Evaluation of a novel monoclonal antibody against tumor-associated MUC1 for diagnosis and prognosis of breast cancer.

There is still a great unmet medical need concerning diagnosis and treatment of breast cancer which could be addressed by utilizing specific molecular targets. Tumor-associated MUC1 is expressed on over 90 % of all breast cancer entities and differs strongly from its physiological form on epithelial cells, therefore presenting a unique target for breast cancer diagnosis and antibody-mediated immune therapy. Utilizing an anti-tumor vaccine based on a synthetically prepared glycopeptide, we generated a monoclonal antibody (mAb) GGSK-1/30, selectively recognizing human tumor-associated MUC1. This antibody targets exclusively tumor-associated MUC1 in the absence of any binding to MUC1 on healthy epithelial cells thus enabling the generation of breast tumor-specific radiolabeled immune therapeutic tools. Methods: MAb GGSK-1/30 was used for immunohistochemical analysis of human breast cancer tissue. Its desferrioxamine (Df')-conjugate was synthesized and labelled with 89Zr. [89Zr]Zr-Df'-GGSK-1/30 was evaluated as a potential PET tracer. Binding and pharmacokinetic properties of [89Zr]Zr-Df'-GGSK-1/30 were analyzed in vitro using human and murine cell lines that express tumor-associated MUC1. Self-generated primary murine breast cancer cells expressing human tumor-associated MUC1 were transplanted subcutaneously in wild type and human MUC1-transgenic mice. The pharmacology of [89Zr]Zr-Df'-GGSK-1/30 was investigated using breast tumor-bearing mice in vivo by PET/MRT imaging as well as by ex vivo organ biodistribution analysis. Results: The mAb GGSK-1/30 stained specifically human breast tumor tissue and can be possibly used to predict the severity of disease progression based on the expression of the tumor-associated MUC1. For in vivo imaging, the Df'-conjugated mAb was radiolabeled with a radiochemical yield of 60 %, a radiochemical purity of 95 % and an apparent specific activity of 6.1 GBq/µmol. After 7 d, stabilities of 84 % in human serum and of 93 % in saline were observed. In vitro cell studies showed strong binding to human tumor-associated MUC1 expressing breast cancer cells. The breast tumor-bearing mice showed an in vivo tumor uptake of >50 %ID/g and clearly visible specific enrichment of the radioconjugate via PET/MRT. Principal conclusions: Tumor-associated MUC1 is a very important biomarker for breast cancer next to the traditional markers estrogen receptor (ER), progesterone receptor (PR) and HER/2-neu. The mAb GGSK-1/30 can be used for the diagnosis of over 90% of breast cancers, including triple negative breast cancer based on biopsy staining. Its radioimmunoconjugate represents a promising PET-tracer for breast cancer imaging selectively targeting breast cancer cells.

Equilibrium, Kinetic and Structural Properties of Gallium(III) and Some Divalent Metal Complexes Formed with the New DATAm and DATA5m Ligands.

The development of 68 Ge/68 Ga generators has made the positron-emitting 68 Ga isotope widely accessible and raised interest in new chelate complexes of Ga3+ . The hexadentate 1,4-di(acetate)-6-methyl[amino(methyl)acetate]perhydro-1,4-diazepane (DATAm ) ligand and its bifunctional analogue, 1,4-di(acetate)-6-pentanoic acid[amino(methyl)acetate]perhydro-1,4-diazepane (DATA5m ), rapidly form complexes with 68 Ga in high radiochemical yield. The stability constants of DATAm and DATA5m complexes formed with Ga3+ , Zn2+ , Cu2+ , Mn2+ and Ca2+ have been determined by using pH potentiometry, spectrophotometry (Cu2+ ) and 1 H and 71 Ga NMR spectroscopy (Ga3+ ). The stability constants of Ga(DATAm ) and Ga(DATA5m ) complexes are slightly higher than those of Ga(AAZTA). The species distribution calculations indicated the predominance of Ga(L)OH mixed-hydroxo complexes at physiological pH. The 1 H and 71 Ga NMR spectroscopy studies provided information about the coordinated functional groups of ligands and on the kinetics of exchange between the Ga(L) and Ga(L)OH complexes. The transmetalation reactions between the Ga(L) complexes and Cu2+ citrate (6

Acupuncture at the auricular branch of the vagus nerve enhances heart rate variability in humans: An exploratory study.

BACKGROUND: Recent animal and human studies have shown antiarrhythmic effects inhibiting inducibility of atrial fibrillation through low-level transcutaneous electrical stimulation at the auricular branch of the vagus nerve (ABVN). OBJECTIVE: The present study investigated effects of acupuncture at the ABVN on the autonomic cardiac nervous system in humans through analysis of heart rate and heart rate variability (HRV) parameters. METHODS: We enrolled 24 healthy male volunteers and compared acupuncture at the ABVN to placebo-acupuncture performed at the Ma-35 point (an acupuncture point used in traditional Chinese medicine to treat pain caused by gonarthrosis). An additional measurement without acupuncture served as control. We analyzed the following heart rate and HRV parameters: standard deviation of normal-to-normal intervals (SDNN), root mean square of successive R-R interval differences (RMSSD), high frequency (HF), low frequency (LF), LF/HF ratio. RESULTS: In comparison to placebo acupuncture, acupuncture at the ABVN led to a significant reduction in heart rate (approximately 4%-6%, P < .05) and an increase in overall HRV demonstrated by SDNN (approximately 19%, P < .05). RMSSD and power spectral density parameters (HF, LF, LF/HF) showed statistical trends (P < .1) induced by auricular acupuncture in favor of vagal tone. No relevant difference was shown between control and placebo group. CONCLUSION: Acupuncture of the region innervated by the ABVN may activate the parasympathetic nervous system, as suggested by reduction in heart rate and increase in SDNN. However, given the lack of clear significant changes in other HRV parameters, this effect seems modest and its evaluation requires further investigation.

AAZTA5/AAZTA5-TOC: synthesis and radiochemical evaluation with 68Ga, 44Sc and 177Lu.

PURPOSE: AAZTA (1,4-bis (carboxymethyl)-6-[bis (carboxymethyl)]amino-6-methylperhydro-1,4-diazepine) based chelators were initially developed in the context of magnetic resonance imaging. First radiochemical studies showed the capability of AAZTA to form stable complexes with radiolanthanides and moderately stable complexes with 68Ga. For a systematic comparison of the labelling capabilities with current diagnostic and therapeutic trivalent radiometals, AAZTA5 (1,4-bis (carboxymethyl)-6-[bis (carboxymethyl)]amino-6-[pentanoic-acid]perhydro-1,4-diazepine) was synthesized representing a bifunctional version with a pentanoic acid at the carbon-6 atom. To evaluate the effect of adding a targeting vector (TV) to the bifunctional chelator on the complex formation, AAZTA5-TOC was synthesized, radiolabeled and tested in comparison to the uncoupled AAZTA5. METHODS: AAZTA5 was synthesized in a 5-step synthesis. It was coupled to the cyclic peptide TOC (Phe1-Tyr3 octreotide) via amide bound formation. AAZTA and AAZTA5-TOC complex formations with 68Ga, 44Sc and 177Lu were investigated at different pH, temperature and precursor amounts. Stability studies against human serum, PBS buffer, EDTA and DTPA were performed. RESULTS: AAZTA5 and AAZTA5-TOC achieved quantitative labelling (> 95%) at room temperature in less than 5 min with all three nuclides at pH ranges from 4 to 5.5 with low precursor amounts of 1 to 10 nmol. [44Sc]Sc-AAZTA5 complexes as well as [44Sc]Sc-AAZTA5-TOC were completely stable. The 177Lu complexes of AAZTA5 and AAZTA5-TOC showed high stability comparable to the 44Sc complexes. In contrast, the [68Ga]Ga-AAZTA5 complex stability was rather low, but interestingly, [68Ga]Ga-AAZTA5-TOC was completely stable. CONCLUSION: AAZTA5 appears to be a promising bifunctional chelator for 68Ga, 44Sc and 177Lu with outstanding labelling capabilities at room temperature. Complex stabilities are high in the case of 44Sc and 177Lu. While [68Ga]Ga-AAZTA complexes alone lacking stability, [68Ga]Ga-AAZTA5-TOC demonstrated high stability. The latter indicates an interesting feature of [68Ga]Ga-AAZTA5-labelled radiopharmaceuticals.

Instant kit preparation of 68Ga-radiopharmaceuticals via the hybrid chelator DATA: clinical translation of [68Ga]Ga-DATA-TOC.

PURPOSE: The widespread use of 68Ga for positron emission tomography (PET) relies on the development of radiopharmaceutical precursors that can be radiolabelled and dispensed in a simple, quick, and convenient manner. The DATA (6-amino-1,4-diazapine-triacetate) scaffold represents a novel hybrid chelator architecture possessing both cyclic and acyclic character that may allow for facile access to 68Ga-labelled tracers in the clinic. We report the first bifunctional DATA chelator conjugated to [Tyr3]octreotide (TOC), a somatostatin subtype 2 receptor (SST2)-targeting vector for imaging and functional characterisation of SSTR2 expressing tumours. METHODS: The radiopharmaceutical precursor, DATA-TOC, was synthesised as previously described and used to complex natGa(III) and 68Ga(III). Competition binding assays of [natGa]Ga-DATA-TOC or [natGa]Ga-DOTA-TOC against [125I-Tyr25]LTT-SS28 were conducted in membranes of HEK293 cells transfected to stably express one of the hSST2,3,5 receptor subtypes (HEK293-hSST2/3/5 cells). First in vivo studies were performed in female NMRI-nude mice bearing SST2-positive mouse phaeochromocytoma mCherry (MPC-mCherry) tumours to compare the in vivo SST2-specific tumour-targeting of [68Ga]Ga-DATA-TOC and its overall pharmacokinetics versus the [68Ga]Ga-DOTA-TOC reference. A direct comparison of [68Ga]Ga-DATA-TOC with the well-established PET radiotracer [68Ga]Ga-DOTA-TOC was additionally performed in a 46-year-old male patient with a well-differentiated NET (neuroendocrine tumour), representing the first in human administration of [68Ga]Ga-DATA-TOC. RESULTS: DATA-TOC was labelled with 68Ga with a radiolabelling efficiency of > 95% in less than 10 min at ambient temperature. A molar activity up to 35 MBq/nmol was achieved. The hSST2-affinities of [natGa]Ga-DATA-TOC and [natGa]Ga-DOTA-TOC were found similar with only sub-nanomolar differences in the respective IC50 values. In mice, [68Ga]Ga-DATA-TOC was able to visualise the tumour lesions, showing standardised uptake values (SUVs) similar to [68Ga]Ga-DOTA-TOC. Direct comparison of the two PET tracers in a NET patient revealed very similar tumour uptake for the two 68Ga-radiotracers, but with a higher tumour-to-liver contrast for [68Ga]Ga-DATA-TOC. CONCLUSION: [68Ga]Ga-DATA-TOC was prepared, to a quality appropriate for in vivo use, following a highly efficient kit type process. Furthermore, the novel radiopharmaceutical was comparable or better than [68Ga]Ga-DOTA-TOC in all preclinical tests, achieving a higher tumour-to-liver contrast in a NET-patient. The results illustrate the potential of the DATA-chelator to facilitate the access to and preparation of 68Ga-radiotracers in a routine clinical radiopharmacy setting.

Novel bifunctional DATA chelator for quick access to site-directed PET 68Ga-radiotracers: preclinical proof-of-principle with [Tyr3]octreotide.

Molecular imaging of tumors with the PET radionuclide 68Ga has gained momentum in clinical oncology due to the expanding availability of commercial 68Ge/68Ga-generators in combination with state-of-the-art PET/CT and PET/MRI hybrid imaging systems. Concurrently, interesting peptide-based or small-size vectors have been developed for theranostic use in cancer patients. Owing to the short half-life of 68Ga (t1/2 = 67.7 min) and the sensitivity of many targeting biomolecules, labeling and kit reconstitution in mild conditions allowing for quick access to ready-for-injection PET-tracers are highly desirable. The novel DATA ((6-pentanoic acid)-6-(amino)methy-1,4-diazepinetriacetate) chelator previously showing promising qualities for kit type labeling, was coupled to TOC ([Tyr3]octreotide). We herein report results from a first proof-of-principle study directly comparing 67Ga-DATA-TOC with the well-established 67Ga-DOTA-TOC in a series of preclinical models. Both analogs were shown to be sst2-preferring and specifically internalized in AR42J and HEK293-hsst2 cells, with 67Ga-DOTA-TOC internalizing faster in both cell lines. Similarly, after injection in mice bearing either AR42J or HEK293-hsst2 tumors, both tracers efficiently and specifically localized in the implants. Whereas 67Ga-DOTA-TOC exhibited higher tumor values, 67Ga-DATA-TOC cleared faster from background tissues. These findings support the suitability of the newly introduced bifunctional chelator DATA as a reliable, quick and convenient means for labeling medically relevant vectors with the PET radiometal 68Ga.