We have recently described the in vivo properties of an iodinated anti-p185 HER2 engineered antibody fragment [minibody (scFv-C H 3) 2 ; 80 kDa], made from the internalizing 10H8 monoclonal antibody. Although the 10H8 minibody showed excellent binding to the target in vitro, only modest tumor uptake [5.6 F 1.7% injected dose per gram (ID/g) of tissue] was achieved in nude mice bearing MCF7/HER2 breast cancer tumors. Here, in an attempt to improve targeting, the 10H8 minibody was conjugated to 1,4,7,10-tetraazacyclododecane-N , N V , N V V , N

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.

Radiolabeled anti-carcinoembryonic antigen (CEA) antibodies have the potential to give excellent images of a wide variety of human tumors, including tumors of the colon, breast, lung, and medullar thyroid. In order to realize the goals of routine and repetitive clinical imaging with anti-CEA antibodies, it is necessary that the antibodies have high affinity for CEA, low cross reactivity and uptake in normal tissues, and low immunogenicity. The humanized anti-CEA antibody hT84.66-M5A (M5A) fulfills these criteria with an affinity constant >10 10 M −1 , no reactivity with CEA crossreacting antigens found in normal tissues, and >90% human protein sequence. A further requirement for routine clinical use of radiolabeled antibodies is a versatile method of radiolabeling that allows the use of multiple radionuclides that differ in their radioemissions and half-lives. We describe a versatile bifunctional chelator, DO3A-VS (1, 4, 7-tris(carboxymethyl)-10-(vinylsulfone)-1, 4, 7, 10-tetraazacyclododecane) that binds a range of radiometals including 111 In for gamma-ray imaging and 64 Cu for Positron Emission Tomography (PET), and which can be conjugated with negligible loss of immunoreactivity either to sulfhydryls (SH) in the hinge region of lightly reduced immunoglobulins or surface lysines (NH) of immunoglobulins.Methods-Athymic mice peripherally xenografted with CEA-positive human colon tumors (LS-174T) were injected with 111 In-labeled or 64 Cu-labeled SH-DO3A-VS-M5A, NH-DO3A-VS-M5A, or DOTA-M5A and sacrificed at various time points for biodistribution measurements. Other mice injected with 64 Cu-labeled SH-DO3A-VS-M5A or NH-DO3A-VS-M5A were imaged serially with small animal PET from 1 to 48 h post injection and then sacrificed for biodistribution measurements.Results-Virtually identical biodistributions were obtained for SH-and NH-DO3A-VS-M5A or DOTA-M5A whether radiolabeled with 111 In or 64 Cu. Rapid tumor uptake of radiolabel was observed, reaching 40% injected dose/gram or more by 48 h. Importantly, excellent PET images of tumor were obtained as early as 22 h after injection of 64 Cu-labeled SH-or NH-DO3A-VS-M5A. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptConclusions-Based on our correlative studies comparing the kinetics of radiolabeled anti-CEA antibodies in murine models with those in man, we predict that 64 Cu-labeled intact, humanized antibodies can be used to image CEA positive tumors in the clinic.Keywords carcinoembryonic antigen; bifunctional chelate; radioimmunoimaging; positron emission tomographyIn order to be effective tumor imaging agents, radiolabeled anti-tumor antibodies must demonstrate both high tumor uptake and high tumor to normal tissue ratios (1). Monoclonal antibodies (150 kDa) have a relatively slow blood clearance (second phase half-life t 1/2 β = 48-72 h), allowing ample time for high accumulation in tumor, but suffer from low tumor to normal tissue contrast due to slow clearance from the vasculature. In order to improve the dynamic biodistribut...

As a growing number of patients with multiple myeloma (MM) respond to upfront therapies while eventually relapsing in a time frame that is often unpredictable, attention has increasingly focused on developing novel diagnostic criteria to also account for disease dissemination. Positron emission tomography/computed tomography (PET/CT) is often used as a noninvasive monitoring strategy to assess cancer cell dissemination, but because the uptake of the currently used radiotracer 18fluorodeoxyglucose (F-FDG) is a function of the metabolic activity of both malignant and nonmalignant cells, the results frequently lack sufficient specificity. Radiolabeled antibodies targeting MM tissue may detect disease irrespective of cell metabolism. Hence, we conjugated the clinically significant CD38-directed human antibody daratumumab (Darzalex [Dara]) to the DOTA chelator and labeled it with the positron-emitting radionuclide copper 64 (Cu; Cu-DOTA-Dara). Here, we show thatCu-DOTA-Dara can efficiently bind CD38 on the surface of MM cells and was mainly detected in the bones associated with tumor in a MM murine model. We also show that PET/CT based on Cu-DOTA-Dara displays a higher resolution and specificity to detect MM cell dissemination than doesF-FDG PET/CT and was even more sensitive than were bioluminescence signals. We therefore have supporting evidence for using Cu-DOTA-Dara as a novel imaging agent for MM.

Diabodies are non-covalent dimers of single chain antibody fragments (scFvs) that retain the avidity of intact IgG but have more favorable blood clearance than intact IgGs. Radiometals offer a wide range of half lives and emissions for matching imaging and therapy requirements and provide facile labeling of chelate-antibody conjugates. However, due to their high retention and metabolism in the kidney, use of radiometal labeled diabodies can be problematic for both imaging and therapy. Methods Having previously shown that 111In-DOTA-PEG3400-anti-CEA-diabody has similarly high tumor uptake and retention and less than 50% as much kidney uptake and retention as non-PEGylated diabody, we synthesized a similar derivative for an anti-TAG-72-diabody. We also reduced the molecular size of the polydispersed PEG3400 to monodispersed PEG27 and PEG12 (nominal masses of 1188 and 528, respectively). We performed biodistributions of their DOTA conjugates radiolabeled with 125I, 111In, or 64Cu in tumor bearing athymic mice. Results Addition of PEG3400 to the diabody reduced kidney uptake to a level (≈10 %ID/g) comparable to that obtained with radiometal labeled intact IgG. The PEG27 and PEG12 diabody conjugates also demonstrated low kidney uptake without reduction of tumor uptake or tumor to blood ratios. When radiolabeled with 64Cu, the DOTA-PEG12- and PEG27-diabody conjugates gave high contrast PET images of colon cancer xenografts in athymic mice. Conclusion PEGylated diabodies may be a valuable platform for delivery of radionuclides and other agents to tumors.