Feasibility of Imaging EpCAM Expression in Ovarian Cancer Using Radiolabeled DARPin Ec1.

Epithelial cell adhesion molecule (EpCAM) is overexpressed in 55%-75% of ovarian carcinomas (OC). EpCAM might be used as a target for a treatment of disseminated OC. Designed ankyrin repeats protein (DARPin) Ec1 is a small (18 kDa) protein, which binds to EpCAM with subnanomolar affinity. We tested a hypothesis that Ec1 labeled with a non-residualizing label might serve as a companion imaging diagnostic for stratification of patients for EpCAM-targeting therapy. Ec1 was labeled with 125I using N-succinimidyl-para-iodobenzoate. Binding affinity, specificity, and cellular processing of [125I]I-PIB-Ec1 were evaluated using SKOV-3 and OVCAR-3 ovarian carcinoma cell lines. Biodistribution and tumor-targeting properties of [125I]I-PIB-Ec1 were studied in Balb/c nu/nu mice bearing SKOV-3 and OVCAR-3 xenografts. EpCAM-negative Ramos lymphoma xenografts served as specificity control. Binding of [125I]I-PIB-Ec1 to ovarian carcinoma cell lines was highly specific and had affinity in picomolar range. Slow internalization of [125I]I-PIB-Ec1 by OC cells confirmed utility of non-residualizing label for in vivo imaging. [125I]I-PIB-Ec1 provided 6 h after injection tumor-to-blood ratios of 30 ± 11 and 48 ± 12 for OVCAR-3 and SKOV-3 xenografts, respectively, and high contrast to other organs. Tumor targeting was highly specific. Saturation of tumor uptake at a high dose of Ec1 in SKOV-3 model provided a rationale for dose selection in further studies using therapeutic conjugates of Ec1 for targeted therapy. In conclusion, [125I]I-PIB-Ec1 is a promising agent for visualizing EpCAM expression in OC.

Pretargeted Imaging and Therapy.

In vivo pretargeting stands as a promising approach to harnessing the exquisite tumor-targeting properties of antibodies for nuclear imaging and therapy while simultaneously skirting their pharmacokinetic limitations. The core premise of pretargeting lies in administering the targeting vector and radioisotope separately and having the 2 components combine within the body. In this manner, pretargeting strategies decrease the circulation time of the radioactivity, reduce the uptake of the radionuclide in healthy nontarget tissues, and facilitate the use of short-lived radionuclides that would otherwise be incompatible with antibody-based vectors. In this short review, we seek to provide a brief yet informative survey of the 4 preeminent mechanistic approaches to pretargeting, strategies predicated on streptavidin and biotin, bispecific antibodies, complementary oligonucleotides, and bioorthogonal click chemistry.

Design, Preparation, and Characterization of PNA-Based Hybridization Probes for Affibody-Molecule-Mediated Pretargeting.

In radioimmunotherapy, the contrast between tumor and normal tissue can be improved by using a pretargeting strategy with a primary targeting agent, which is conjugated to a recognition tag, and a secondary radiolabeled molecule binding specifically to the recognition tag. The secondary molecule is injected after the targeting agent has accumulated in the tumor and is designed to have a favorable biodistribution profile, with fast clearance from blood and low uptake in normal tissues. In this study, we have designed and evaluated two complementary peptide nucleic acid (PNA)-based probes for specific and high-affinity association in vivo. An anti-HER2 Affibody-PNA chimera, Z(HER2:342)-SR-HP1, was produced by a semisynthetic approach using sortase A catalyzed ligation of a recombinantly produced Affibody molecule to a PNA-based HP1-probe assembled using solid-phase chemistry. A complementary HP2 probe carrying a DOTA chelator and a tyrosine for dual radiolabeling was prepared by solid-phase synthesis. Circular dichroism (CD) spectroscopy and UV thermal melts showed that the probes can hybridize to form a structured duplex with a very high melting temperature (T(m)), both in HP1:HP2 and in Z(HER2:342)-SR-HP1:HP2 (T(m) = 86-88 °C), and the high binding affinity between Z(HER2:342)-SR-HP1 and HP2 was confirmed in a surface plasmon resonance (SPR)-based binding study. Following a moderately fast association (1.7 × 10(5) M(-1) s(-1)), the dissociation of the probes was extremely slow and <5% dissociation was observed after 17 h. The equilibrium dissociation constant (K(D)) for Z(HER2:342)-SR-HP1:HP2 binding to HER2 was estimated by SPR to be 212 pM, suggesting that the conjugation to PNA does not impair Affibody binding to HER2. The biodistribution profiles of (111)In- and (125)I-labeled HP2 were measured in NMRI mice, showing very fast blood clearance rates and low accumulation of radioactivity in kidneys and other organs. The measured radioactivity in blood was 0.63 ± 0.15 and 0.41 ± 0.15%ID/g for (125)I- and (111)In-HP2, respectively, at 1 h p.i., and at 4 h p.i., the kidney accumulation of radioactivity was 0.17 ± 0.04%ID/g for (125)I-HP2 and 3.83 ± 0.39%ID/g for (111)In-HP2. Taken together, the results suggest that a PNA-based system has suitable biophysical and in vivo properties and is a promising approach for pretargeting of Affibody molecules.

188Re-ZHER2:V2, a promising affibody-based targeting agent against HER2-expressing tumors: preclinical assessment.

UNLABELLED: Affibody molecules are small (7 kDa) nonimmunoglobulin scaffold proteins with favorable tumor-targeting properties. Studies concerning the influence of chelators on biodistribution of (99m)Tc-labeled Affibody molecules demonstrated that the variant with a C-terminal glycyl-glycyl-glycyl-cysteine peptide-based chelator (designated ZHER2:V2) has the best biodistribution profile in vivo and the lowest renal retention of radioactivity. The aim of this study was to evaluate (188)Re-ZHER2:V2 as a potential candidate for radionuclide therapy of human epidermal growth factor receptor type 2 (HER2)-expressing tumors. METHODS: ZHER2:V2 was labeled with (188)Re using a gluconate-containing kit. Targeting of HER2-overexpressing SKOV-3 ovarian carcinoma xenografts in nude mice was studied for a dosimetry assessment. RESULTS: Binding of (188)Re-ZHER2:V2 to living SKOV-3 cells was demonstrated to be specific, with an affinity of 6.4 ± 0.4 pM. The biodistribution study showed a rapid blood clearance (1.4 ± 0.1 percentage injected activity per gram [%ID/g] at 1 h after injection). The tumor uptake was 14 ± 2, 12 ± 2, 5 ± 2, and 1.8 ± 0.5 %IA/g at 1, 4, 24, and 48 h after injection, respectively. The in vivo targeting of HER2-expressing xenografts was specific. Already at 4 h after injection, tumor uptake exceeded kidney uptake (2.1 ± 0.2 %IA/g). Scintillation-camera imaging showed that tumor xenografts were the only sites with prominent accumulation of radioactivity at 4 h after injection. Based on the biokinetics, a dosimetry evaluation for humans suggests that (188)Re-ZHER2:V2 would provide an absorbed dose to tumor of 79 Gy without exceeding absorbed doses of 23 Gy to kidneys and 2 Gy to bone marrow. This indicates that future human radiotherapy studies may be feasible. CONCLUSION: (188)Re-ZHER2:V2 can deliver high absorbed doses to tumors without exceeding kidney and bone marrow toxicity limits.

Development and preclinical characterisation of 99mTc-labelled Affibody molecules with reduced renal uptake.

PURPOSE: Affibody molecules are low molecular weight proteins (7 kDa), which can be selected to bind to tumour-associated target proteins with subnanomolar affinity. Because of rapid tumour localisation and clearance from nonspecific compartments, Affibody molecules are promising tracers for molecular imaging. Earlier, (99m)Tc-labelled Affibody molecules demonstrated specific targeting of tumour xenografts. However, the biodistribution was suboptimal either because of hepatobiliary excretion or high renal uptake of the radioactivity. The goal of this study was to optimise the biodistribution of Affibody molecules by chelator engineering. MATERIALS AND METHODS: Anti-HER2 Z(HER2:342) Affibody molecules, carrying the mercaptoacetyl-glutamyl-seryl-glutamyl (maESE), mercaptoacetyl-glutamyl-glutamyl-seryl (maEES) and mercaptoacetyl-seryl-glutamyl-glutamyl (maSEE) chelators, were prepared by peptide synthesis and labelled with (99m)Tc. The tumour-targeting capacity of these conjugates was compared with each other and with the best previously available conjugate, (99m)Tc-maEEE-Z(HER2:342,) in nude mice bearing SKOV-3 xenografts. The tumour-targeting capacity of the most promising conjugate, (99m)Tc-maESE-Z(HER2:342,) was compared with radioiodinated Z(HER2:342). RESULTS: All novel conjugates demonstrated successful tumour targeting and a low degree of hepatobiliary excretion. The renal uptakes of serine-containing conjugates, 33 +/- 5, 68 +/- 21 and 71 +/- 10%IA/g, for(99m)Tc-maESE-Z(HER2:342), (99m)Tc-maEES-Z(HER2:342) and (99m)Tc-maSEE-Z(HER2:342), respectively, were significantly reduced in comparison with (99m)Tc-maEEE-Z(HER2:342) (102 +/- 13%IA/g). For (99m)Tc-maESE-Z(HER2:342), a tumour uptake of 9.6 +/- 1.8%IA/g and a tumour-to-blood ratio of 58 +/- 6 were reached at 4 h p.i. CONCLUSIONS: A combination of serine and glutamic acid residues in the chelator sequence confers increased renal excretion and relatively low renal uptake of (99m)Tc-labelled Affibody molecules. In combination with preserved targeting capacity, this improved imaging of targets in abdominal area.

Pre-clinical evaluation of [111In]-benzyl-DOTA-Z(HER2:342), a potential agent for imaging of HER2 expression in malignant tumors.

Imaging of expression of human epidermal growth factor receptor type 2 (HER2) in breast carcinomas may help to select patients eligible for trastuzumab therapy. The Affibody molecule Z(HER2:342) is a small (7-kDa) non-immunoglobulin affinity protein, which binds to HER2 with a picomolar affinity. Previously, a benzyl-DTPA conjugate of Z(HER2:342) was labeled with 111In and demonstrated good targeting in murine xenografts. We considered that the use of the macrocyclic chelator DOTA could increase the label stability and enhance a choice of nuclides, which could be used as a label for Z(HER2:342). The goal of this study was the preparation and pre-clinical evaluation of the indium-111- labeled DOTA-derivative of Z(HER2:342). Isothiocyanate-benzyl-DOTA was coupled to recombinant Z(HER2:342), and the conjugate was efficiently labeled with 111In at 60 degrees C. The specificity of 111In-benzyl-DOTA-Z(HER2:342) binding to HER2 was confirmed in vitro using HER2-expressing breast carcinoma BT474 and ovarian carcinoma SKOV-3 cell lines. Biodistribution of 111In-benzyl-DOTA-Z(HER2:342) was performed in nude mice bearing LS174T xenografts and compared directly with the biodistribution of 111In-benzyl-DTPA-Z(HER2:342). In vivo, 111In-benzyl-DOTA-Z(HER2:342) demonstrated quick clearance from blood and non-specific organs except the kidneys. Four hours post injection (pi), the tumor uptake of 111In-benzyl-DOTA-Z(HER2:342) (4.4+/-1.0% IA/g) was specific and the tumor-to-blood ratio was 23. The use of benzyl-DTPA provided higher tumor-to-blood and tumor-to-liver ratios. gamma-camera imaging showed clear visualization of HER2-expressing xenografts using 111In-benzyl-DOTA-Z(HER2:342). 111In-benzyl-DOTA-Z(HER2:342) has a potential for imaging of HER2 expression in malignant tumors.

Labelling chemistry and characterization of [90Y/177Lu]-DOTA-ZHER2:342-3 Affibody molecule, a candidate agent for locoregional treatment of urinary bladder carcinoma.

The direct instillation of radiolabelled conjugates in the urinary bladder is a promising path for the treatment of bladder carcinoma. The targeting of HER2/neu receptors expressed on the surface of many bladder carcinoma cells shows potential to be developed as a therapeutic strategy, and patients identified with a high risk of progression may benefit from adjuvant targeted radionuclide therapy. A phage-display selected Affibody molecule (Z(HER2:342)) which binds to HER2/neu with picomolar affinity, can be used for targeting HER2/neu-expressing bladder carcinomas. A DOTA-derivative of Z(HER2:342), designated as DOTA-Z(HER2:342)-3, is considered as a suitable targeting agent for therapy. The DOTA chelator provides stable labelling with radiometals, and the low molecular weight (7.2 kDa) of the DOTA-Z(HER2:342)-3 compound is expected to enable efficient tumor penetration. DOTA-Z(HER2:342)-3 was radiolabelled with 90Y and 177Lu in 1 M ammonium acetate buffer, at pH 5.5, and in the presence of ascorbic acid. Nearly quantitative labelling yields were achieved for both nuclides after 15 min of incubation at 60 degrees C. After chelation, the conjugates retained their capacity to specifically bind to HER2/neu-expressing SKOV-3 cells. The radiolabelled affibody conjugate (DOTA-Z(HER2:342)-3) demonstrated high antigen-binding capacity and good cellular retention. Biodistribution in normal mice demonstrated low uptake in all organs and tissues except for kidneys.

Preparation and evaluation of (68)Ga-DOTA-hEGF for visualization of EGFR expression in malignant tumors.

UNLABELLED: Detection of epidermal growth factor receptor (EGFR) overexpression in many carcinomas provides important diagnostic information, which can influence patient management. The use of PET may enable such detection in vivo by a noninvasive procedure with high sensitivity. The aim of this study was to develop a method for preparation of a positron-emitting tracer based on a natural ligand to EGFR, the recombinant human epidermal growth factor (hEGF), and to perform a preclinical evaluation of the tracer. METHODS: DOTA-hEGF (DOTA is 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid) was prepared by coupling of a N-sulfosuccinimide ester of DOTA to hEGF. The conjugate was labeled with a generator-produced positron-emitting nuclide, (68)Ga (half-life = 68 min), using microwave heating. Binding specificity, affinity, internalization, and retention of (68)Ga-DOTA-hEGF was studied in 2 EGFR-expressing cell lines, U343 glioma cells and A431 cervical carcinoma cells. Biodistribution and microPET visualization studies were performed in BALB/c nu/nu mice bearing A431 carcinoma xenografts. RESULTS: A 1-min-long microwave-assisted labeling provided radioactivity incorporation of 77% +/- 4%. Both cell lines demonstrated receptor-specific uptake of the conjugate, rapid internalization of the tracer, and good retention of radioactivity. Binding to both cell lines occurred with high affinity, approximately 2 nmol/L. The biodistribution study demonstrated accumulation of radioactivity in xenografts and in EGFR-expressing organs. The microPET imaging study enabled visualization of tumors and demonstrated quick--within 5 min--localization of radioactivity in tumors. CONCLUSION: (68)Ga-DOTA-hEGF has potential for imaging EGFR overexpression in tumors.

[99mTc] HYNIC-hEGF, a potential agent for imaging of EGF receptors in vivo: preparation and pre-clinical evaluation.

Expression of epidermal growth factor receptors (EGFR) has prognostic and predictive value in many kinds of tumors. Imaging of expression of EGFR in vivo may give valuable diagnostic information. The epidermal growth factor (EGF), a natural ligand, is a possible candidate for the targeting of EGFR. The present study describes a method for preparation of (99m)Tc-EGF via the hydrazinopyridine-3-carboxylic acid (HYNIC) conjugation using tricine and ethylenediamine-N,N'-diacetic acid (EDDA) as co-ligands. Both conjugates bound EGFR expressing cells with nanomolar affinity, and demonstrated good intracellular retention. The complex with EDDA demonstrated much higher stability in blood serum and during cysteine challenge. Biodistribution of (99m)Tc-EDDA-HYNIC-EGF in normal mice demonstrated fast blood clearance of conjugate, and its ability to bind EGFR in vivo. (99m)Tc-EDDA-HYNIC-EGF is a promising candidate for visualization of EGFR expression in vivo.

Targeting of a head and neck squamous cell carcinoma xenograft model using the chimeric monoclonal antibody U36 radioiodinated with a closo-dodecaborate-containing linker.

OBJECTIVE: High rates of local recurrence and distant metastases following surgery of high-grade head and neck squamous cell carcinoma (HNSCC) necessitate the use of adjuvant systemic treatment. Radioimmunotargeting might be a possible treatment modality in this case. The nuclear properties of 131I make it a suitable isotope for treatment of minimal residual disease and small metastases, but the conventional radioiodine label has poor cellular retention and its radiocatabolites accumulate in the thyroid. We attempted to overcome these problems by using closo-dodecaborate derivatives for attachment of radioiodine. MATERIAL AND METHODS: We investigated the feasibility of targeting an SCC25 HNSCC xenograft in vivo using a benzylisothiocyanate derivative of closo-dodecaborate (DABI) as radioiodine linker and the chimeric anti-CD44v6 antibody U36. 125I was used in biodistribution studies. RESULTS: The use of DABI enabled tumor targeting and decreased the radioactivity uptake of the thyroid. CONCLUSION: Tumor localization of DABI-labeled U36 was similar to its para-iodobenzoate-labeled counterpart, presumably due to the strong dependence of targeting efficiency on tumor size.

[177Lu]Bz-DTPA-EGF: Preclinical characterization of a potential radionuclide targeting agent against glioma.

Patients with glioblastoma multiforme have a poor prognosis due to recurrences originating from spread cells. The use of radionuclide targeting might increase the chance of inactivating single tumor cells with minimal damage to surrounding healthy tissue. As a target, overexpressed epidermal growth factor receptors (EGFR) may be used. A natural ligand to EGFR, the epidermal growth factor (EGF) is an attractive targeting agent due to its low molecular weight (6 kDa) and high affinity for EGFR. 177Lu (T(1/2) = 6.7 days) is a radionuclide well suited for treatment of small tumor cell clusters, since it emits relatively low-energy beta particles. The goal of this study was to prepare and preclinically evaluate both in vitro and in vivo the [177Lu]Bz-DTPA-EGF conjugate. The conjugate was characterized in vitro for its cell-binding properties, and in vivo for its pharmacokinetics and ability to target EGFR. [177Lu]Bz-DTPA-EGF bound to cultured U343 glioblastoma cells with an affinity of 1.9 nM. Interaction with EGFR led to rapid internalization, and more than 70% of the cell-associated radioactivity was internalized after 30 minutes of incubation. The retention of radioactivity was good, with more than 65% of the 177Lu still cell-associated after 2 days. Biodistribution studies of i.v. injected [177Lu]Bz-DTPA-EGF in NMRI mice demonstrated a rapid blood clearance. Most of the radioactivity was found in the liver and kidneys. The liver uptake was receptor-mediated, since it could be significantly reduced by preinjection of unlabeled EGF. In conclusion, [177Lu]Bz-DTPA-EGF seems to be a promising candidate for locoregional treatment of glioblastoma due to its high binding affinity, low molecular weight, and ability to target EGFR in vivo.

Elimination of stabilised hyaluronan from the knee joint in healthy men.

OBJECTIVE: To investigate the elimination of stabilised hyaluronan following intra-articular injection into the knee joint. DESIGN: This was a single-centre, single-dose study in healthy human volunteers. PARTICIPANTS: Six healthy men, aged 28 to 47 (mean 38) years, were enrolled in the study. METHODS: Subjects received a single intra-articular injection (3ml; 20 mg/ml) of (131)I-labelled non-animal stabilised hyaluronic acid (NASHA). Radioactivity in the knee, blood, urine and over the liver was measured with gamma counters for 3 weeks post-injection; magnetic resonance and gamma camera imaging of the knee were also performed at 24 hours post-injection. Radioactivity uptake data were tested for conformity of fit to different mathematical models. RESULTS: Elimination of (131)I-labelled NASHA from the knee was characterised by a fast initial phase and a slow late phase, and conformed to a three-exponential-function model with elimination half-lives of 1.5 hours, 1.5 days and 4 weeks. Radioactivity distribution within the knee joint was homogenous, and no local leakage was observed. Hepatic radioactivity uptake was low, but significantly above background levels, for the first 2 days post-injection, before declining to background levels. Visual imaging indicated an increase in intra-articular fluid volume at 24 hours post-injection. CONCLUSIONS: The elimination kinetics of (131)I-labelled NASHA from the human knee joint were described by three distinct phases, with half-times of 1.5 hours, 1.5 days and 4 weeks. Most likely, the last value reflects the true half-life of NASHA following intra-articular injection since the labelling method used causes minimal modification of hyaluronan.