High radiolabeling efficiency, preferably to high specific activity, and good stability of the radioimmunoconjugate are essential features for a successful immunoconjugate for imaging or therapy. In this study, the radiolabeling efficiency, in vitro stability and biodistribution of immunoconjugates with eight different bifunctional chelators labeled with 64 Cu were compared. The anti-CD20 antibody, rituximab, was conjugated to four macrocyclic bifunctional chelators (p-SCN-Bn-DOTA, p- , three DTPA derivatives (p-SCN-Bn-DTPA, p-SCN-CHX-A"-DTPA and ITC-2B3M-DTPA) and a macrobicyclic hexamine ("sarcophagine") chelator (sar-CO 2 H) = (1-NH 2 -8-NHCO(CH 2 ) 3 CO 2 H)sar where sar = sarcophagine = 3, 6,10,13,16,19-hexaazabicyclo[6.6.6]icosane). Radiolabeling efficiency under various conditions, in vitro stability in serum at 37°C and in vivo biodistribution and imaging in normal mice over 48 h were studied. All chelators except sar-CO 2 H were conjugated to rituximab by thiourea bond formation with an average of 4.9 +/− 0.9 chelators per antibody molecule. Sar-CO 2 H was conjugated to rituximab by amide bond formation with 0.5 chelators per antibody molecule. Efficiencies of 64 Cu radiolabeling were dependent on the concentration of immunoconjugate. Notably, the 64 Cu-NOTA-rituximab conjugate demonstrated highest radiochemical yield (95%) under very dilute conditions (31 nM NOTA-rituximab conjugate). Similarly, sar-CO-rituximab, containing 1/10 th the number of chelators per antibody compared to other conjugates retained high labeling efficiency (98 %) at an antibody concentration of 250 nM. In contrast to the radioimmunoconjugates containing DTPA derivatives, which demonstrated poor serum stability, all macrocyclic radioimmunoconjugates were very stable in serum with <6 % dissociation of 64 Cu over 48 h. In vivo biodistribution profiles in normal female Balb/C mice were similar for all the macrocyclic radioimmunoconjugates with most of the activity remaining in the blood pool up to 48 h. Whilst all the macrocyclic bifunctional chelators are suitable for molecular imaging using 64 Cu-labeled antibody conjugates, NOTA and sar-CO 2 H show significant advantages over the others in that they can be radiolabeled rapidly at room temperature, under dilute conditions resulting in high specific activity. Europe PMC Funders Group
Novel approaches for the treatment of multidrug-resistant Gram-negative bacterial infections are urgently required. One approach is to potentiate the efficacy of existing antibiotics whose spectrum of activity is limited by the permeability barrier presented by the Gram-negative outer membrane. Cationic peptides derived from polymyxin B have been used to permeabilize the outer membrane, granting antibiotics that would otherwise be excluded access to their targets. We assessed the in vitro efficacies of combinations of SPR741 with conventional antibiotics against Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii. Of 35 antibiotics tested, the MICs of 8 of them were reduced 32-to 8,000-fold against E. coli and K. pneumoniae in the presence of SPR741. The eight antibiotics, azithromycin, clarithromycin, erythromycin, fusidic acid, mupirocin, retapamulin, rifampin, and telithromycin, had diverse targets and mechanisms of action. Against A. baumannii, similar potentiation was achieved with clarithromycin, erythromycin, fusidic acid, retapamulin, and rifampin. Susceptibility testing of the most effective antibiotic-SPR741 combinations was extended to 25 additional multidrug-resistant or clinical isolates of E. coli and K. pneumoniae and 17 additional A. baumannii isolates in order to rank the potentiated antibiotics. SPR741 was also able to potentiate antibiotics that are substrates of the AcrAB-TolC efflux pump in E. coli, effectively circumventing the contribution of this pump to intrinsic antibiotic resistance. These studies support the further development of SPR741 in combination with conventional antibiotics for the treatment of Gram-negative bacterial infections.
BackgroundTo date, the majority of protein-based radiopharmaceuticals have been radiolabelled using non-site-specific conjugation methods, with little or no control to ensure retained protein function post-labelling. The incorporation of a hexahistidine sequence (His-tag) in a recombinant protein can be used to site-specifically radiolabel with 99mTc-tricarbonyl ([99mTc(CO)3]+). This chemistry has been made accessible via a technetium tricarbonyl kit; however, reports of radiolabelling efficiencies and specific activities have varied greatly from one protein to another. Here, we aim to optimise the technetium tricarbonyl radiolabelling method to produce consistently >95% radiolabelling efficiencies with high specific activities suitable for in vivo imaging.MethodsFour different recombinant His-tagged proteins (recombinant complement receptor 2 (rCR2) and three single chain antibodies, α-CD33 scFv, α-VCAM-1 scFv and α-PSMA scFv), were used to study the effect of kit volume, ionic strength, pH and temperature on radiolabelling of four proteins.ResultsWe used 260 and 350 μL [99mTc(CO)3]+ kits enabling us to radiolabel at higher [99mTc(CO)3]+ and protein concentrations in a smaller volume and thus increase the rate at which maximum labelling efficiency and specific activity were reached. We also demonstrated that increasing the ionic strength of the reaction medium by increasing [Na+] from 0.25 to 0.63 M significantly increases the rate at which all four proteins reach a >95% labelling efficiency by at least fourfold, as compared to the conventional IsoLink® kit (Covidien, Petten, The Netherlands) and 0.25 M [Na+].ConclusionWe have found optimised kit and protein radiolabelling conditions suitable for the reproducible, fast, efficient radiolabelling of proteins without the need for post-labelling purification.
BackgroundSensitive and specific detection of nodal status, sites of metastases and low-volume recurrent disease could greatly improve management of patients with advanced prostate cancer. Prostate-specific membrane antigen (PSMA) is a well-established marker for prostate carcinoma with increased levels of expression in high-grade, hormone-refractory and metastatic disease. The monoclonal antibody (mAb) J591 is directed against an extracellular epitope of PSMA and has been shown to efficiently target disseminated disease including metastases in lymph nodes and bone. Its use as a diagnostic imaging agent however is limited due to its slow pharmacokinetics. In this study a diabody derived from mAb J591 was developed as a single photon emission computed tomography (SPECT) tracer with improved pharmacokinetics for the detection of PSMA expression in prostate cancer.MethodsA diabody in VH-VL orientation and with a C-terminal cysteine was expressed in HEK293T cells and purified by a combination of metal ion affinity and size exclusion chromatography. Specificity and affinity were determined in cell binding studies. For SPECT imaging, the diabody was site-specifically labelled with [99mTc(CO)3]+ via the C-terminal His tag and evaluated in a subcutaneous DU145/DU145-PSMA prostate carcinoma xenograft model.ResultsJ591C diabody binds to PSMA-expressing cells with low nanomolar affinity (3.3 ± 0.2 nM). SPECT studies allowed imaging of tumour xenografts with high contrast from 4 h post injection (p.i.). Ex vivo biodistribution studies showed peak tumour uptake of the tracer of 12.1% ± 1.7% injected dose (ID)/g at 8 h p.i. with a tumour to blood ratio of 8.0. Uptake in PSMA-negative tumours was significantly lower with 6.3% ± 0.5% at 8 h p.i. (p < 0.001).ConclusionThe presented diabody has favourable properties required to warrant its further development for antibody-based imaging of PSMA expression in prostate cancer, including PSMA-specific uptake, favourable pharmacokinetics compared to the parental antibody and efficient site-specific radiolabelling with 99mTc.
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