Optimal His-Tag Design for Efficient [99mTc(CO)3]+ and [188Re(CO)3]+ Labeling of Proteins for Molecular Imaging and Radionuclide Therapy by Analysis of Peptide Arrays.

Hexahistidine tags (His-tags), incorporated into recombinant proteins to facilitate purification using metal-affinity chromatography, are useful binding sites for radiolabeling with [99mTc(CO)3]+ and [188Re(CO)3]+ for molecular imaging and radionuclide therapy. Labeling efficiencies vary unpredictably, and the method is therefore not universally useful. To overcome this, we have made quantitative comparisons of radiolabeling of a bespoke Celluspots array library of 382 His-tag-containing peptide sequences with [99mTc(CO)3]+ and [188Re(CO)3]+ to identify key features that enhance labeling. A selected sequence with 10-fold enhanced labeling efficiency compared to the most effective literature-reported sequences was incorporated into an exemplar protein and compared biologically with non-optimized analogues, in vitro and in vivo. Optimal labeling with either [99mTc(CO)3]+ or [188Re(CO)3]+ required six consecutive His residues in the protein sequence, surrounded by several positively charged residues (Arg or Lys), and the presence of phosphate in the buffer. Cys or Met residues in the sequence were beneficial, to a lesser extent. Negatively charged residues were deleterious to labeling. His-tags with adjacent positively charged residues could be labeled as much as 40 times more efficiently than those with adjacent negatively charged residues. 31P NMR of [Re(CO)3(H2O)3]+ and electrophoresis of solutions of [99mTc(CO)3(H2O)3]+ suggest that phosphate bridges form between cationic residues and the cationic metal synthon during labeling. The trial optimized protein, a scFv targeted to the PSMA antigen expressed in prostate cancer, was readily labeled in >95% radiochemical yield, without the need for subsequent purification. Labeling occurred more quickly and to higher specific activity than comparable non-optimized proteins, while retaining specific binding to PSMA and prostate cancer in vivo. Thus, optimized His-tags greatly simplify radiolabeling of recombinant proteins making them potentially more widely and economically available for imaging and treating patients.

Design and preclinical evaluation of a 99mTc-labelled diabody of mAb J591 for SPECT imaging of prostate-specific membrane antigen (PSMA).

BACKGROUND: Sensitive 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. METHODS: A 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. RESULTS: J591C 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). CONCLUSION: The 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.

Comparison of (64)Cu-complexing bifunctional chelators for radioimmunoconjugation: labeling efficiency, specific activity, and in vitro/in vivo stability.

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-SCN-Bn-Oxo-DO3A, p-SCN-NOTA, and p-SCN-PCTA), three DTPA derivatives (p-SCN-Bn-DTPA, p-SCN-CHX-A″-DTPA, and ITC-2B3M-DTPA), and a macrobicyclic hexamine (sarcophagine) chelator (sar-CO2H) = (1-NH2-8-NHCO(CH2)3CO2H)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-CO2H were conjugated to rituximab by thiourea bond formation with an average of 4.9 ± 0.9 chelators per antibody molecule. Sar-CO2H 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 the highest radiochemical yield (95%) under very dilute conditions (31 nM NOTA-rituximab conjugate). Similarly, sar-CO-rituximab, containing 1/10th the number of chelators per antibody compared to that of 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. While all the macrocyclic bifunctional chelators are suitable for molecular imaging using (64)Cu-labeled antibody conjugates, NOTA and sar-CO2H show significant advantages over the others in that they can be radiolabeled rapidly at room temperature, under dilute conditions, resulting in high specific activity.