UNLABELLED: Quantitative imaging and dosimetry are crucial for individualized treatment during peptide receptor radionuclide therapy (PRRT). (177)Lu-DOTATATE and (68)Ga-DOTATOC/(68)Ga-DOTATATE are used, respectively, for PRRT and PET examinations targeting somatostatin receptors (SSTRs) in patients affected by neuroendocrine tumors. The aim of the study was to quantitatively and qualitatively compare the performance of (68)Ga-DOTATOC and (68)Ga-DOTATATE in the context of subsequent PRRT with (177)Lu-DOTATATE under standardized conditions in the same patient as well as to investigate the sufficiency of standardized uptake value (SUV) for estimation of SSTR expression.

METHODS: Ten patients with metastatic neuroendocrine tumors underwent one 45-min dynamic and 3 whole-body PET/CT examinations at 1, 2, and 3 h after injection with both tracers. The number of detected lesions, SUVs in lesions and normal tissue, total functional tumor volume, and SSTR volume (functional tumor volume multiplied by mean SUV) were investigated for each time point. Net uptake rate (Ki) was calculated according to the Patlak method for 3 tumors per patient.

RESULTS: There were no significant differences in lesion count, lesion SUV, Ki, functional tumor volume, or SSTR volume between (68)Ga-DOTATOC and (68)Ga-DOTATATE at any time point. The detection rate was similar, although with differences for single lesions in occasional patients. For healthy organs, marginally higher uptake of (68)Ga-DOTATATE was observed in kidneys, bone marrow, and liver at 1 h. (68)Ga-DOTATOC uptake was higher in mediastinal blood pool at the 1-h time point (P = 0.018). The tumor-to-liver ratio was marginally higher for (68)Ga-DOTATOC at the 3-h time point (P = 0.037). Blood clearance was fast and similar for both tracers. SUV did not correlate with Ki linearly and achieved saturation for a Ki of greater than 0.2 mL/cm(3)/min, corresponding to an SUV of more than 25.

CONCLUSION: (68)Ga-DOTATOC and (68)Ga-DOTATATE are suited equally well for staging and patient selection for PRRT with (177)Lu-DOTATATE. However, the slight difference in the healthy organ distribution and excretion may render (68)Ga-DOTATATE preferable. SUV did not correlate linearly with Ki and thus may not reflect the SSTR density accurately at its higher values, whereas Ki might be the outcome measure of choice for quantification of SSTR density and assessment of treatment outcome.

Therapies targeting human epidermal growth factor receptor type 2 (HER2) have revolutionized breast cancer treatment, but require invasive biopsies and rigorous histopathology for optimal patient stratification. A non-invasive and quantitative diagnostic method such as positron emission tomography (PET) for the pre-therapeutic determination of the presence and density of the HER2 would significantly improve patient management efficacy and treatment cost. The essential part of the PET methodology is the production of the radiopharmaceutical in compliance with good manufacturing practice (GMP). The use of generator produced positron emitting (68)Ga radionuclide would provide worldwide accessibility of the agent. GMP compliant, reliable and highly reproducible production of [(68)Ga]Ga-ABY-025 with control over the product peptide concentration and amount of radioactivity was accomplished within one hour. Two radiopharmaceuticals were developed differing in the total peptide content and were validated independently. The specific radioactivity could be kept similar throughout the study, and it was 6-fold higher for the low peptide content radiopharmaceutical. Intrapatient comparison of the two peptide doses allowed imaging optimization. The high peptide content decreased the uptake in healthy tissue, in particular liver, improving image contrast. The later imaging time points enhanced the contrast. The combination of high peptide content radiopharmaceutical and whole-body imaging at 2 hours post injection appeared to be optimal for routine clinical use.

Objectives: Various radionuclide-labeled somatostatin analogues are used currently for diagnosis and therapy of neuroendocrine tumors. In particular, [Ga-68]Ga-DOTA-TOC is commonly used for diagnosis, while [Lu-177]Lu-DOTA-TATE is used for therapy. With the development of theranostics and personalized medicine where the imaging diagnosis is tailored to the subsequent radiotherapy, it is of paramount importance to investigate the relevance of the ligand exchange. The aim of this study was to compare binding capacity of [Ga-67/68]Ga-DOTA-TOC ([Ga-67/68]Ga-N-(4,7,10-(tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)acetyl-D-Phe-c[Cys-D-Tyr-Trp-Lys-Thr-Cys]-Thr(ol)) and [Ga-67/68]Ga-DOTA-TATE ([Ga-67/68]Ga-N-(4,7, 10-(tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)acetyl-D-Phe-c[Cys-D-Tyr-Trp-Lys-Thr-Cys]-Thr) in vitro in monkey brain cryosections and in vivo in the rat, where, in contrast to transfected cell lines, there is a heterogeneous distribution of somatostatin receptor (SSTR) subtypes. The influence of various production methods of [Ga-68]Ga-DOTA-TOC and [Ga-68]Ga-DOTA-TATE on the biological performance of the tracers was also studied. Material and Methods: [Ga-67]Ga-DOTA-TOC, [Ga-68]Ga-DOTA-TOC, [Ga-67]Ga-DOTA-TATE and [Ga-68]Ga-DOTA-TATE were synthesized including preconcentration and purification of the generator eluate. The binding of the radioligands was assessed in vitro using autoradiography on cryosections of Rhesus monkey brains and in vivo/ex vivo using organ distribution studies in rats. Results and Discussion: The tracer production method was improved in terms of higher robustness, simplification and good manufacturing practice (GMP) relevance. The synthesis variation did not influence the biological performance of the tracers. There was no statistically significant difference observed in the binding of [Ga-67/68]Ga-DOTA-TOC and [Ga-67/68]Ga-DOTA-TATE either in brain cortex in vitro or in rat biodistribution and uptake in SSTR-positive tissues such as pancreas, adrenals and pituitary. The uptake in these organs was precluded by the excess of octreotide (Sandostatin). The 10-fold higher affinity to SSTR2 of DOTA-TATE as compared to DOTA-TOC known from studies in transfected cells was reflected in a slightly more intense binding of [Ga-67/68]Ga-DOTA-TATE than of [Ga-67/68]Ga-DOTA-TOC in the monkey brain sections in vitro, but not in vivo in the rat. Conclusion: A robust Ga-68-labeling method was introduced. The difference in the uptake of [Ga-67/68]Ga-DOTA-TOC and [Ga-67/68]Ga-DOTA-TATE in SSTR2-positive organs was not statistically significant either in vitro in tissue studies or in vivo/ex vivo in rat experiments. The results indicate that the more complex environment in vitro and in vivo diminishes the difference observed in transfected cell line binding. 

Background The number of radiolabeled monoclonal antibodies (mAbs) used for medical imaging and cancer therapy is increasing. The required chemical modification for attaching a radioactive label and all associated treatment may lead to a damaged mAb subpopulation. This paper describes a novel method, concentration through kinetics (CTK), for rapid assessment of the concentration of immunoreactive mAb and the specific radioactivity, based on monitoring binding kinetics. Methods The interaction of radiolabeled mAb with either the antigen or a general mAb binder such as Protein A was monitored in real time using the instrument LigandTracer. As the curvature of the binding trace has a distinct shape based on the interaction kinetics and concentration of the functional mAb, the immunoreactive mAb concentration could be calculated through reverse kinetic fitting of the binding curves, using software developed for this project. The specific activity, describing the degree of radioactive labeling, was determined through the use of calibrated signal intensities. Results The performance of the CTK assay was evaluated on the basis of various mAb-based interaction systems and assay formats, and it was shown that the assay can provide accurate and repeatable results for immunoreactive concentration and specific activity, with both accuracy and relative SD values below 15%. Conclusion By applying reverse kinetics on real-time binding traces it is possible to estimate the functional concentration and specific activity of radiolabeled mAb. The CTK assay may in the future be included as a complement to current quality assessment methods of radiolabeled mAbs.

Resistance has been reported to human epidermal growth factor receptor 2 (HER2)-targeted therapy with the tyrosine kinase inhibitor lapatinib and the antibody trastuzumab in metastatic gastric cancer. An alternative or complement might be to target the extracellular domain of HER2 with therapy-effective radionuclides. The fraction of patients with HER2 expression in primary tumors and major metastatic sites, e.g., lymph nodes and liver, was analyzed to evaluate the potential for such therapy. Samples from primary tumors and lymph node and liver metastases were taken from each patient within a few hours, and to our knowledge, such sampling is unique. The number of analyzed cases was therefore limited, since patients that had received preoperative radiotherapy, chemotherapy, or HER2-targeted therapy were excluded. From a large number of considered patients, only 29 could be included for HER2 analysis. Intracellular mutations were not analyzed since they are assumed to have no or minor effect on the extracellular binding of molecules that deliver radionuclides. HER2 was positive in nearly 52 % of the primary tumors, and these expressed HER2 in corresponding lymph node and liver metastases in 93 and 100 % of the cases, respectively. Similar values for primary tumors and also good concordance with metastases have been indicated in the literature. Thus, relevant radionuclides and targeting molecules for nuclear medicine-based noninvasive, whole-body receptor analysis, dose planning, and therapy can be applied for many patients; see "Discussion" Hopefully, more patients can then be treated with curative instead of palliative intention.

Affibody molecules are a recently developed class of targeting proteins based on a nonimmunoglobulin scaffold. The small size (7 kDa) and subnanomolar affinity of Affibody molecules enables high-contrast imaging of tumor-associated molecular targets, particularly human epidermal growth factor receptor type 2 (HER2). ^99mTc as a label offers advantages in clinical practice, and earlier studies demonstrated that ^99mTc-labeled recombinant Affibody molecules with a C-terminal cysteine could be used for HER2 imaging. However, the renal retention of radioactivity exceeded tumor uptake, which might complicate imaging of metastases in the lumbar region. The aim of this study was to develop an agent with low renal uptake and preserved tumor targeting.

Methods:

A series of recombinant derivatives of the HER2-binding Z_HER2:342 Affibody molecule with a C-terminal chelating sequence, –GXXC (X denoting glycine, serine, lysine, or glutamate), was designed. The constructs were labeled with ^99mTc and evaluated in vitro and in vivo.

Results:

All variants were stably labeled with ^99mTc, with preserved capacity to bind specifically to HER2-expressing cells in vitro and in vivo. The composition of the chelating sequence had a clear influence on the cellular processing and biodistribution properties of the Affibody molecules. The best variant, ^99mTc-Z_HER2:V2, with the C-terminal chelating sequence –GGGC, provided the lowest radioactivity retention in all normal organs and tissues including the kidneys. ^99mTc-Z_HER2:V2 displayed high uptake of radioactivity in HER2-expressing xenografts, 22.6 ± 4.0 and 7.7 ± 1.5 percentage injected activity per gram of tissue at 4 h after injection in SKOV-3 (high HER2 expression) and DU-145 (low HER2 expression) tumors, respectively. In both models, the tumor uptake exceeded the renal uptake.

Conclusion:

These results demonstrate that the biodistribution properties of recombinant ^99mTc-labeled Affibody molecules can be optimized by modification of the C-terminal cysteine-containing chelating sequence. ^99mTc-Z_HER2:V2 is a promising candidate for further development as a diagnostic radiopharmaceutical for imaging of HER2-expressing tumors. These results may be useful for the development of imaging agents based on other Affibody molecules and, hopefully, other scaffolds.

Affibody molecules generated by combinatorial protein engineering to bind the human epidermal growth factor receptor 2 (HER2) have in earlier studies proven to be promising tracers for HER2-mediated molecular imaging of cancer. Amino acid extensions either at the N- or C-terminus of these Z(HER2) affibody molecules, have been successfully employed for site-specific radiolabeling of the tracer candidates. Hexahistidyls or other tags, which would be convenient for recovery purposes, should be avoided since they could negatively influence the tumor targeting efficacy and biodistribution properties of the tracer. Using a new ß-lactamase-based protein fragment complementation assay (PCA), an affibody molecule was isolated which bound a Z(HER2) affibody molecule with sub-micromolar affinity, but not unrelated affibody molecules. This suggests that the interacting area include the HER2-binding surface of Z(HER2). This novel anti-idiotypic affibody molecule Z(E01) was produced in Escherichia coli, purified, and chemically coupled to a chromatography resin in order to generate an affibody-based affinity column, suitable for recovery of different variants of Z(HER2) affibody molecules, having a common binding surface for HER2. Eight such Z(HER2) affibody molecules, designed for future radioimaging investigations, having different C-terminal peptide extensions aimed for radioisotope ((⁹⁹m)Tc)-chelation, were successfully produced and recovered in a single step to high purity using the anti-idiotypic affibody ligand for the affinity purification. These results clearly suggest a potential for the development of anti-idiotypic affibody-based resins for efficient recovery of related variants of a target protein that might have altered biochemical properties, thus avoiding the cumbersome design of specific recovery schemes for each variant of a target protein.

These results demonstrate that the biodistribution properties of recombinant (99m)Tc-labeled Affibody molecules can be optimized by modification of the C-terminal cysteine-containing chelating sequence. (99m)Tc-Z(HER2)(:V2) is a promising candidate for further development as a diagnostic radiopharmaceutical for imaging of HER2-expressing tumors. These results may be useful for the development of imaging agents based on other Affibody molecules and, hopefully, other scaffolds.

G protein-coupled receptors (GPCRs) are a large group of receptors of great biological and clinical relevance. Despite this, the tools for a detailed analysis of ligand-GPCR interactions are limited. The aim of this paper was to demonstrate how ligand binding to GPCRs can be followed in real-time on living cells. This was conducted using two model systems, the radiolabeled porcine peptide YY (pPYY) interacting with transfected human Y2 receptor (hY2R) and the bombesin antagonist RM26 binding to the naturally expressed gastrin-releasing peptide receptor (GRPR). By following the interaction over time, the affinity and kinetic properties such as association and dissociation rate were obtained. Additionally, data were analyzed using the Interaction Map method, which can evaluate a real-time binding curve and present the number of parallel interactions contributing to the curve. It was found that pPYY binds very slowly with an estimated time to equilibrium of approximately 12 h. This may be problematic in standard end-point assays where equilibrium is required. The RM26 binding showed signs of heterogeneity, observed as two parallel interactions with unique kinetic properties. In conclusion, measuring binding in real-time using living cells opens up for a better understanding of ligand interactions with GPCRs.