Roger M. Bjerke

Detection of colorectal polyps in humans using an intravenously administered fluorescent peptide targeted against c-Met

Colon cancer prevention currently relies on colonoscopy using white light to detect and remove polyps, but small and flat polyps are difficult to detect and frequently missed when using this technique. Fluorescence colonoscopy combined with a fluorescent probe specific for a polyp biomarker may improve polyp detection. Here we describe GE-137, a water-soluble probe consisting of a 26–amino acid cyclic peptide that binds the human tyrosine kinase c-Met conjugated to a fluorescent cyanine dye. Intravenous administration of GE-137 leads to its accumulation specifically in c-Met–expressing tumors in mice, and it is safe and well tolerated in humans. Fluorescence colonoscopy in patients receiving intravenous GE-137 enabled visualization of all neoplastic polyps that were visible with white light (38), as well as an additional nine polyps that were not visible with white light. This first-in-human pilot study shows that molecular imaging using an intravenous fluorescent agent specific for c-Met is feasible and safe, and that it may enable the detection of polyps missed by other techniques.

Design and Characterization of Targeted Ultrasound Microbubbles for Diagnostic Use

Targeted ultrasound (US) contrast agents represent, because of their size (1 to 5 μm), a unique class of diagnostic imaging agents enabling true vascular imaging of conditions like inflammation and tumor angiogenesis. The objective of this study was to develop technology for preparing targeted microbubbles with binding and acoustic properties compatible with diagnostic use. Phosphatidylcholine (PC) was shown to represent the most favorable wall material. Various thiolated peptide binders were effectively conjugated to PC-based microbubbles containing maleimide functionalized lipids (95:5) without the need for biotin-streptavidin or antibody technology. By optimizing the technology, specific targeting of the inflammatory target E-selectin and the angiogenic target VEGFR2 in the presence of 100% serum was achieved. Increased phospholipid chain length from 18 carbons to 22 carbons improved the stability of the microbubbles during US exposure, without compromising binding or acoustic properties.

Whole-body section fluorescence imaging--a novel method for tissue distribution studies of fluorescent substances.

The present study demonstrates the usefulness of whole body section fluorescence imaging, a novel technique used in optical imaging drug discovery. This method is in principle an analog of whole body autoradiography, except that fluorescence is measured instead of radioactivity. The method was shown to have a linear concentration-response relationship over a 1000-fold concentration range. Densitometric image analysis allowed semiquantitative studies of drug disposition and selective tumor retention of an optical imaging drug candidate.

Targeted alpha therapy using a novel CD70 targeted thorium-227 conjugate in in vitro and in vivo models of renal cell carcinoma

The cell surface receptor CD70 has been previously reported as a promising target for B-cell lymphomas and several solid cancers including renal cell carcinoma. We describe herein the characterization and efficacy of a novel CD70 targeted thorium-227 conjugate (CD70-TTC) comprising the combination of the three components, a CD70 targeting antibody, a chelator moiety and the short-range, high-energy alpha-emitting radionuclide thorium-227 (227Th). In vitro analysis demonstrated that the CD70-TTC retained binding affinity to its target and displayed potent and specific cytotoxicity compared to an isotype control-TTC. A biodistribution study in subcutaneous tumor-bearing nude mice using the human renal cell carcinoma cell line 786-O demonstrated significant uptake and retention with 122 ± 42% of the injected dose of 227Th per gram (% ID/g) remaining in the tumor seven days post dose administration compared to only 3% ID/g for the isotype control-TTC. Tumor accumulation correlated with a dose dependent and statistically significant inhibition in tumor growth compared to vehicle and isotype control-TTC groups at radioactivity doses as low as 50 kBq/kg. The CD70-TTC was well tolerated as evidenced by only modest changes in hematology and normal gain in body weight of the mice. To our knowledge, this is the first report describing molecular targeting of CD70 expressing tumors using a targeted alpha-therapy (TAT).The cell surface receptor CD70 has been previously reported as a promising target for B-cell lymphomas and several solid cancers including renal cell carcinoma. We describe herein the characterization and efficacy of a novel CD70 targeted thorium-227 conjugate (CD70-TTC) comprising the combination of the three components, a CD70 targeting antibody, a chelator moiety and the short-range, high-energy alpha-emitting radionuclide thorium-227 (227Th). In vitro analysis demonstrated that the CD70-TTC retained binding affinity to its target and displayed potent and specific cytotoxicity compared to an isotype control-TTC. A biodistribution study in subcutaneous tumor-bearing nude mice using the human renal cell carcinoma cell line 786-O demonstrated significant uptake and retention with 122 ± 42% of the injected dose of 227Th per gram (% ID/g) remaining in the tumor seven days post dose administration compared to only 3% ID/g for the isotype control-TTC. Tumor accumulation correlated with a dose dependent and statistically significant inhibition in tumor growth compared to vehicle and isotype control-TTC groups at radioactivity doses as low as 50 kBq/kg. The CD70-TTC was well tolerated as evidenced by only modest changes in hematology and normal gain in body weight of the mice. To our knowledge, this is the first report describing molecular targeting of CD70 expressing tumors using a targeted alpha-therapy (TAT).

A targeted molecular probe for colorectal cancer imaging

Colorectal cancer is a major cause of cancer death. Morbidity, mortality and healthcare costs can be reduced if the disease can be detected at an early stage. Screening is a viable approach as there is a clear link to risk factors such as age. We have developed a fluorescent contrast agent for use during colonoscopy. The agent is administered intravenously and is targeted to an early stage molecular marker for colorectal cancer. The agent consists of a targeting section comprising a peptide, and a fluorescent reporter molecule. Clinical imaging of the agent is to be performed with a far red fluorescence imaging channel (635 nm excitation/660-700 nm emission) as an adjunct to white light colonoscopy. Preclinical proof of mechanism results are presented. The compound has a Kd of ~3nM. Two human xenograft tumour models were used. Tumour cells were implanted and grown subcutaneously in nude mice. Imaging using a fluorescence reflectance imaging system and quantitative biodistribution studies were performed. Substances tested include the targeted agent, and a scrambled sequence of the peptide (no binding) used as a negative control. Competition studies were also performed by co-administration of 180 times excess unlabelled peptide. Positive imaging contrast was shown in the tumours, with a clear relationship to expression levels (confirmed with quantitative biodistribution data). There was a significant difference between the positive and negative control substances, and a significant reduction in contrast in the competition experiment.

Abstract 357: Nonclinical tumor efficacy studies of [18F]AH113804, a novel PET imaging agent with high affinity for the human c-Met receptor

c-Met, the tyrosine-kinase receptor for hepatocyte growth factor (HGF)/scatter factor, is involved in tumour growth, invasion and metastasis in many human cancers of epithelial origin. Various tumour types are reported to overexpress c-Met, whilst expression in most normal tissues is relatively low. [18F]AH113804 is a peptide-based molecular imaging agent being developed by GE Healthcare for the in vivo evaluation of tumour and metastatic c-Met expression by PET imaging. In vitro affinity studies with fluorescently labelled analogues confirmed that whilst the AH113804 peptide had high affinity for human c-Met (hc-Met), there was limited or no affinity for dog or rodent c-Met, respectively. In mice, relatively high uptake of [18F]AH113804 was observed in human xenograft tumours known to express high levels of hc-Met, with rapid clearance from key background tissues such as muscle (tumour to muscle ratio of >5 achieved by 30 minutes post-injection). This biodistribution profile allowed the tumour to be clearly visualised by micro-PET imaging. Tumour uptake was significantly reduced by co-administration of excess non-radioactive peptide, confirming tumour uptake was specific. Tumour uptake of [18F]AH113804 was also shown to correlate with expression of hcMet, with a relative retention of 2.0±0.1, 1.2±0.2 and 0.7±0.2% retained dose per gram normalised for body weight (% rd/g/100g bw) 60 minutes post-injection in xenograft tumours with relatively high (HT-29 tumours), lower (U87 tumours) and no (LLC tumours) expression of hc-Met respectively (as assayed by ELISA). Finally, 111InCl3 was used in a dual tumour model as a non-specific marker of blood pool to confirm differences in tumour uptake were not related to differences in tumour blood pool or delivery. There was a significant difference in [18F]AH113804 uptake between HT-29 (2.2±0.8% rd/g/100 g bw) and LLC (0.9±0.2% rd/g/100 g bw) tumours (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 357. doi:1538-7445.AM2012-357

Abstract 5200: Preclinical pharmacology of the PSMA-targeted thorium-227 conjugate PSMA-TTC: a novel targeted alpha therapeutic for the treatment of prostate cancer

Prostate-specific membrane antigen (PSMA, FOLH1) is a type II transmembrane glycoprotein of the M28 peptidase family that acts as a glutamate carboxypeptidase on various substrates. PSMA is well established as a target antigen in prostate cancer due to its high and specific overexpression on the surface of prostate cancer cells at all tumor stages, including metastatic and hormone-refractory disease. Several PSMA targeting antibodies and ligands are currently in clinical development or compassionate use therapeutically or as imaging agents. Targeted alpha therapy (TAT) has an established clinical profile with the successful transition of Ra223, an alpha-particle emitter, from bench to bedside in prostate cancer. Thorium-227 is the immediate precursor for Ra223 via alpha-particle emission. We herein describe the generation of a novel TAT, a high energy, alpha-particle emitting PSMA-targeted thorium-227 conjugate (PSMA-TTC). PSMA-TTC consists of a fully human PSMA targeting IgG1 antibody covalently linked via an amide bond to a chelator moiety (3,2 HOPO), enabling radiolabeling with thorium-227 (227Th). PSMA-TTC was prepared in high radiochemical yield and purity and tested for binding affinity to PSMA target (ELISA) as well as PSMA expressing cell lines (FACS). In vitro cytotoxicity experiments were carried out on prostate CA cell lines with different PSMA levels (from 3.000 to 150.000 mAbs bound/ cell). In vivo biodistribution and anti-tumor efficacy were analyzed after i.v. injection of 100-500 kBq/kg at protein doses of 0.14 mg/kg to mice bearing s.c. prostate cancer xenograft models. Additionally, anti-tumor efficacy was evaluated in a PSMA expressing orthotopic bone xenograft model (LNCaP-Luc) monitored by bioluminescence imaging, micro CT and x-ray. PSMA-TTC retains binding affinities to PSMA target and PSMA positive cancer cells similar to the PSMA antibody. Strong in vitro potency and selectivity of PSMA-TTC was shown on different PSMA positive cells. Biodistribution studies in C4-2 xenografts demonstrated specific tumor uptake of PSMA-TTC with a maximum of 50 % of ID/g at t = 72h post dose administration. Selective significant antitumor efficacy was shown for PSMA-TTC in s.c. prostate CA xenograft models with high (C4-2) and medium/low (22Rv1) PSMA protein levels at doses of 250 and 500 kBq/kg. Furthermore, statistically significant prevention of tumor growth was observed after treatment with PSMA-TTC at a dose of 100 kBq/kg in an orthotopic bone xenograft model (LNCaP-Luc). The promising preclinical antitumor activity of PSMA-TTC supports its development for the treatment of patients with metastatic prostate cancer. Citation Format: Stefanie Hammer, Aasmund Larssen, Christine Ellingsen, Solene Geraudie, Derek Grant, Baard Indrevoll, Oliver von Ahsen, Alexander Kristian, Urs B Hagemann, Jenny Karlsson, Roger M Bjerke, Olav B Ryan, Dominik Mumberg, Bertolt Kreft, Alan Cuthbertson. Preclinical pharmacology of the PSMA-targeted thorium-227 conjugate PSMA-TTC: a novel targeted alpha therapeutic for the treatment of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5200. doi:10.1158/1538-7445.AM2017-5200

Imaging efficacy of a targeted imaging agent for fluorescence endoscopy

Colorectal cancer is a major cause of cancer death. A significant unmet clinical need exists in the area of screening for earlier and more accurate diagnosis and treatment. We have identified a fluorescence imaging agent targeted to an early stage molecular marker for colorectal cancer. The agent is administered intravenously and imaged in a far red imaging channel as an adjunct to white light endoscopy. There is experimental evidence of preclinical proof of mechanism for the agent. In order to assess potential clinical efficacy, imaging was performed with a prototype fluorescence endoscope system designed to produce clinically relevant images. A clinical laparoscope system was modified for fluorescence imaging. The system was optimised for sensitivity. Images were recorded at settings matching those expected with a clinical endoscope implementation (at video frame rate operation). The animal model was comprised of a HCT-15 xenograft tumour expressing the target at concentration levels expected in early stage colorectal cancer. Tumours were grown subcutaneously. The imaging agent was administered intravenously at a dose of 50nmol/kg body weight. The animals were killed 2 hours post administration and prepared for imaging. A 3-4mm diameter, 1.6mm thick slice of viable tumour was placed over the opened colon and imaged with the laparoscope system. A receiver operator characteristic analysis was applied to imaging results. An area under the curve of 0.98 and a sensitivity of 87% [73, 96] and specificity of 100% [93, 100] were obtained.

Abstract 5859: HER2-targeted thorium-227 conjugate (HER2-TTC): Efficacy in preclinical models of trastuzumab and T-DM1 resistance

The human epidermal growth factor receptor 2 (HER2) is encoded by the proto-oncogene c-erbB-2 and initiates downstream signaling pathways leading to cell proliferation and tumorigenesis. HER2 is overexpressed in several cancer (Ca) types and is one of the most strongly validated targets for the treatment of breast and gastric cancer serving as both a prognostic and predictive biomarker. Several HER2-targeting antibodies as well as antibody-drug conjugates are either approved or are in clinical development. Prolonged treatment with monoclonal antibodies and antibody drug conjugates have resulted in development of resistance and so there is still an unmet medical need for drugs of new mechanism of action targeting this important receptor system. We describe herein the generation of a high energy, alpha-particle emitting HER2 targeted thorium-227 antibody-chelator conjugate. HER2-TTC consists of the humanized HER2 targeting IgG1 antibody (trastuzumab) covalently linked via an amide bond to a 3,2-hydroxypyridino-based chelator moiety, enabling efficient radiolabeling with the alpha particle emitting radionuclide thorium-227 (Th-227). HER2-TTC was prepared at high radiochemical yield and purity. When tested for binding to recombinant HER2, HER2-TTC was shown to retain comparable binding affinity to trastuzumab. In vitro cytotoxicity experiments were performed on 8 cell lines with different HER2 expression levels (from 7 000 - 500 000 mAbs bound/ cell as determined by FACS) of breast, ovarian, gastric and lung cancer origin. HER2-TTC demonstrated target mediated in vitro cytotoxicity in the pM-range. In vivo biodistribution and anti-tumor efficacy of HER2-TTC was evaluated in the dose range 100-500 kBq/kg at a protein dose of 0.14 mg/kg and i.v. injection in the s.c. KPL-4 breast and Calu-3 lung model previously described to be resistant to trastuzumab. The biodistribution study demonstrated specific tumor accumulation of HER2-TTC in both models with a maximum of 77 and 50 %ID/g 227Th at t = 168 h post dose (decay corrected to T0), respectively. Significant antitumor efficacy was shown for HER2-TTC in the JIMT-1 s.c. breast Ca xenograft model resistant to trastuzumab and T-DM1. The promising preclinical anti-tumor activity supports the development of the targeted alpha therapeutic HER2-TTC for the treatment of trastuzumab and T-DM1 resistant patients. Citation Format: Jenny Karlsson, Urs B. Hagemann, Christoph Schatz, Derek Grant, Alexander Kristian, Christine Ellingsen, Dessislava Mihaylova, Solene Geraudie, Bard Indrevoll, Uta Wirnitzer, Roger M. Bjerke, Olav B. Ryan, Carl F. Nising, Dominik Mumberg, Alan Cuthbertson. HER2-targeted thorium-227 conjugate (HER2-TTC): Efficacy in preclinical models of trastuzumab and T-DM1 resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5859. doi:10.1158/1538-7445.AM2017-5859

Abstract 5857: HER2-targeted thorium-227 conjugate (HER2-TTC): Efficacy in a HER2 positive orthotopic bone model

In 2015, the estimated incidence of new breast cancer (Ca) cases in the US was 234.190 and number of deaths 40.730. Human epidermal growth factor receptor 2 (HER2) is encoded by the proto-oncogene c-erbB-2 and initiates downstream signaling pathways leading to cell proliferation and tumorigenesis. HER2 is overexpressed in several cancer types and has emerged as one of the most strongly validated targets for the treatment of breast and gastric cancer serving as both a prognostic and predictive biomarker. Given that 20% of breast Ca patients are HER2 positive and 70% of patients with metastatic disease will develop bone metastases and associated morbidities, there is still an unmet medical need for improved therapies targeting HER2. Radium-223 (Ra-223) is a novel targeted alpha therapeutic for treatment of patients with castration-resistant prostate cancer and bone metastases. Localized high energy alpha particle emission induces double-stranded DNA breaks and cellular apoptosis. Thorium-227 (Th-227) is the immediate precursor for Ra-223 which, in contrast to Ra-223, can be complexed by chelating agents at high affinity, allowing targeted delivery to tumor cells via antibodies. We describe herein the generation of a novel HER2-targeted Th-227 conjugate (HER2-TTC). HER2-TTC consists of the humanized HER2 targeting IgG1 antibody trastuzumab covalently linked via an amide bond to a 3,2-hydroxypyridino-based chelator moiety, enabling efficient radiolabeling with the alpha particle emitting radionuclide Th-227. Anonymized samples of consenting breast cancer patients were analyzed by Immunohistochemistry (IHC). The IHC data demonstrated HER2 positive expression in breast tumor and matched bone metastases, supporting the preclinical evaluation of the anti-tumor efficacy of HER2-TTC in the BT-474 orthotopic bone mouse model. HER2-TTC was prepared at high radiochemical yield and purity. When tested for binding to recombinant HER2, HER2-TTC was shown to retain comparable binding affinity to trastuzumab. In vitro cytotoxicity experiment of HER2-TTC demonstrated target mediated in vitro cytotoxicity in the pM-range on breast cancer cell line BT-474 (430 000 mAbs bound/ cell as determined by FACS). Anti-tumor efficacy of HER2-TTC was evaluated at 250 and 500 kBq/kg at a protein dose of 0.14 mg/kg. X-ray imaging, serum bone formation marker PINP, micro CT 3D reconstruction imaging and histological analysis demonstrated significantly reduced bone lesions and tumor induced bone remodeling. The promising preclinical anti-tumor activity supports the development of the HER2-TTC as a novel targeted alpha therapeutic for the treatment of patients with HER2 positive bone metastatic disease. Citation Format: Jenny Karlsson, Urs B. Hagemann, Christoph Schatz, Derek Grant, Christine Ellingsen, Alexander Kristian, Dessislava Mihaylova, Steinar R. Uran, Mari Suominen, Roger M. Bjerke, Olav B. Ryan, Carl F. Nising, Dominik Mumberg, Alan Cuthbertson. HER2-targeted thorium-227 conjugate (HER2-TTC): Efficacy in a HER2 positive orthotopic bone model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5857. doi:10.1158/1538-7445.AM2017-5857