Desiree Crow

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.

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 (t(1/2) = 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 (64)Cu (t(1/2) = 12 h). 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 preconjugated to the chelator DOTA, the conjugates were able to bind radiometals such as (111)In and (64)Cu 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 24 h when radiolabeled with (111)In and 37.9% ID/g of tumor uptake (T/B = 8) at 44 h when radiolabeled with (64)Cu in PET imaging in an animal model. Tumor uptake was significantly improved from the 50% ID/g at 24 h 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.

A series of anti-CEA/anti-DOTA bispecific antibody formats evaluated for pre-targeting: comparison of tumor uptake and blood clearance

A series of anti-tumor/anti-chelate bispecific antibody formats were developed for pre-targeted radioimmunotherapy. Based on the anti-carcinoembryonic antigen humanized hT84.66-M5A monoclonal antibody and the anti-DOTA C8.2.5 scFv antibody fragment, this cognate series of bispecific antibodies were radioiodinated to determine their tumor targeting, biodistribution and pharmacokinetic properties in a mouse xenograft tumor model. The in vivo biodistribution studies showed that all the bispecific antibodies exhibited specific high tumor uptake but the tumor targeting was approximately one-half of the parental anti-CEA mAb due to faster blood clearance. Serum stability and FcRn studies showed no apparent reason for the faster blood clearance. A dual radiolabel biodistribution study revealed that the (111)In-DOTA bispecific antibody had increased liver and spleen uptake, not seen for the (125)I-version due to metabolism and release of the radioiodine from the cells. These data suggest increased clearance of the antibody fusion formats by the mononuclear phagocyte system. Importantly, a pre-targeted study showed specific tumor uptake of (177)Lu-DOTA and a tumor : blood ratio of 199 : 1. This pre-targeted radiotherapeutic and substantial reduction in the radioactive exposure to the bone marrow should enhance the therapeutic potential of RIT.

Monodispersed DOTA-PEG–Conjugated Anti-TAG-72 Diabody Has Low Kidney Uptake and High Tumor-to-Blood Ratios Resulting in Improved 64Cu PET

Diabodies are noncovalent dimers of single-chain antibody fragments that retain the avidity of intact IgG but have more favorable blood clearance than intact IgG. 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, because of their high retention and metabolism in the kidney, the use of radiometal-labeled diabodies can be problematic for both imaging and therapy. Methods: Having previously shown that 111In-DOTA-polyethylene glycol (PEG)3400-anti–carcinoembryonic antigen diabody has less than half the kidney uptake and retention of non-PEGylated diabody and that the two have similarly high tumor uptake and retention, we synthesized a similar derivative for an anti–tumor-associated glycoprotein 72 diabody. We also reduced the molecular size of the polydispersed PEG3400 to monodispersed PEG27 and PEG12 (nominal masses of 1,321 and 617, respectively). We performed biodistributions of their DOTA conjugates radiolabeled with 125I, 111In, or 64Cu in tumor-bearing athymic mice. Results: The addition of PEG3400 to the diabody reduced kidney uptake to a level (≈10 percentage injected dose/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.

Copper-64-labeled daratumumab as a PET/CT imaging tracer for multiple myeloma

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 (18F-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 (64Cu; 64Cu-DOTA-Dara). Here, we show that 64Cu-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 64Cu-DOTA-Dara displays a higher resolution and specificity to detect MM cell dissemination than does 18F-FDG PET/CT and was even more sensitive than were bioluminescence signals. We therefore have supporting evidence for using 64Cu-DOTA-Dara as a novel imaging agent for MM.

PET imaging of 64Cu-DOTA-scFv-anti-PSMA lipid nanoparticles (LNPs): Enhanced tumor targeting over anti-PSMA scFv or untargeted LNPs

INTRODUCTION Single chain (scFv) antibodies are ideal targeting ligands due to their modular structure, high antigen specificity and affinity. These monovalent ligands display rapid tumor targeting but have limitations due to their fast urinary clearance. METHODS An anti-prostate membrane antigen (PSMA) scFv with a site-specific cysteine was expressed and evaluated in a prostate cancer xenograft model by Cu-64 PET imaging. To enhance tumor accumulation, the scFv-cys was conjugated to the co-polymer DSPE-PEG-maleimide that spontaneously assembled into a homogeneous multivalent lipid nanoparticle (LNP). RESULTS The targeted LNP exhibited a 2-fold increase in tumor uptake compared to the scFv alone using two different thiol ester chemistries. The anti-PSMA scFv-LNP exhibited a 1.6 fold increase in tumor targeting over the untargeted LNP. CONCLUSIONS The targeted anti-PSMA scFv-LNP showed enhanced tumor accumulation over the scFv alone or the untargeted DOTA-micelle providing evidence for the development of this system for drug delivery. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE Anti-tumor scFv antibody fragments have not achieved their therapeutic potential due to their fast blood clearance. Conjugation to an LNP enables multivalency to the tumor antigen as well as increased molecular size for chemotherapy drug delivery.

Erratum: Copper 64–labeled daratumumab as a PET/CT imaging tracer for multiple myeloma (Blood (2018) 131:7 (741-745) DOI: 10.1182/blood-2017-09-807263)

1Department of Hematologic Malignancies Translational Science, Beckman Research Institute, City of Hope, Duarte, CA; 2Briskin Myeloma Center, Department of Hematology and Hematopoietic Cell Transplantation, City of Hope, Duarte, CA; 3Department of Molecular Immunology and 4Center for Comparative Medicine, Beckman Research Institute, City of Hope, Duarte, CA; 5Department of Information Sciences, Division of Biostatistics, City of Hope, Duarte, CA; 6Department of Radiation Oncology, Beckman Research Institute, City of Hope, Duarte, CA; 7Translational Genomics Research Institute, Phoenix, AZ; and 8Department of Pathology, City of Hope, Duarte, CA