 1.7 +/- 0.3, respectively. A reduction in the surviving fraction (SF) in MDA-MB-468 (0.013 +/- 0.001) and MDA-MB-231 (0.5 +/- 0.1) but not in MCF-7 cells after exposure to 111In-DTPA-hEGF (5.2 MBq/mL, 43 nM) for 20 h has been demonstrated. The SF of MDA-MB-468 cells after exposure to DTPA-EGF (43 nM) and 111In-acetate (5.2 MBq/mL) for 20 h was 0.5 +/- 0.1 and 0.53 +/- 0.05, respectively. MDA-MB-468 was the most sensitive of the cell lines to gamma-irradiation, with an SF after 2 Gy of 0.45 +/- 0.04, compared with 0.7 +/- 0.1 and 0.8 +/- 0.1 for MCF-7 and MDA-MB-231, respectively. The number of gamma-H2AX foci per nucleus in MDA-MB-468 cells correlated with the concentration, specific activity, and incubation time of 111In-DTPA-hEGF. CONCLUSION: DNA damage caused by 111In-DTPA-hEGF correlates with the EGFR expression level of the exposed cells and with concentration, specific activity, and incubation time of 111In-DTPA-hEGF. The gamma-H2AX assay may be a useful biomarker to predict and monitor the outcome of treatment with 111In-DTPA-hEGF.

Properties of [(111)In]-labeled HIV-1 tat peptide radioimmunoconjugates in tumor-bearing mice following intravenous or intratumoral injection.

INTRODUCTION: Our objective was to evaluate the tumor and normal tissue distribution and nuclear importation properties of [(111)In]-mouse IgG (mIgG) conjugated to tat peptides (GRKKRRQRRRPPQGYG) in athymic mice with subcutaneous BT-474 human breast cancer xenografts. METHODS: Tumor and normal tissue uptake was compared after intravenous (iv) or intratumoral injection of [(111)In]-mIgG-tat and [(111)In]-mIgG. Area under the curve (AUC) was estimated for blood, liver, spleen, kidneys and tumor. Nuclear localization was measured by subcellular fractionation and estimated by microdosimetry. Imaging studies were performed with a gamma-camera. RESULTS: [(111)In]-mIgG-tat was eliminated from the blood and normal tissues two- to threefold more rapidly after iv injection than [(111)In]-mIgG. Tumor uptake was 4-5% injected dose per gram (%ID/g). Tumor radioactivity after intratumoral injection was initially very high (146-154 %ID/g), but declined 12- to 14-fold by 144 h postinjection. There was greater retention of [(111)In]-mIgG-tat in BT-474 tumors after intratumoral than iv injection, and the AUC (610+/-157 %ID h) was threefold greater than for intratumorally injected [(111)In]-mIgG (200+/-37 %ID h). Tat peptides increased nuclear localization of [(111)In]-mIgG after iv injection in tumor, kidney and liver cells, but only in tumor cells after intratumoral injection. Tumors were not imaged after iv administration but were predominant with intratumorally injected [(111)In]-mIgG and [(111)In]-mIgG-tat. Estimated radiation doses to the nucleus of tumor cells from intratumoral [(111)In]-mIgG-tat were 2.8x10(3) mGy/MBq and were 15-fold higher than for iv injection. CONCLUSION: [(111)In]-labeled tat immunoconjugates may have potential for imaging intracellular epitopes or localized Auger electron radiotherapy of tumors.