Katelyn E. McCabe

Cys-diabody Quantum Dot Conjugates (ImmunoQdots) for Cancer Marker Detection

The present work demonstrates the use of small bivalent engineered antibody fragments, cys-diabodies, for biological modification of nanoscale particles such as quantum dots (Qdots) for detection of target antigens. Novel bioconjugated quantum dots known as immunoQdots (iQdots) were developed by thiol-specific oriented coupling of tumor specific cys-diabodies, at a position away from the antigen binding site to amino PEG CdSe/ZnS Qdots. Initially, amino PEG Qdot 655 were coupled with reduced anti-HER2 cys-diabody by amine-sulfhydryl-reactive linker [N-ε-maleimidocaproyloxy] succinimide ester (EMCS) to produce anti-HER2 iQdot 655. Spectral characterization of the conjugate revealed that the spectrum was symmetrical and essentially identical to unconjugated Qdot. Specific receptor binding activity of anti-HER2 iQdot 655 was confirmed by flow cytometry on HER2 positive and negative cells. Immunofluorescence results showed homogeneous surface labeling of the cell membrane with Qdot 655 conjugate. In addition, cys-diabodies specific for HER2, as well as prostate stem cell antigen (PSCA), were conjugated successfully with amino PEG Qdot 800. All of these iQdots retain the photoluminescence properties of the unconjugated Qdot 800 as well as the antigen binding specificity of the cys-diabody as demonstrated by flow cytometry. Simultaneous detection of two tumor antigens on LNCaP/PSCA prostate cancer cells (which express PSCA and HER2) in culture was possible using two iQdots, anti-HER2 iQdot 655 and anti-PSCA iQdot 800. Thus, these iQdots are potentially useful as optical probes for sensitive, multiplexed detection of surface markers on tumor cells. The present thiol-specific conjugation method demonstrates a general approach for site-specific oriented coupling of cys-diabodies to a wide variety of nanoparticles without disturbing the antigen binding site and maintaining small size compared to intact antibody.

Positive progress in immunoPET--not just a coincidence.

The identification of tumor tissue biomarkers has led to the production, validation, and Food and Drug Administration-approval of a number of antibody-based targeted therapeutics in the past two decades. As a result of the significant role that these immunotherapeutics play in the management of cancer, and the potential utility of complementary imaging agents, immunoPET imaging has generated considerable interest. This update discusses the important factors to consider when designing a PET (positron emission tomography) imaging agent from the molecular target to the biological targeting molecule and radionuclide combination and also reviews recent preclinical and clinical findings in the immunoPET field. Although there are a variety of radionuclides that are currently utilized in PET studies, this update focuses on four of the positron emitters commonly used in labeling proteins: iodine-124, zirconium-89, copper-64, and fluorine-18. Notable advances in the preclinical setting include the continued development of immunoPET probes to predict the biodistribution of related radioimmunotherapeutics, the success of nontraditional radionuclide and antibody fragment combinations, the broader use of zirconium-89, and the recent emergence of (18)F-labeled diabodies for same-day imaging. Antibody-based PET probes constitute a valuable class of molecular imaging agents, and the progress made preclinically should expedite the transition of these targeted diagnostics to clinical applications.

Targeting CEA in Pancreas Cancer Xenografts with a Mutated scFv-Fc Antibody Fragment

BackgroundSensitive antibody-based tumor targeting has the potential not only to image metastatic and micrometastatic disease, but also to be the basis of targeted therapy. The vast majority of pancreas cancers express carcinoembryonic antigen (CEA). Thus, we sought to evaluate the potential of CEA as a pancreatic cancer target utilizing a rapidly clearing engineered anti-CEA scFv-Fc antibody fragment with a mutation in the Fc region [anti-CEA scFv-Fc H310A].MethodsImmunohistochemistry (IHC) with the antibody fragment was used to confirm expression of CEA on human pancreas cancer specimens. In vivo tumor targeting was evaluated by tail vein injection of I124-labeled anti-CEA scFv-Fc(H310A) into mice harboring CEA-positive and -negative xenografts. MicroPET/CT imaging was performed at successive time intervals. Radioactivity in blood and tumor was measured after the last time point. Additionally, unlabeled anti-CEA scFv-Fc(H310A) was injected into CEA-positive tumor bearing mice and ex vivo IHC was performed to identify the presence of the antibody to define the microscopic intratumoral pattern of targeting.ResultsModerate to strong staining by IHC was noted on 84% of our human pancreatic cancer specimens and was comparable to staining of our xenografts. Pancreas xenograft imaging with the radiolabeled anti-CEA scFv-Fc(H310A) antibody demonstrated average tumor/blood ratios of 4.0. Immunolocalization demonstrated peripheral antibody fragment penetration of one to five cell diameters (0.75 to 1.5 μm).ConclusionsWe characterized a preclinical xenograft model with respect to CEA expression that was comparable to human cases. We demonstrated that the anti-CEA scFv-Fc(H310A) antibody exhibited antigen-specific tumor targeting and shows promise as an imaging and potentially therapeutic agent.

An engineered anti-CA19-9 cys-diabody for positron emission tomography imaging of pancreatic cancer and targeting of polymerized liposomal nanoparticles

BACKGROUND Antibody-based therapeutics is a rapidly growing field. Small engineered antibody fragments demonstrate similar antigen affinity compared with the parental antibody but have a shorter serum half-life and possess the ability to be conjugated to nanoparticles. The goal of this study was to engineer an anti-carbohydrate antigen 19-9 (CA19-9) cys-diabody fragment in hopes of targeting nanoparticles to pancreatic cancer. METHODS The anti-CA19-9 cys-diabody was created by engineering a C-terminal cysteine residue into the DNA single-chain Fv construct of the anti-CA19-9 diabody and expressed in NS0 cells. Maleimide chemistry was used to conjugate the cys-diabody to polymerized liposomal nanoparticles (PLNs) through the cysteine residues. Flow cytometry was used to evaluate targeting of cys-diabody and cys-diabody-PLN conjugate to human pancreatic cancer cell lines. The cys-diabody was radiolabeled with a positron emitter ((124)I) and evaluated in a mouse model of CA19-9-positive and CA19-9-negative xenografts with micro-positron emission tomography/micro-computed tomography at successive time intervals after injection. Percentage of injected dose per gram of radioactivity was measured in blood and tumor to provide objective confirmation of the micro-positron emission tomographic images. RESULTS Tumor xenograft imaging of the anti-CA19-9 cys-diabody demonstrated an average tumor-to-blood ratio of 3.0 and positive-to-negative tumor ratio of 7.4. Successful conjugation of the cys-diabody to PLNs was indicated by flow cytometry showing specific binding of cys-diabody-PLN conjugate to human pancreatic cancer cells in vitro. CONCLUSIONS Our results show that the anti-CA19-9 cys-diabody targets pancreatic cancer providing specific molecular imaging in tumor xenograft models. Furthermore, the cys-diabody-PLN conjugate demonstrates target-specific binding of human pancreatic cancer cells with the potential to deliver targeted treatment.

CA19-9 as a Potential Target for Radiolabeled Antibody-Based Positron Emission Tomography of Pancreas Cancer

Introduction. Sensitive and specific imaging of pancreas cancer are necessary for accurate diagnosis, staging, and treatment. The vast majority of pancreas cancers express the carbohydrate tumor antigen CA19-9. The goal of this study was to determine the potential to target CA19-9 with a radiolabeled anti-CA19-9 antibody for imaging pancreas cancer. Methods. CA19-9 was quantified using flow cytometry on human pancreas cancer cell lines. An intact murine anti-CA19-9 monoclonal antibody was labeled with a positron emitting radionuclide (Iodine-124) and injected into mice harboring antigen positive and negative xenografts. MicroPET/CT were performed at successive time intervals (72 hours, 96 hours, 120 hours) after injection. Radioactivity was measured in blood and tumor to provide objective confirmation of the images. Results. Antigen expression by flow cytometry revealed approximately 1.3 × 106 CA19-9 antigens for the positive cell line and no expression in the negative cell line. Pancreas xenograft imaging with Iodine-124-labeled anti-CA19-9 mAb demonstrated an average tumor to blood ratio of 5 and positive to negative tumor ratio of 20. Conclusion. We show in vivo targeting of our antigen positive xenograft with a radiolabeled anti-CA19-9 antibody. These data demonstrate the potential to achieve anti-CA19-9 antibody based positron emission tomography of pancreas cancer.

Targeting cancer with cys-diabody conjugated polymerized liposomal nanoparticles (PLNs)

Background: Polymerized liposomal nanoparticles (PLNs) are a promising new technology for delivery of chemotherapeutic drugs to cancer cells. They are easy to prepare, biocompatible, and can carry a wide variety of therapeutics. Additionally, PLNs have the potential to be conjugated to a tumor targeting agent in hopes of delivering a cytotoxic payload directly to the cancer cells, thus minimizing systemic toxicity. Cys-diabodies are the smallest tumor antibody fragment (55 Kd) that retain the bivalency of an intact antibody. The goal of this study was to determine our ability to conjugate various engineered cys-diabody fragments to PLNs and to evaluate the ability to specifically target tumor cells based on the specificity of the diabody-tumor antigen interaction. Methods: Cys-diabody constructs against tumor antigens (ALCAM, HER2/neu, Ca19.9) were engineered, expressed in NSO myeloma cells and purified using HPLC. A cysteine residue was inserted at the C-terminus to provide the free sulhydryl group necessary for the conjugation reaction. Using malemide chemistry, cys-diabodies were conjugated to PLNs. Immunofluorescence and flow cytometry were performed to evaluate targeting of conjugated PLNs to human pancreatic and breast cancer cell lines. Unconjugated PLNs and tumor antigen negative cell lines were used as controls. Results: Anti-HER2/neu, ALCAM, and CA19.9 cys-diabodies were characterized by flow cytometry showing specific binding to MCF7 (HER2/neu positive), HPAF (ALCAM positive), and BxPC3 (CA19.9 positive) cell lines, respectively. No binding was observed to antigen-negative cell lines nor with the unconjugated PLNs. Conjugation of cys-diabodies to PLNs was indicated by immunofluorescence showing specific binding of PLN-cys-diabody conjugates to breast and pancreatic cancer cells in vitro (Figure 1). These data were also confirmed by flow cytometry. Conclusions: We have demonstrated the ability to conjugate our PLN with engineered anti-tumor cys-diabodies. Furthermore, we have demonstrated that after conjugation, the PLNs demonstrate specific tumor-cell targeting based on the specificity of the diabody-tumor antigen affinity. These studies represent an important first step in the development of targeted PLNs with the ultimate goal of delivering high doses of a chemotherapeutic directly to the cancer cells.