Site-Specific Conjugation of Monodispersed DOTA-PEGn to a Thiolated Diabody Reveals the Effect of Increasing PEG Size on Kidney Clearance and Tumor Uptake with Improved 64-Copper PET Imaging

Abstract

Optimal PET imaging of tumors with radiolabeled engineered antibodies requires, among other parameters, matching blood clearance and tumor uptake with the half-life of the engineered antibody. Although diabodies have favorable molecular sizes (50 kDa) for rapid blood clearance (t1/2= 30-60 min) and are bivalent, thereby increasing tumor uptake, they exhibit substantial kidney uptake as their major route of clearance, which is especially evident when they are labeled with the PET isotope 64Cu (t1/2= 12 hr). To overcome this drawback, diabodies may be conjugated to PEG, a modification that increases the apparent molecular size of the diabody and reduces kidney uptake without adversely affecting tumor uptake or the tumor to blood ratio. We show here that site specific attachment of monodispersed PEGn of increasing molecular size (n= 12, 24, and 48) can uniformly increase the apparent molecular size of the PEG-diabody conjugate, decrease kidney uptake and increase tumor uptake, the latter due to the increased residence time of the conjugate in the blood. Since the monodispersed PEGs were pre-conjugated to the chelator DOTA, the conjugates were able to bind radiometals such as 111In and 64Cu that can be used for SPECT and PET imaging, respectively. To allow conjugation of the DOTA-PEG to the diabody, the DOTA-PEG incorporated a terminal Cysteine conjugated to a vinyl sulfone moiety. In order to control the conjugation chemistry, we have engineered a surface thiolated diabody that incorporates two cysteines per monomer (four per diabody). The thiolated diabody was expressed and purified from bacterial fermentation and only needs to be reduced prior to conjugation to the DOTA-PEGn-Cys-VS. This novel imaging agent (a diabody with DOTA-PEG48-Cys-VS attached to introduced thiols) gave up to 80 %ID/g of tumor uptake with a tumor to blood ratio (T/B) of 8 at 24h when radiolabeled with 111In and 37.9% ID/g of tumor uptake (T/B= 8) at 44h when radiolabeled with 64Cu in PET imaging in an animal model. Tumor uptake was significantly improved from the 50% ID/g at 24 hours observed with diabodies that were pegylated on surface Lysine residues. Importantly, there was no loss of immunoreactivity of the site-specific Cys-conjugated diabody to its antigen (TAG-72) compared to the parent, unconjugated diabody. We propose that thiolated diabodies conjugated to DOTAylated monodisperse PEGs have the potential for superior SPECT and PET imaging in a clinical setting.

Mapping biological behaviors by application of longer-lived positron emitting radionuclides

“…Numerous attempts have been made to modify the structure of the radiotracers to increase tumor uptake and decrease kidney uptake concurrently. For example, by altering the size of a diabody with a PEG component, a high tumor uptake (37.9% ID/g) was obtained with a PEG 48 conjugate at a later time point (44 hour) than with a smaller- or non-PEG diabody, along with a high tumor-to-background ratio [117]. Importantly, no interference from the kidneys was observed in PET imaging.…”

Different radiolabelling methods alter the pharmacokinetic and biodistribution properties of Plasminogen Activator Inhibitor Type 2 (PAI-2) forms

“…Addition of PEG groups to targeting moieties has the significant effect of increasing the residence time in the blood stream (due to retardation of renal clearance), hence increasing the bioavailability and clinical potency of the drug [25,26]. This approach has also been used to increase tumour uptake while decreasing kidney uptake of radiolabeled diabodies for improved SPECT imaging purposes [27].…”

A novel bifunctional chelating agent based on bis(hydroxamamide) for 99mTc labeling of polypeptides

“…Many polypeptides such as antibodies, single‐chain Fv fragments, diabodies, affibodies, minibodies, and bioactive peptides have been used as scaffolds of radiolabeled probes for targeted imaging of sites of tumors, infection, and thrombosis . Among radionuclides for radiolabeling of these polypeptides, 99m Tc is ideal for scintigraphic imaging because of its excellent physical properties, low cost, and ready availability .…”