Significantly lower tumour accumulation was observed in competitive assays and DU145 xenografts
Significantly lower tumour accumulation was observed in competitive assays and DU145 xenografts. lower tumour accumulation was observed in competitive assays and DU145 xenografts. SPECT/CT imaging could clearly visualize the subcutaneous and intra-tibial LNCaP xenografts. Conclusions Our study demonstrates the potential of 111In-DTPA-11B6 for the detection of metastatic prostate cancer and monitoring anti-androgen therapy, as it exhibits an increased uptake and accumulation in viable tumour when compared to normal tissue. A humanised version of the 11B6 monoclonal antibody is currently under evaluation. Electronic supplementary material The online version of this article (doi:10.1186/s13550-014-0051-5) contains supplementary material, which is available to authorized users. and located on chromosome 19, respectively, and are Engeletin well characterized as AR-regulated genes [14-16]. These kallikreins are produced by the same secretory luminal cells in the prostate and share an 80% amino acid homology, as well as several structural similarities [15-17]. Importantly, the PSA and hK2 antigens are abundantly expressed in malignant prostate tissue throughout all clinical stages. Recent publications on the 5A10  and PSA30 antibodies  have explored the concept of PSA imaging, including targeting the free, unbound forms of prostate-specific antigen (fPSA). Results from animal studies using the PSA30 antibody showed a selective uptake in LNCaP tumours hK2 imaging with 111In-labelled 11B6 in AR-positive LNCaP xenografts. Methods Antibody conjugation and radiolabelling The murine monoclonal antibody, 11B6, was first described and characterized by Vaisanen et al.  and was provided by the University of Turku (Turku, Finland) for this study. Conjugation and radiolabelling was performed as previously described by Tolmachev et al. . Briefly, 2 mg of 11B6 was conjugated with the chelator CHX-A-DTPA (B-355, Macrocyclics; Dallas, TX, USA) through the isothiocyanate functional group. A solution of 11B6 (4 to 5 mg/mL in PRKCG PBS) was adjusted to pH 9.2 using 0.07 M sodium borate buffer (Sigma Aldrich; St. Louis, MO, USA). CHX-A-DTPA was then added to the protein solution at a molar ratio of 3:1 (chelator to antibody) and incubated at 40C with gentle shaking. The reaction was terminated after 4 h, and CHX-A-DTPA-11B6, henceforth referred to as DTPA-11B6, was separated from the free chelate by size-exclusion chromatography on a NAP-5 column (GE Healthcare; Uppsala, Sweden) equilibrated with 20 mL of 0.2 M ammonium acetate buffer (Sigma Aldrich), pH 5.5. Conjugated 11B6 was eluted with 1 mL of ammonium acetate buffer, and aliquoted samples were stored at ?20C. For radiolabelling, approximately 125 L of DTPA-11B6 (approximately 1 g/L in 0.2 M ammonium acetate buffer pH 5.5) was mixed with a predetermined amount (approximately 50 to 100 MBq) of 111InCl3 (Mallinckrodt Medical; Dublin, Ireland), incubated at room temperature for 1.5 to 2 h and then purified on a NAP-5 column (GE Healthcare) equilibrated with PBS (Thermo Scientific; Waltham, MA, USA). Labelling efficiency and kinetics were monitored by instant thin-layer chromatography (ITLC) (Biodex, Shirley, NY, USA) eluted with 0.2 M citric acid (Sigma Aldrich). In this system, the radiolabelled Engeletin conjugate remains at the origin line, while free 111In and 111In-DTPA migrate with the solvent front. The radioactive distribution was determined using a Cyclone Storage Phosphor System with Optiquant quantification software (Perkin Elmer; Waltham, MA, USA). Binding kinetics with surface plasmon resonance The 11B6 binding kinetics were analysed by surface plasmon resonance using a Biacore 2000 (Biacore AB; Uppsala, Sweden). The affinity of 11B6 to hK2 before and after CHX-A-DTPA conjugation was determined. The hK2 antigen, provided by the University of Turku (Department of Biotechnology; Turku, Finland), was produced and purified as previously Engeletin described . hK2 antigen (25.9 g/mL in 10 mM sodium acetate buffer pH 4.0 (Sigma Aldrich)) was immobilized on a CM4 research grade chip (Biacore AB) by amino coupling using N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 1 M ethanolamine hydrochloride-NaOH, pH 8.5, in a Biacore 2000 system. Samples were flown over two flow cells, one being a blank reference, in five different concentrations ranging from 0.5 to 100 nM to detect eventual binding. One of the two flow cells contained immobilized hK2, while the other was served as a blank reference. The binding kinetics were studied in a 3-min-long association phase and a 15-min-long dissociation phase with a flow rate of 30 L/min, followed by regeneration with 25 mM glycine buffer pH 2.7. Kinetic constants were calculated using a 1:1 Langmuir binding model with correction for mass transfer. BIAEvaluation 4.1 software (Biacore AB) was used for calculations. Stability studies The stability of.