Glenn A Cartwright

18F-THK523: a novel in vivo tau imaging ligand for Alzheimer’s disease

While considerable effort has focused on developing positron emission tomography β-amyloid imaging radiotracers for the early diagnosis of Alzheimers disease, no radiotracer is available for the non-invasive quantification of tau. In this study, we detail the characterization of (18)F-THK523 as a novel tau imaging radiotracer. In vitro binding studies demonstrated that (18)F-THK523 binds with higher affinity to a greater number of binding sites on recombinant tau (K18Δ280K) compared with β-amyloid(1-42) fibrils. Autoradiographic and histofluorescence analysis of human hippocampal serial sections with Alzheimers disease exhibited positive THK523 binding that co-localized with immunoreactive tau pathology, but failed to highlight β-amyloid plaques. Micro-positron emission tomography analysis demonstrated significantly higher retention of (18)F-THK523 (48%; P < 0.007) in tau transgenic mice brains compared with their wild-type littermates or APP/PS1 mice. The preclinical examination of THK523 has demonstrated its high affinity and selectivity for tau pathology both in vitro and in vivo, indicating that (18)F-THK523 fulfils ligand criteria for human imaging trials.

The antitumor monoclonal antibody 806 recognizes a high-mannose form of the EGF receptor that reaches the cell surface when cells over-express the receptor

Overexpression of the EGFR is commonly caused by EGFR gene amplification and is sometimes associated with expression of a variant EGFR (de2‐7 EGFR or EGFRvIII) bearing an internal deletion in its extracellular domain. mAb 806 is a novel EGFR antibody with significant antitumor activity that recognizes both the de2‐7 EGFR and a subset of the wild‐type (wt) EGFR when overexpressed, but does not bind the EGFR expressed in normal tissues. Recently, we demonstrated that the mAb 806 epitope is restricted to a short cysteine loop of the EGFR (amino acids 287–302) that is only available for antibody binding in a transitional form of the receptor, which occurs as the receptor changes from its inactive tethered conformation to a dimeric untethered form. The truncation associated with the de2‐7 EGFR mutation renders this receptor constitutively untethered, leading to increased binding of mAb 806. We now show that mAb 806 preferentially binds the immature high‐mannose wt and de2‐7 EGFR precursors normally located in the endoplasmic reticulum, indicating that this form of the wt EGFR is also constitutively untethered. Using the unique specificity of mAb 806, we clearly demonstrated the presence of these high‐mannose EGFR precursors on the cell surface. Given that the high‐mannose forms of the wt EGFR must be untethered they may contribute to the spontaneous EGFR signaling reported in cells overexpressing the receptor. These precursor forms of the EGFR thus represent novel tumor targets and contribute to the exceptional selectivity of mAb 806 for EGFR when overexpressed in cancer cells. As our observations are likely to apply to other receptors overexpressed in cancer, they suggest a strategy for developing antitumor antibodies even when the target receptor is expressed in normal tissue.

Antibodies specifically targeting a locally misfolded region of tumor associated EGFR

Epidermal Growth Factor Receptor (EGFR) is involved in stimulating the growth of many human tumors, but the success of therapeutic agents has been limited in part by interference from the EGFR on normal tissues. Previously, we reported an antibody (mab806) against a truncated form of EGFR found commonly in gliomas. Remarkably, it also recognizes full-length EGFR on tumor cells but not on normal cells. However, the mechanism for this activity was unclear. Crystallographic structures for Fab:EGFR287–302 complexes of mAb806 (and a second, related antibody, mAb175) show that this peptide epitope adopts conformations similar to those found in the wtEGFR. However, in both conformations observed for wtEGFR, tethered and untethered, antibody binding would be prohibited by significant steric clashes with the CR1 domain. Thus, these antibodies must recognize a cryptic epitope in EGFR. Structurally, it appeared that breaking the disulfide bond preceding the epitope might allow the CR1 domain to open up sufficiently for antibody binding. The EGFRC271A/C283A mutant not only binds mAb806, but binds with 1:1 stoichiometry, which is significantly greater than wtEGFR binding. Although mAb806 and mAb175 decrease tumor growth in xenografts displaying mutant, overexpressed, or autocrine stimulated EGFR, neither antibody inhibits the in vitro growth of cells expressing wtEGFR. In contrast, mAb806 completely inhibits the ligand-associated stimulation of cells expressing EGFRC271A/C283A. Clearly, the binding of mAb806 and mAb175 to the wtEGFR requires the epitope to be exposed either during receptor activation, mutation, or overexpression. This mechanism suggests the possibility of generating antibodies to target other wild-type receptors on tumor cells.

Recombinant NY‐ESO‐1 Cancer Antigen: Production and Purification under cGMP Conditions

Abstract The cancer‐testis antigen, NY‐ESO‐1, has been engineered into a bacterial expression plasmid which incorporates a His6‐tag. The plasmid was transfected into E. coli strain BL21 and Master and Working cell banks generated from this expression system. Three 15‐litre fermentations were performed under cGMP (code of Good Manufacturing Practice) conditions and the crude NY‐ESO‐1 tagged protein isolated as solubilised inclusion bodies. A three‐step cGMP chromatography process (immobilised metal affinity, anion exchange, and hydrophobic interaction) was utilised to purify the protein. The purified NY‐ESO‐1 is being used in early stage human cancer vaccine trials in Australia and the U.S.A.

Targeting EphA3 Inhibits Cancer Growth by Disrupting the Tumor Stromal Microenvironment

Eph receptor tyrosine kinases are critical for cell-cell communication during normal and oncogenic tissue patterning and tumor growth. Somatic mutation profiles of several cancer genomes suggest EphA3 as a tumor suppressor, but its oncogenic expression pattern and role in tumorigenesis remain largely undefined. Here, we report unexpected EphA3 overexpression within the microenvironment of a range of human cancers and mouse tumor xenografts where its activation inhibits tumor growth. EphA3 is found on mouse bone marrow-derived cells with mesenchymal and myeloid phenotypes, and activation of EphA3(+)/CD90(+)/Sca1(+) mesenchymal/stromal cells with an EphA3 agonist leads to cell contraction, cell-cell segregation, and apoptosis. Treatment of mice with an agonistic α-EphA3 antibody inhibits tumor growth by severely disrupting the integrity and function of newly formed tumor stroma and microvasculature. Our data define EphA3 as a novel target for selective ablation of the tumor microenvironment and demonstrate the potential of EphA3 agonists for anticancer therapy.

Generation of Anti-Idiotype Antibodies for Application in Clinical Immunotherapy Laboratory Analyses

The chimeric monoclonal antibody ch806 specifically targets the tumor-associated mutant epidermal growth factor receptor (de 2-7EGFR or EGFRVIII) and is currently under investigation for its potential use in cancer therapy. The humanised monoclonal antibody hu3S193 specifically targets the Lewis Y epithelial antigen and is currently in Phase I clinical trials in patients with advanced breast, colon, and ovarian carcinomas. To assist the clinical evaluation of ch806 and hu3S193, laboratory assays are required to monitor their serum pharmacokinetics and quantitate any immune responses to the antibodies. Mice immunized with ch806 or hu3S193 were used to generate hybridomas producing antibodies with specific binding to ch806 or hu3S193 and competitive for antigen binding. These anti-idiotype antibodies (designated Ludwig Melbourne Hybridomas, LMH) were investigated as reagents suitable for use as positive controls for HAHA or HACA analyses and for measuring hu3S193 or ch806 in human serum. Anti-idiotypes with the ability to concurrently bind two target antibody molecules were identified, which enabled the development of highly reproducible, sensitive, specific ELISA assays for determining serum concentrations of hu3S193 and ch806 with a 3 ng/mL limit of quantitation using LMH-3 and LMH-12, respectively. BIAcore analyses determined high apparent binding affinity for both idiotypes: LMH-3 binding immobilized hu3S193, Ka = 4.76 x 10(8) M(-1); LMH-12 binding immobilised ch806, Ka = 1.74 x 10(9) M(-1). Establishment of HAHA or HACA analysis of sera samples using BIAcore was possible using LMH-3 and LMH-12 as positive controls for quantitation of immune responses to hu3S193 or ch806 in patient sera. These anti-idiotypes could also be used to study the penetrance and binding of ch806 or hu3S193 to tumor cells through immunohistochemical analysis of tumor biopsies. The generation of anti-idiotype antibodies capable of concurrently binding a target antibody on each variable domain provides reagents with high sensitivity for the assessment of safety and pharmacokinetic profiles of target antibodies administered clinically.

Radiolabelling and evaluation of novel haloethylsulfoxides as PET imaging agents for tumor hypoxia

The significance of imaging hypoxia with the PET ligand [(18)F]FMISO has been demonstrated in a variety of cancers. However, the slow kinetics of [(18)F]FMISO require a 2-h delay between tracer administration and patient scanning. Labelled chloroethyl sulfoxides have shown faster kinetics and higher contrast than [(18)F]FMISO in a rat model of ischemic stroke. However, these nitrogen mustard analogues are unsuitable for routine production and use in humans. Here we report on the synthesis and in vitro and in vivo evaluation of two novel sulfoxides which we synthesised from a single precursor molecule via either 2-[(18)F]fluoroethyl azide click chemistry or conventional nucleophilic displacement of a chloride leaving group. The yields of the click chemistry approach were 90±5% of [(18)F]2 based on 2-[(18)F]fluoroethyl azide, and the yields for the S(N) reaction were 15±5% of [(18)F]1 based on K[(18)F]F. Both radiotracers underwent metabolism in an in vitro assay using S9 liver fractions with biological half-lives of 32.39 and 43.32 min, respectively. Imaging studies using an SK-RC-52 tumor model in BALB/c nude mice have revealed that only [(18)F]1 is retained in hypoxic tumors, whereas [(18)F]2 is cleared from those tumors at a rate similar to that of muscle tissue. [(18)F]1 has emerged as a promising new lead structure for further development of sulfoxide-based hypoxia imaging agents. In particular, the mechanism of uptake needs to be elucidated and changes to the chemical structure need to be made in order to reduce metabolism and improve radiotracer kinetics.

Molecular Imaging of Death Receptor 5 Occupancy and Saturation Kinetics In Vivo by Humanized Monoclonal Antibody CS-1008

Purpose: CS-1008 (tigatuzumab; phase I/II), an antihuman death receptor 5 (DR5) agonist, induces apoptosis and has cytotoxic activity against human cancer cell lines. This study reports on the preclinical validation of 111In-labeled anti-DR5 humanized antibody CS-1008 as a diagnostic tool to study the DR5 occupancy in patients with cancer and establish dose ranges for receptor saturation kinetics in vivo. Experimental Design: CS-1008 was radiolabeled and characterized for DR5 binding and labeling efficiency on TRAIL-sensitive DR5–positive colorectal cancer cells (COLO 205 and WiDr). Pharmacokinetic and biodistribution studies were conducted in BALB/c nu/nu mice bearing COLO 205, WiDr, or DR5-negative CT26 colon tumors. Planar gamma camera imaging and computerized tomography (CT) images were obtained to study receptor occupancy in vivo. Results: Scatchard analysis showed high and specific binding affinity (Kd, 1.05 ± 0.12 nmol/L) of 111In-labeled CS-1008. 111In-labeled CS-1008 was specifically taken up in mice bearing COLO 205 and WiDr tumors with prolonged tumor retention (26.25 ± 2.85%ID/g vs. 12.20 ± 2.24 at 168 hours post injection; n = 5, SD), and uptake correlated both with DR5 expression on tumor cells and antitumor activity. DR5 saturation was shown in vivo via both biodistribution studies and planar gamma camera imaging/CT imaging of 111In-labeled CS-1008. Saturation of DR5 corresponded to maximal in vivo antitumor efficacy. Conclusions: Imaging of DR5 receptor occupancy in vivo correlates with tumor concentration and in vivo efficacy, and is a novel molecular imaging technique that can be used to determine receptor occupancy and effective dose levels of DR5 agonist antibodies in the clinic. Clin Cancer Res; 19(21); 5984–93. ©2013 AACR.

Radiolabelling and evaluation of a novel sulfoxide as a PET imaging agent for tumor hypoxia

[¹⁸F]FMISO is the most widely validated PET radiotracer for imaging hypoxic tissue. However, as a result of the pharmacokinetics of [¹⁸F]FMISO a 2h wait between tracer administration and patient scanning is required for optimal image acquisition. In order to develop hypoxia imaging agents with faster kinetics, we have synthesised and evaluated several F-18 labelled anilino sulfoxides. In this manuscript we report on the synthesis, in vitro and in vivo evaluation of a novel fluoroethyltriazolyl propargyl anilino sulfoxide. The radiolabelling of the novel tracer was achieved via 2-[¹⁸F]fluoroethyl azide click chemistry. Radiochemical yields were 23 ± 4% based on 2-[¹⁸F]fluoroethyl azide and 7 ± 2% based on K[¹⁸F]F. The radiotracer did not undergo metabolism or defluorination in an in vitro assay using S9 liver fractions. Imaging studies using SK-RC-52 tumors in BALB/c nude mice have indicated that the tracer may have a higher pO₂ threshold than [¹⁸F]FMISO for uptake in hypoxic tumors. Although clearance from muscle was faster than [¹⁸F]FMISO, uptake in hypoxic tumors was slower. The average tumor to muscle ratio at 2h post injection in large, hypoxic tumors with a volume greater than 686 mm³ was 1.7, which was similar to the observed ratio of 1.75 for [¹⁸F]FMISO. Although the new tracer showed improved pharmacokinetics when compared with the previously synthesised sulfoxides, further modifications to the chemical structure need to be made in order to offer significant in vivo imaging advantages over [¹⁸F]FMISO.

Abstract 5318: Optimizing pharmacokinetics and targeting properties of recombinant anti-Lewis Y antibody hu3S193 in A431 tumor-bearing mice

Aim: The humanized monoclonal antibody 3S193 (hu3S193) specifically binds the Lewis Y (Le y ) antigen and has shown a long half-life in Phase I first-in-man trials. Previous results have shown that mutations in residues I253, H310 and H435 of the Fc region interfere with the neonatal receptor (FcRn) binding. We have generated a hu3S193 antibody with an alanine mutation in residue I253 (I253A) that shows a shorter half-life than parental hu3S193. The objective of this pilot study was to evaluate the radiolabeling properties of 89 Zr-labelled hu3S193_I253A compared to its parent hu3S193 and evaluate them as small-animal positron emission tomography (PET) imaging agents. Methods: Site-directed mutagenesis was performed using a QuickChange XL II site-directed mutagenesis kit (Stratagene) to generate the hu3S193_I253A. The LONZA Glutamine Synthetase expression system was used for eukaryotic expression of antibody heavy and kappa light chain vectors. Mutants were produced in transient (Freestyle 293, Invitrogen) cells and tested for Le y binding using ELISA, FACS and BIAcore analysis. Parental hu3S193 and hu3S193_I253A were radiolabelled with 89 Zr after conjugation to deferroxamine-p-SCN (Df). The 89 Zr-Df-labelled antibodies were evaluated in small-animal PET studies at 4, 24, 48, and 72 hours post injection. At 72 hours after injection, animals were sacrificed. Blood samples were taken via cardiac puncture and organs were removed. Blood and organ samples were counted for radioactivity. Results: Analysis of hu3S193_I253A mutant by ELISA, BIAcore and FACS showed retention of Le y antigen binding ability. PET imaging demonstrated specific tumor uptake of both 89 Zr-Df-labeled parental and hu3S193_I253A at 24 hours post injection and this uptake was maintained for 6 days after injection. Lesions as small as 16 mm 3 were clearly detected with both agents. Higher uptake in the liver was detected when using 89 Zr-Df-labeled hu3S193_I253A. The 72 hour biodistribution data displayed a higher non-specific uptake in the liver with hu3S193_I253A versus parental (i.e. 15 %ID/g versus 6 %ID/g respectively). For comparison, using 111 In-CHX-A”-hu3S193_I253A liver uptake was 7 ± 0.3 %ID/g. This observation indicates that improvement to the 89 Zr-labelling chemistry could reduce the liver uptake of faster clearing hu3S193 constructs. Conclusion: The pilot study of 89 Zr-labelled hu3S193 and hu3S193_I253A demonstrated excellent tumor uptake of both agents, with high-contrast images generated of lesions as small as 16mm 3 . Improvements in 89 Zr radiolabelling chemistry may be required to optimize imaging of fast clearing recombinant antibodies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5318. doi:10.1158/1538-7445.AM2011-5318