Daniel Bradley

Molecular imaging of aurora kinase A (AURKA) expression: Synthesis and preclinical evaluation of radiolabeled alisertib (MLN8237).

INTRODUCTION Survival of patients after resection of colorectal cancer liver metastasis (CRCLM) is 36%-58%. Positron emission tomography (PET) tracers, imaging the expression of prognostic biomarkers, may contribute to assign appropriate management to individual patients. Aurora kinase A (AURKA) expression is associated with survival of patients after CRCLM resection. METHODS We synthesized [(3)H]alisertib and [(11)C]alisertib, starting from [(3)H]methyl nosylate and [(11)C]methyl iodide, respectively. We measured in vitro uptake of [(3)H]alisertib in cancer cells with high (Caco2), moderate (A431, HCT116, SW480) and low (MKN45) AURKA expression, before and after siRNA-mediated AURKA downmodulation, as well as after inhibition of P-glycoprotein (P-gp) activity. We measured in vivo uptake and biodistribution of [(11)C]alisertib in nude mice, xenografted with A431, HCT116 or MKN45 cells, or P-gp knockout mice. RESULTS [(3)H]Alisertib was synthesized with an overall yield of 42% and [(11)C]alisertib with an overall yield of 23%±9% (radiochemical purity ≥99%). Uptake of [(3)H]alisertib in Caco2 cells was higher than in A431 cells (P=.02) and higher than in SW480, HCT116 and MKN45 cells (P<.01). Uptake in A431 cells was higher than in SW480, HCT116 and MKN45 cells (P<.01). Downmodulation of AURKA expression reduced [(3)H]alisertib uptake in Caco2 cells (P<.01). P-gp inhibition increased [(3)H]alisertib uptake in Caco2 (P<.01) and MKN45 (P<.01) cells. In vivo stability of [(11)C]alisertib 90min post-injection was 94.7%±1.3% and tumor-to-background ratios were 2.3±0.8 (A431), 1.6±0.5 (HCT116) and 1.9±0.5 (MKN45). In brains of P-gp knockout mice [(11)C]alisertib uptake was increased compared to uptake in wild-type mice (P<.01) CONCLUSIONS: Radiolabeled alisertib can be synthesized and may have potential for the imaging of AURKA, particularly when AURKA expression is high. However, the exact mechanisms underlying alisertib accumulation need further investigation. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE Radiolabeled alisertib may be used for non-invasively measuring AURKA protein expression and to stratify patients for treatment accordingly.

Dual-Isotope Cryoimaging Quantitative Autoradiography: Investigating Antibody–Drug Conjugate Distribution and Payload Delivery Through Imaging

In vitro properties of antibody–drug conjugates (ADCs) such as binding, internalization, and cytotoxicity are often well characterized before in vivo studies. Interpretation of in vivo studies might be significantly enhanced by molecular imaging tools. We present here a dual-isotope cryoimaging quantitative autoradiography (CIQA) methodology combined with advanced 3-dimensional imaging and analysis allowing for the simultaneous study of both antibody and payload distribution in tissues of interest in a preclinical setting. Methods: TAK-264, an investigational ADC targeting anti–guanylyl cyclase C (GCC), was synthesized using tritiated monomethyl auristatin E. The tritiated ADC was then conjugated to diethylenetriaminepentaacetic acid, labeled with 111In, and evaluated in vivo in animals bearing GCC-positive and GCC-negative tumors. Results: CIQA revealed the time course of drug release from ADC and its distribution into various tumor regions that are less accessible to the antibody. For GCC-positive tumors, a representative section obtained 96 h after tracer injection showed only 0.8% of the voxels to have colocalized signal, versus over 15% of the voxels for a GCC-negative tumor section, suggesting successful and specific cleaving of the toxin in the GCC-positive lesions. Conclusion: The combination of a veteran established autoradiography technology with advanced image analysis methodologies affords an experimental tool that can support detailed characterization of ADC tumor penetration and pharmacokinetics.

Abstract LB-185: A dual-isotope 3D cryo-imaging quantitative autoradiography (CIQA) method for simultaneous and quantitative assessment of both antibody and drug conjugate tumor distribution and kinetics

Antibody-drug conjugates (ADCs) are optimized extensively in in vitro studies. Including drug-conjugate selection, linker stability and antibody affinity, most ADC characteristics have been studied to improve affinity, stability, efficacy and the bystander effect. Most nuclear medicine molecular imaging modalities’ resolution is too low to enable accurate in vivo intratumoral tracer distribution analysis. Thus, they have focused on understanding in vivo PK profiles and antigen-dependent tumor accumulation. We set out to develop a novel method for studying the intratumoral distribution of both antibody and drug conjugate simultaneously ex vivo. MLN0264, an ADC targeting guanylyl cyclase C (GCC) currently in phase 2 clinical trials (NCT02391038), was labeled with both 3H (MMAE drug conjugate) and 111In (DTPA-mAb) and injected into GCC-positive (GCC-293) and GCC-negative (HEK-293) subcutaneous tumor bearing female SCID mice. The tumors were excised 1, 8, 24, and 96 hours post injection (n = 2 per tumor line per time point), blocked and sectioned (30 μm) for analysis. High resolution optical images were acquired for all sections and every 10th section was evaluated for radioactivity content via autoradiography, first to evaluate the distribution of 111In immediately after tumor excision and again following 111In decay to evaluate the 3H-specific signal. Analysis of accumulation, distribution and overlap of the two signals enables the estimation of antigen-mediated metabolism of the ADC, the tumoral distribution of the drug metabolites and the time course of the bystander effect (Fig 1A). The distribution of 3H and 111In signals at 1 h was very similar for the two cell lines. At 24 and 96 hours, substantial differences in the co-localization of signals were observed between the antigen-positive and antigen-negative tumors. Quantitatively, of the top 3% of pixel values in the 3H and 111In images at 96 hours, only 0.8% of GCC-293 tumor voxels shows an overlap of signals while over 15% of voxels in the HEK-293 tumors express both signals suggesting increased ADC metabolism and bystander effect in antigen positive tumors (Fig 1B). Cryo-Imaging Quantitative Autoradiography (CIQA) is a novel technique to extend conventional autoradiography by combining it with digital imaging and advanced 3D image analysis. For the first time, we demonstrate here the use of CIQA to quantify and visualize the two major components of an ADC, the mAb and small molecule drug simultaneously in three dimensions over time (4D). We believe this powerful and unique tool will allow for increased insight into the influence of ADC properties on tumor spatial distribution, in vivo bystander effect, off-target ADC metabolism and correlation of 3D distribution and heterogeneity with immunohistochemical markers to enable more accurate pharmacodynamic profiles. Citation Format: Ohad Ilovich, Mohammed Qutaish, Jacob Hesterman, Kelly Orcutt, Jack Hoppin, Ildiko Polyak, Marc Seaman, Paige Czarnecki, Vijay Gottumukkala, Mihaela Plesescu, Ozlem Yardibi, Daniel Bradley. A dual-isotope 3D cryo-imaging quantitative autoradiography (CIQA) method for simultaneous and quantitative assessment of both antibody and drug conjugate tumor distribution and kinetics. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-185.

Abstract 112: Synthesis and preclinical evaluation of radiolabeled alisertib as an investigational aurora kinase A PET tracer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: High expression of the aurora kinase A (AURKA) protein is associated with colorectal adenoma-to-carcinoma progression and with poor prognosis of patients with stage III colorectal cancer (CRC) and patients with CRC liver metastases. Several agents have been developed that specifically target AURKA kinase activity, among those the investigational agent alisertib. To identify CRC patients with high AURKA expression levels a dedicated imaging method would be preferred, since only then a whole body assessment of AURKA expression in primary tumors as well as in metastases can be achieved. Positron emission tomography (PET) using a dedicated radiotracer allows for this. To this end, [3H]alisertib has been synthesized for in vitro experiments and [11C]alisertib for in vivo imaging with PET in xenograft mouse models. Methods: [3H]alisertib was synthesized starting from [3H]methyl nosylate under similar conditions as [11C]alisertib, which was synthesized starting from [11C]CH3I in a two-step procedure, purified and formulated within 45 minutes. Four CRC cell lines with different levels of AURKA expression were selected, HCT116, SW480, SW1398 and Caco-2, and the in vitro dynamic uptake of [3H]alisertib in these cell lines was measured before and after siRNA-mediated AURKA downmodulation. Next, the uptake of [11C]alisertib was assessed with PET in xenografted mice using the same four CRC cell lines. Results: The synthesis of [3H]alisertib was optimized to an overall yield of 42%, while an overall yield of 23 ± 4%, starting from [11C]CH3I, was obtained in an optimized synthesis of [11C]alisertib. The CRC cell line with high expression of AURKA, Caco-2, showed a significantly higher uptake of [3H]alisertib compared to the lower expressing AURKA CRC cell lines, HCT116, SW480 and SW1398. In addition, the uptake of [3H]alisertib was reduced in all cell lines upon downmodulation of AURKA. The stability of [11C]alisertib in rodents was determined at 97.8% ± 1.3% intact tracer in blood at 45 minutes after iv injection (n=4). Preliminary data using a HCT116 xenograft mouse model indicated a tumor-to-background ratio of 1.56 ± 0.12, with an uptake of 1.00 %ID/g at 90 minutes after injection. PET studies with the SW480, SW1398 and Caco-2 xenografts are currently in progress. Conclusions: Both [3H]alisertib and [11C]alisertib were obtained with good purity and yield. In vitro studies using [3H]alisertib indicate good correlation of cellular uptake with AURKA expression. [11C]alisertib is stable in vivo and HCT116 xenografted mice show a fair tumor uptake of [11C]alisertib. Citation Format: Joost Verbeek, Jeroen ACM Goos, Albert A. Geldof, Annemieke C. Hiemstra, Otto S. Hoekstra, Gerrit A. Meijer, Steven Stroud, Daniel Bradley, Remond JA Fijneman, Albert D. Windhorst. Synthesis and preclinical evaluation of radiolabeled alisertib as an investigational aurora kinase A PET tracer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 112. doi:10.1158/1538-7445.AM2014-112

Abstract 365: Using radionuclear imaging and mechanistic modeling to assess the therapeutic potential of antibody-drug conjugates (ADCs)

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Antibody-drug conjugates (ADCs) comprise a drug class that allows for direct delivery of a cytotoxic agent to the target tumor cell. The successful development of an ADC involves the assessment of patient characteristics such as tumor antigen expression and tumor vascularity. There is a need to develop a mechanistic understanding of the effect these tumor parameters have on ADC biological activity. This mechanistic insight can be derived from a mathematical model integrating quantitative preclinical data, and will support ADC development. MLN0264 is an investigational ADC consisting of a human anti-guanylyl cyclase C (GCC) antibody linked to the microtubule-disrupting agent monomethyl auristatin (MMAE). Here, we use mathematical modeling, in conjunction with in vivo imaging, to decouple the contribution of different tumor parameters to overall ADC biological activity. We constructed a mathematical model of ADC biological activity by integrating experimental results from (1) in vivo antibody imaging studies, (2) in vitro viability assays, and (3) in vivo xenograft biological activity studies. First, blood pharmacokinetics and tumor disposition were quantitatively constrained using in vivo radiolabeled antibody single-photon emission computed tomography (SPECT) data for both blood and tumor tissues. SPECT data from three xenografts with various antigen expression levels were used to link antigen expression level to ADC uptake. Second, the relationship between bound GCC receptor concentration and cell viability was established using viability assays run on an engineered cell line (293-GCC) with high antigen expression and high sensitivity to MMAE. Finally, using this relationship, we built a tumor growth dynamics model to describe in vivo xenograft biological activity, and to estimate the growth inhibition coefficient of 293-GCC. This mechanistic model can be used to gain insights into the factors driving response of a tumor that is intrinsically sensitive to MMAE. Our results indicate the process of ADC vascular permeability is one of the limiting factors of ADC disposition. This outcome is reasonable given that a large ADC molecular weight decreases the permeability. Furthermore, the model simulations suggest some tumors that are intrinsically sensitive to MMAE may not be affected by the ADC if GCC antigen expression levels are very low. Taken together, the mechanistic model developed here forms the basis of a quantitative understanding for several factors influencing MLN0264 patient selection. Citation Format: Shu-Wen Teng, Ozlem Yardibi, Julie Zhang, Donna Cvet, Johnny Yang, Kelly Orcutt, Melissa Gallery, Arijit Chakravarty, Wen Chyi Shyu, Jerome Mettetal, Daniel Bradley, Petter Veiby. Using radionuclear imaging and mechanistic modeling to assess the therapeutic potential of antibody-drug conjugates (ADCs). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 365. doi:10.1158/1538-7445.AM2014-365

Abstract 4949: In vitro and in vivo investigation of the novel, first-in-class, Guanylyl Cyclase C (GCC) targeted 68Ga labeled heat stable peptide MLN6907 ([68Ga]MLN6907) for tumor imaging

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA A novel, first-in-class, 68Ga labeled DOTA-para-bn-SCN-Ahx-STp(5-18) 2.2kDa peptide \[MLN6907\] ([68Ga]MLN6907) with high affinity to the guanalyl cyclase C (GCC) receptor has been developed to select cancer patients for treatment with GCC targeted therapies using PET/CT. GCC is sequestered exclusively in the gastrointestinal luminal compartment except under malignant transformation where it is then made accessible to intravenous agents. Conventional patient selection strategies often rely on an analytical IHC or total protein based assessment of a tumor biopsy which can be limited to an archival tissue sample from a single region of a single lesion. Imaging may offer whole body, real time, multi-region and multi lesion assessment of target levels. In addition, functional parameters associated with receptor kinetics may also be explored in vivo. 68Ga has a high avidity for the common chelating moiety DOTA. Furthermore, its short radioactive half life (half-life, 68.3 minutes) matches well to a biological targeting moiety like a peptide with its rapid biological clearance and fast diffusion to target thus providing optimal tumour-to-normal tissue contrast. In vitro: Cellular competitive binding studies confirmed high affinity of non-radiolabeled MLN6907 for its cognate receptor, GCC, with a KD of 3.2 nM. Upon ligand-recpetor binding, MLN6907 is internalized rapidly with a half-life of 56 min. In vivo: Similar to other radiolabeled peptides, [68Ga]MLN6907 clears rapidly from blood (t1/2 = 26 min) through renal excretion as investigated in Long Evans rats and non-human primate studies. The radiation exposure from [68Ga]MLN6907 was highest in the kidney and bladder indicating that renal excretion was a primary route of elimination. Using OLINDA/EXM software, the effective dose was estimated to be 0.013 mSv/MBq in man. In tumor bearing C.B-17 SCID mice, both ex vivo and in vivo signal was measured in both human tumor cell lines and primary human tumor xenografts with varied GCC levels and compared to non tumor bearing tissues. [68Ga]MLN6907 total uptake (%I.D./g) varied across the different tumors investigated. Using the unlabeled precursor, no toxicity was observed in repeat dose rat and monkey studies. Using the rat as the more conservative species for dose estimation, it was calculated that a human equivalent dose of 282 μg would be safe. However, using a saturable effect PK/PD compartment model for mice and humans, we estimated a single human dose of less than 100μg would provide resolution of tumors with different GCC levels. Collectively, biological, pharmacokinetic and safety data obtained with [68Ga]MLN6907 are consistent with an effective GCC PET imaging agent. [68Ga]MLN6907 is being developed clinically as a single, i.v., microdose GCC PET imaging agent in a Phase 1 investigation in patients with surgically resectable metastatic colorectal carcinoma Citation Format: Donna Cvet, Robert Robertson, Melissa Saylor, Jennifer Terkelsen, Ozlem Yardibi, Maria Borland, Nicolas Salem, Petter Veiby, Todd Sells, Mary Carsillo, Johnny Yang, Shu-Wen Teng, John Hoppin, Kelly Orcutt, Jacob Hesterman, Jeffery Norenberg, Tamara Anderson, Mike Schulz, Mary Ruscowski, Marc Berridge, Steven Mather, Daniel P. Bradley. In vitro and in vivo investigation of the novel, first-in-class, Guanylyl Cyclase C (GCC) targeted 68Ga labeled heat stable peptide MLN6907 ([68Ga]MLN6907) for tumor imaging. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4949. doi:10.1158/1538-7445.AM2014-4949

Abstract 4948: PET/CT clinical protocol design for the novel, first in class 68Ga labeled guanylyl cyclase C targeted peptide MLN6907 ([68Ga]MLN6907)

Acquisition and interpretation guidelines for clinical PET/CT imaging in oncology have been designed for whole-body 18F-FDG imaging and may not be optimized for assessment of other PET imaging tracers. Here we describe a methodology of PET/CT study design for the novel first in class 68Ga-labeled Guanylyl cyclase C (GCC) targeted peptide, [68Ga]MLN6907, based on a combination of in vitro, ex vivo, and in vivo preclinical imaging studies and model-based estimation of tumor parameters from simulated clinical PET data. GCC, a protein expressed in GI malignancies, is being targeted by the antibody drug conjugate MLN0264. GCC is also expressed on the healthy apical surface of the intestinal epithelium, which should be inaccessible to intravascular treatment. [68Ga]MLN6907 binds GCC with high affinity and is being developed as an imaging biomarker in an effort to help identify patients likely to respond to GCC-targeted therapy. In a series of experiments, the peptide affinity, internalization rate, and clearance were determined in patient-derived CRC xenografts with varied tumor microenvironmental phenotype. In addition to supporting the clinical development of the imaging agent, this data was used in combination with simulated clinical list-mode PET data to evaluate tumor parameter estimability under several clinically viable acquisition and reconstruction conditions. Specifically, liver CRC metastases of varying tumor diameter, antigen density, and vascularity were simulated in combination with PET imaging acquisition duration and reconstruction with and without partial-volume correction. Tumor, liver, and background time-activity curves (TACs) were generated from the reconstructed data and analyzed using a distributed tumor model to estimate the known tumor antigen density and vascularity. Analysis of the simulation studies revealed: 1) Partial volume correction is required for accurate antigen density and vascularity estimation; 2) Parameter estimation was most accurate within a tumor size range of 1-5 cm; 3) Parameter estimation was robust for all tested TAC reconstruction durations (e.g., 2, 3, 5, and 10 min); 4) Parameter estimation was optimal for common clinical acquisition times of 30-90 minutes; 5) Antigen density estimation was less accurate in poorly vascularized tumors. For the translation of a novel clinical biomarker, well controlled preclinical studies are critical; and, in this case, the findings combined with the distributed tumor model simulations directly guided the clinical image protocol. This rational and data-driven approach has the ability to not only improve the estimation of tumor properties in human subjects but also to guide the design of first-in-human oncology clinical imaging protocols with novel biomarkers. Citation Format: Jacob Y. Hesterman, Kelly D. Orcutt, Ozlem Yardibi, Jerome T. Mettetal, Shu-Wen Teng, Donna Cvet, Jack Hoppin, Thea Kalebic, Daniel P. Bradley. PET/CT clinical protocol design for the novel, first in class 68Ga labeled guanylyl cyclase C targeted peptide MLN6907 ([68Ga]MLN6907). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4948. doi:10.1158/1538-7445.AM2014-4948