Ohad Ilovich

A systematic comparison of 18F-C-SNAT to established radiotracer imaging agents for the detection of tumor response to treatment

Purpose: An early readout of tumor response to therapy through measurement of drug or radiation-induced cell death may provide important prognostic indications and improved patient management. It has been shown that the uptake of 18F-C-SNAT can be used to detect early response to therapy in tumors by positron emission tomography (PET) via a mechanism of caspase-3–triggered nanoaggregation. Experimental Design: Here, we compared the preclinical utility of 18F-C-SNAT for the detection of drug-induced cell death to clinically evaluated radiotracers, 18F-FDG, 99mTc-Annexin V, and 18F-ML-10 in tumor cells in culture, and in tumor-bearing mice in vivo. Results: In drug-treated lymphoma cells, 18F-FDG, 99mTc-Annexin V, and 18F-C-SNAT cell-associated radioactivity correlated well to levels of cell death (R2 > 0.8; P < 0.001), with no correlation measured for 18F-ML-10 (R2 = 0.05; P > 0.05). A similar pattern of response was observed in two human NSCLC cell lines following carboplatin treatment. EL-4 tumor uptake of 99mTc-Annexin V and 18F-C-SNAT were increased 1.4- and 2.1-fold, respectively, in drug-treated versus naïve control animals (P < 0.05), although 99mTc-Annexin V binding did not correlate to ex vivo TUNEL staining of tissue sections. A differential response was not observed with either 18F-FDG or 18F-ML-10. Conclusions: We have demonstrated here that 18F-C-SNAT can sensitively detect drug-induced cell death in murine lymphoma and human NSCLC. Despite favorable image contrast obtained with 18F-C-SNAT, the development of next-generation derivatives, using the same novel and promising uptake mechanism, but displaying improved biodistribution profiles, are warranted for maximum clinical utility. Clin Cancer Res; 21(17); 3896–905. ©2015 AACR.

Development and Validation of an Immuno-PET Tracer as a Companion Diagnostic Agent for Antibody-Drug Conjugate Therapy to Target the CA6 Epitope

PURPOSE To develop and compare three copper 64 ((64)Cu)-labeled antibody fragments derived from a CA6-targeting antibody (huDS6) as immuno-positron emission tomography (immuno-PET)-based companion diagnostic agents for an antibody-drug conjugate by using huDS6. MATERIALS AND METHODS Three antibody fragments derived from huDS6 were produced, purified, conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and evaluated in the following ways: (a) the affinity of the fragments and the DOTA conjugates was measured via flow cytometry, (b) the stability of the labeled fragments was determined ex vivo in human serum over 24 hours, and (c) comparison of the in vivo imaging potential of the fragments was evaluated in mice bearing subcutaneous CA6-positive and CA6-negative xenografts by using serial PET imaging and biodistribution. Isotype controls with antilysozyme and anti-DM4 B-Fabs and blocking experiments with an excess of either B-Fab or huDS6 were used to determine the extent of the antibody fragment (64)Cu-DOTA-B-Fab binding specificity. Immunoreactivity and tracer kinetics were evaluated by using cellular uptake and 48-hour imaging experiments, respectively. Statistical analyses were performed by using t tests, one-way analysis of variance, and Wilcoxon and Mann-Whitney tests. RESULTS The antibody fragment (64)Cu-DOTA-B-Fab was more than 95% stable after 24 hours in human serum, had an immunoreactivity of more than 70%, and allowed differentiation between CA6-positive and CA6-negative tumors in vivo as early as 6 hours after injection, with a 1.7-fold uptake ratio between tumors. Isotype and blocking studies experiments showed tracer-specific uptake in antigen-positive tumors, despite some nonspecific uptake in both tumor models. CONCLUSION Three antibody fragments were produced and examined as potential companion diagnostic agents. (64)Cu-DOTA-B-Fab is a stable and effective immuno-PET tracer for CA6 imaging in vivo.

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.

Imaging B cells in a mouse model of multiple sclerosis using 64Cu-Rituximab-PET

B lymphocytes are a key pathologic feature of multiple sclerosis (MS) and are becoming an important therapeutic target for this condition. Currently, there is no approved technique to noninvasively visualize B cells in the central nervous system (CNS) to monitor MS disease progression and response to therapies. Here, we evaluated 64Cu-rituximab, a radiolabeled antibody specifically targeting the human B cell marker CD20, for its ability to image B cells in a mouse model of MS using PET. Methods: To model CNS infiltration by B cells, experimental autoimmune encephalomyelitis (EAE) was induced in transgenic mice that express human CD20 on B cells. EAE mice were given subcutaneous injections of myelin oligodendrocyte glycoprotein fragment1–125 emulsified in complete Freund adjuvant. Control mice received complete Freund adjuvant alone. PET imaging of EAE and control mice was performed 1, 4, and 19 h after 64Cu-rituximab administration. Mice were perfused and sacrificed after the final PET scan, and radioactivity in dissected tissues was measured with a γ-counter. CNS tissues from these mice were immunostained to quantify B cells or were further analyzed via digital autoradiography. Results: Lumbar spinal cord PET signal was significantly higher in EAE mice than in controls at all evaluated time points (e.g., 1 h after injection: 5.44 ± 0.37 vs. 3.33 ± 0.20 percentage injected dose [%ID]/g, P < 0.05). 64Cu-rituximab PET signal in brain regions ranged between 1.74 ± 0.11 and 2.93 ± 0.15 %ID/g for EAE mice, compared with 1.25 ± 0.08 and 2.24 ± 0.11 %ID/g for controls (P < 0.05 for all regions except striatum and thalamus at 1 h after injection). Similarly, ex vivo biodistribution results revealed notably higher 64Cu-rituximab uptake in the brain and spinal cord of huCD20tg EAE, and B220 immunostaining verified that increased 64Cu-rituximab uptake in CNS tissues corresponded with elevated B cells. Conclusion: B cells can be detected in the CNS of EAE mice using 64Cu-rituximab PET. Results from these studies warrant further investigation of 64Cu-rituximab in EAE models and consideration of use in MS patients to evaluate its potential for detecting and monitoring B cells in the progression and treatment of this disease. These results represent an initial step toward generating a platform to evaluate B cell–targeted therapeutics en route to the clinic.

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.