Matthew K. Robinson

Quantitative Immuno-Positron Emission Tomography Imaging of HER2-Positive Tumor Xenografts with an Iodine-124 Labeled Anti-HER2 Diabody

Positron emission tomography (PET) provides an effective means of both diagnosing/staging several types of cancer and evaluating efficacy of treatment. To date, the only U.S. Food and Drug Administration-approved radiotracer for oncologic PET is (18)F-fluoro-deoxyglucose, which measures glucose accumulation as a surrogate for malignant activity. Engineered antibody fragments have been developed with the appropriate targeting specificity and systemic elimination properties predicted to allow for effective imaging of cancer based on expression of tumor associated antigens. We evaluated a small engineered antibody fragment specific for the HER2 receptor tyrosine kinase (C6.5 diabody) for its ability to function as a PET radiotracer when labeled with iodine-124. Our studies revealed HER2-dependent imaging of mouse tumor xenografts with a time-dependent increase in tumor-to-background signal over the course of the experiments. Radioiodination via an indirect method attenuated uptake of radioiodine in tissues that express the Na/I symporter without affecting the ability to image the tumor xenografts. In addition, we validated a method for using a clinical PET/computed tomography scanner to quantify tumor uptake in small-animal model systems; quantitation of the tumor targeting by PET correlated with traditional necropsy-based analysis at all time points analyzed. Thus, diabodies may represent an effective molecular structure for development of novel PET radiotracers.

Regulation of Antibody-Dependent Cellular Cytotoxicity by IgG Intrinsic and Apparent Affinity for Target Antigen

Unconjugated mAbs have emerged as useful cancer therapeutics. Ab-dependent cellular cytotoxicity (ADCC) is believed to be a major antitumor mechanism of some anticancer Abs. However, the factors that regulate the magnitude of ADCC are incompletely understood. In this study, we described the relationship between Ab affinity and ADCC. A series of human IgG1 isotype Abs was created from the anti-HER2/neu (also named c-erbB2) C6.5 single-chain Fv (scFv) and its affinity mutants. The scFv affinities range from 10−7 to 10−11 M, and the IgG Abs retain the affinities of the scFv from which they were derived. The apparent affinity of the Abs ranged from nearly 10−10 M (the lowest affinity variant) to almost 10−11 M (the other variants). The IgG molecules were tested for their ability to elicit ADCC in vitro against three tumor cell lines with differing levels of HER2/neu expression using unactivated human PBMC from healthy donors as the effector cells. The results demonstrated that both the apparent affinity and intrinsic affinity of the Abs studied regulate ADCC. High-affinity tumor Ag binding by the IgGs led to the most efficient and powerful ADCC. Tumor cells expressing high levels of HER2/neu are more susceptible to the ADCC triggered by Abs than the cells expressing lower amounts of HER2/neu. These findings justify the examination of high affinity Abs for ADCC promotion. Because high affinity may impair in vivo tumor targeting, a careful examination of Ab structure to function relationships is required to develop optimized therapeutic unconjugated Abs.

Hybrids of a Genetically Engineered Antibody and a Carbon Nanotube Transistor for Detection of Prostate Cancer Biomarkers

We developed a novel detection method for osteopontin (OPN), a new biomarker for prostate cancer, by attaching a genetically engineered single-chain variable fragment (scFv) protein with high binding affinity for OPN to a carbon nanotube field-effect transistor (NT-FET). Chemical functionalization using diazonium salts is used to covalently attach scFv to NT-FETs, as confirmed by atomic force microscopy, while preserving the activity of the biological binding site for OPN. Electron transport measurements indicate that functionalized NT-FET may be used to detect the binding of OPN to the complementary scFv protein. A concentration-dependent increase in the source-drain current is observed in the regime of clinical significance, with a detection limit of approximately 30 fM. The scFv-NT hybrid devices exhibit selectivity for OPN over other control proteins. These devices respond to the presence of OPN in a background of concentrated bovine serum albumin, without loss of signal. On the basis of these observations, the detection mechanism is attributed to changes in scattering at scFv protein-occupied defect sites on the carbon nanotube sidewall. The functionalization procedure described here is expected to be generalizable to any antibody containing an accessible amine group and to result in biosensors appropriate for detection of corresponding complementary proteins at fM concentrations.

Highly Sensitive Detection of HER2 Extracellular Domain in the Serum of Breast Cancer Patients by Piezoelectric Microcantilevers

Rapid and sensitive detection of serum tumor biomarkers are needed to monitor cancer patients for disease progression. Highly sensitive piezoelectric microcantilever sensors (PEMS) offer an attractive tool for biomarker detection; however, their utility in the complex environment encountered in serum has yet to be determined. As a proof of concept, we have functionalized PEMS with antibodies that specifically bind to HER2, a biomarker (antigen) that is commonly overexpressed in the blood of breast cancer patients. The function and sensitivity of these anti-HER2 PEMS biosensors was initially assessed using recombinant HER2 spiked into human serum. Their ability to detect native HER2 present in the serum of breast cancer patients was then determined. We have found that the anti-HER2 PEMS were able to accurately detect both recombinant and naturally occurring HER2 at clinically relevant levels (>2 ng/mL). This indicates that PEMS-based biosensors provide a potentially effective tool for biomarker detection.

Effective Treatment of Established Human Breast Tumor Xenografts in Immunodeficient Mice with a Single Dose of the α- Emitting Radioisotope Astatine-211 Conjugated to Anti-HER2/ neu Diabodies

Purpose: Successful radioimmunotherapy strategies depend on selecting radioisotopes with physical properties complementary to the biological properties of the targeting vehicle. Small, engineered antitumor antibody fragments are capable of rapid, highly specific tumor targeting in immunodeficient mouse models. We hypothesized that the C6.5 diabody, a noncovalent anti-HER2 single-chain Fv dimer, would be an ideal radioisotope carrier for the radioimmunotherapy of established tumors using the short-lived α-emitting radioisotope 211At. Experimental Design: Immunodeficient nude mice bearing established HER2/neu–positive MDA-MB-361/DYT2 tumors treated with N-succinimidyl N-(4-[211At]astatophenethyl)succinamate (211At-SAPS)-C6.5 diabody. Additional cohorts of mice were treated with 211At-SAPS T84.66 diabody targeting the carcinoembryonic antigen or 211At-SAPS on a diabody specific for the Müllerian inhibiting substance type II receptor, which is minimally expressed on this tumor cell line. Results: A single i.v. injection of 211At-SAPS C6.5 diabody led to a 30-day delay in tumor growth when a 20 μCi dose was administered and a 57-day delay in tumor growth (60% tumor-free after 1 year) when a 45 μCi dose was used. Treatment of mice bearing the same tumors with 211At-SAPS T84.66 diabody at the same doses led to a delay in tumor growth, but no complete responses, likely due to substantially lower expression of this antigen on the MDA-MB-361/DYT2 tumors. In contrast, a dose of 20 μCi of 211At-SAPS on the anti–Müllerian-inhibiting substance type II receptor diabody did not affect tumor growth rate, demonstrating specificity of the therapeutic effect. Conclusions: These findings indicate that diabody molecules can be effective agents for targeted radioimmunotherapy of solid tumors using powerful, short-lived α-emitting radioisotopes.

Protein-intrinsic and signaling network-based sources of resistance to EGFR- and ErbB family-targeted therapies in head and neck cancer

Agents targeting EGFR and related ErbB family proteins are valuable therapies for the treatment of many cancers. For some tumor types, including squamous cell carcinomas of the head and neck (SCCHN), antibodies targeting EGFR were the first protein-directed agents to show clinical benefit, and remain a standard component of clinical strategies for management of the disease. Nevertheless, many patients display either intrinsic or acquired resistance to these drugs; hence, major research goals are to better understand the underlying causes of resistance, and to develop new therapeutic strategies that boost the impact of EGFR/ErbB inhibitors. In this review, we first summarize current standard use of EGFR inhibitors in the context of SCCHN, and described new agents targeting EGFR currently moving through pre-clinical and clinical development. We then discuss how changes in other transmembrane receptors, including IGF1R, c-Met, and TGF-β, can confer resistance to EGFR-targeted inhibitors, and discuss new agents targeting these proteins. Moving downstream, we discuss critical EGFR-dependent effectors, including PLC-γ; PI3K and PTEN; SHC, GRB2, and RAS and the STAT proteins, as factors in resistance to EGFR-directed inhibitors and as alternative targets of therapeutic inhibition. We summarize alternative sources of resistance among cellular changes that target EGFR itself, through regulation of ligand availability, post-translational modification of EGFR, availability of EGFR partners for hetero-dimerization and control of EGFR intracellular trafficking for recycling versus degradation. Finally, we discuss new strategies to identify effective therapeutic combinations involving EGFR-targeted inhibitors, in the context of new system level data becoming available for analysis of individual tumors.

Evaluation of the Anti-HER2 C6.5 Diabody as a PET Radiotracer to Monitor HER2 status and Predict Response to Trastuzumab Treatment

Purpose: The rapid tumor targeting and pharmacokinetic properties of engineered antibodies make them potentially suitable for use in imaging strategies to predict and monitor response to targeted therapies. This study aims to evaluate C6.5 diabody (C6.5db), a noncovalent anti-HER2 single-chain Fv dimer, as a radiotracer for predicting response to HER2-targeted therapies such as trastuzumab. Experimental Design: Immunodeficient mice bearing established HER2-positive tumor xenografts were injected with radioiodinated C6.5db and imaged by PET/CT. Radiotracer biodistribution was quantified by biopsied tumor and normal tissues. Potential competition between trastuzumab and C6.5db was examined in vitro by flow cytometry and coimmunoprecipitations. Results: Biodistribution analysis of mice bearing xenografts with varying HER2 density revealed that the tumor uptake of 125I-C6.5db correlates with HER2 tumor density. In vitro competition experiments suggest that the C6.5db targets an epitope on HER2 that is distinct from that bound by trastuzumab. Treatment of mice affected with SK-OV-3 tumor with trastuzumab for 3 days caused a 42% (P = 0.002) decrease in tumor uptake of 125I-C6.5db. This is consistent with a dramatic decrease in the tumor PET signal of 124I-C6.5db after trastuzumab treatment. Furthermore, mice affected with BT-474 tumor showed an approximately 60% decrease (P = 0.0026) in C6.5db uptake after 6 days of trastuzumab treatment. Immunohistochemistry of excised xenograft sections and in vitro flow cytometry revealed that the decreased C6.5db uptake on trastuzumab treatment is not associated with HER2 downregulation. Conclusions: These studies suggest that 124I-C6.5db–based imaging can be used to evaluate HER2 levels as a predictor of response to HER2-directed therapies. Clin Cancer Res; 17(6); 1509–20. ©2010 AACR.

Immuno-Positron Emission Tomography in Cancer Models

Positron emission tomography (PET) is playing an increasingly important role in the diagnosis, staging, and monitoring response to treatment in a variety of cancers. Recent efforts have focused on immuno-PET, which uses antibody-based radiotracers, to image tumors based on expression of tumor-associated antigens. It is postulated that the specificity afforded by antibody targeting should both improve tumor detection and provide phenotypic information related to primary and metastatic lesions that will guide therapy decisions. Advances in antibody-engineering are providing the tools to develop antibody-based molecules with pharmacokinetic properties optimized for use as immuno-PET radiotracers. Coupled with technical advances in the design of PET scanners, immuno-PET holds promise to improve diagnostic imaging and to guide the use of targeted therapies. An overview of the preclinical immuno-PET studies in cancer models is reviewed here.

Isolation of anti-MISIIR scFv molecules from a phage display library by cell sorter biopanning

While cell surface antigens represent the most common targets for antibody-based cancer therapy, isolation of new antibodies specific for these targets from single-chain Fv phage display libraries has been hindered by limitations associated with traditional selection techniques. Solid phase panning is often associated with conformational changes to the target protein due to its immobilization on plastic tubes that can limit the ability of the isolated scFv to bind to conformational epitopes and solution panning methods require the use of secondary tags that often mask desired sequences and create unintended epitopes. Commonly utilized cell-based panning methods typically yield a panel of single-chain Fv (scFv) molecules that are specific for numerous cell surface antigens, often obscuring the desired clones. Here, we describe a novel cell sorter-based system to isolate single-chain Fv molecules specific for defined antigen targets expressed on stably-transformed mammalian cells. We employed these methods to isolate promising scFv clones that bind specifically to the Müllerian inhibiting substance type II receptor, a cell surface ovarian cancer antigen that has proven to be a difficult target for selection strategies.

Inhibition of Cancer Cell Proliferation and Breast Tumor Targeting of pHLIP–Monomethyl Auristatin E Conjugates

Localized delivery is vital for the successful development of novel and effective therapeutics for the treatment of cancer. The targeting and delivery described herein is based on the pH (low) insertion peptide (pHLIP), a unique delivery peptide that can selectively target tumors in mice and translocate and release cargo molecules intracellularly based solely on the low extracellular pH intrinsic to cancer cells. In this study, we investigate the efficacy of pHLIP to target and deliver the highly potent and clinically validated microtubule inhibitor monomethyl auristatin E (MMAE) to cancer cells and breast tumors. We show that pHLIP-MMAE conjugates induce a potent cytotoxic effect (>90% inhibition of cell growth) in a concentration- and pH-dependent manner after only 2 h incubation without any apparent disruption of the plasma membrane. pHLIP-MMAE conjugates exhibit between an 11- and 144-fold higher antiproliferative effect at low pH than that at physiological pH and a pronounced pH-dependent cytotoxicity as compared to that of free drug. Furthermore, we demonstrate that a pHLIP-MMAE drug conjugate effectively targets triple-negative breast tumor xenografts in mice. These results indicate that pHLIP-based auristatin conjugates may have an enhanced therapeutic window as compared to that of free drug, providing a targeting mechanism to attenuate systemic toxicity.