Erasmus Poku

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.

Tumor Uptake of 64Cu-DOTA-Trastuzumab in Patients with Metastatic Breast Cancer

The goal of this study was to characterize the relationship between tumor uptake of 64Cu-DOTA-trastuzumab as measured by PET/CT and standard, immunohistochemistry (IHC)-based, histopathologic classification of human epidermal growth factor receptor 2 (HER2) status in women with metastatic breast cancer (MBC). Methods: Women with biopsy-confirmed MBC and not given trastuzumab for 2 mo or more underwent complete staging, including 18F-FDG PET/CT. Patients were classified as HER2-positive (HER2+) or -negative (HER2−) based on fluorescence in situ hybridization (FISH)–supplemented immunohistochemistry of biopsied tumor tissue. Eighteen patients underwent 64Cu-DOTA-trastuzumab injection, preceded in 16 cases by trastuzumab infusion (45 mg). PET/CT was performed 21–25 (day 1) and 47–49 (day 2) h after 64Cu-DOTA-trastuzumab injection. Radiolabel uptake in prominent lesions was measured as SUVmax. Average intrapatient SUVmax (pt) was compared between HER2+ and HER2− patients. Results: Eleven women were HER2+ (8 immunohistochemistry 3+; 3 immunohistochemistry 2+/FISH amplified), whereas 7 were HER2− (3 immunohistochemistry 2+/FISH nonamplified; 4 immunohistochemistry 1+). Median pt for day 1 and day 2 was 6.6 and 6.8 g/mL for HER 2+ and 3.7 and 4.3 g/mL for HER2− patients (P < 0.005 either day). The distributions of pt overlapped between the 2 groups, and interpatient variability was greater for HER2+ than HER2− disease (P < 0.005 and 0.001, respectively, on days 1 and 2). Conclusion: By 1 d after injection, uptake of 64Cu-DOTA-trastuzumab in MBC is strongly associated with patient HER2 status and is indicative of binding to HER2. The variability within and among HER2+ patients, as well as the overlap between the HER2+ and HER2− groups, suggests a role for 64Cu-DOTA-trastuzumab PET/CT in optimizing treatments that include trastuzumab.

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.

PET of Adoptively Transferred Chimeric Antigen Receptor T Cells with 89Zr-Oxine

Chimeric antigen receptor (CAR) T cell therapy is a promising clinical approach for reducing tumor progression and prolonging patient survival. However, improvements in both the safety and the potency of CAR T cell therapy demand quantitative imaging techniques to determine the distribution of cells after adoptive transfer. The purpose of this study was to optimize 89Zr-oxine labeling of CAR T cells and evaluate PET as a platform for imaging adoptively transferred CAR T cells. Methods: CAR T cells were labeled with 0–1.4 MBq of 89Zr-oxine per 106 cells and assessed for radioactivity retention, viability, and functionality. In vivo trafficking of 89Zr-oxine–labeled CAR T cells was evaluated in 2 murine xenograft tumor models: glioblastoma brain tumors with intracranially delivered IL13Rα2-targeted CAR T cells, and subcutaneous prostate tumors with intravenously delivered prostate stem cell antigen (PSCA)–targeted CAR T cells. Results: CAR T cells were efficiently labeled (75%) and retained more than 60% of the 89Zr over 6 d. In vitro cytokine production, migration, and tumor cytotoxicity, as well as in vivo antitumor activity, were not significantly reduced when labeled with 70 kBq/106 cells. IL13Rα2-CAR T cells delivered intraventricularly were detectable by PET for at least 6 d throughout the central nervous system and within intracranial tumors. When intravenously administered, PSCA-CAR T cells also showed tumor tropism, with a 9-fold greater tumor-to-muscle ratio than for CAR-negative T cells. Conclusion: 89Zr-oxine can be used for labeling and imaging CAR T cells while maintaining cell viability and function. On the basis of these studies, we conclude that 89Zr-oxine is a clinically translatable platform for real-time assessment of cell therapies.

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

Diagnostic PET Imaging of Mammary Microcalcifications Using 64 Cu-DOTA-Alendronate in a Rat Model of Breast Cancer

The development of improved breast cancer screening methods is hindered by a lack of cancer-specific imaging agents and effective small-animal models to test them. The purpose of this study was to evaluate 64Cu-DOTA-alendronate as a mammary microcalcification-targeting PET imaging agent, using an ideal rat model. Our long-term goal is to develop 64Cu-DOTA-alendronate for the detection and noninvasive differentiation of malignant versus benign breast tumors with PET. Methods: DOTA-alendronate was synthesized, radiolabeled with 64Cu, and administered to normal or tumor-bearing aged, female, retired breeder Sprague–Dawley rats for PET imaging. Mammary tissues were subsequently labeled and imaged with light, confocal, and electron microscopy to verify microcalcification targeting specificity of DOTA-alendronate and elucidate the histologic and ultrastructural characteristics of the microcalcifications in different mammary tumor types. Tumor uptake, biodistribution, and dosimetry studies were performed to evaluate the efficacy and safety of 64Cu-DOTA-alendronate. Results: 64Cu-DOTA-alendronate was radiolabeled with a 98% yield. PET imaging using aged, female, retired breeder rats showed specific binding of 64Cu-DOTA-alendronate in mammary glands and mammary tumors. The highest uptake of 64Cu-DOTA-alendronate was in malignant tumors and the lowest uptake in benign tumors and normal mammary tissue. Confocal analysis with carboxyfluorescein-alendronate confirmed the microcalcification binding specificity of alendronate derivatives. Biodistribution studies revealed tissue alendronate concentrations peaking within the first hour, then decreasing over the next 48 h. Our dosimetric analysis demonstrated a 64Cu effective dose within the acceptable range for clinical PET imaging agents and the potential for translation into human patients. Conclusion: 64Cu-DOTA-alendronate is a promising PET imaging agent for the sensitive and specific detection of mammary tumors as well as the differentiation of malignant versus benign tumors based on absolute labeling uptake.