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Purpose: The clinical success of the first-in-class proteasome inhibitor bortezomib (VELCADE) has validated the proteasome as a therapeutic target for treating human cancers. MLN9708 is an investigational proteasome inhibitor that, compared with bortezomib, has improved pharmacokinetics, pharmacodynamics, and antitumor activity in preclinical studies. Here, we focused on evaluating the in vivo activity of MLN2238 (the biologically active form of MLN9708) in a variety of mouse models of hematologic malignancies, including tumor xenograft models derived from a human lymphoma cell line and primary human lymphoma tissue, and genetically engineered mouse (GEM) models of plasma cell malignancies (PCM).Experimental Design: Both cell line–derived OCI-Ly10 and primary human lymphoma–derived PHTX22L xenograft models of diffuse large B-cell lymphoma were used to evaluate the pharmacodynamics and antitumor effects of MLN2238 and bortezomib. The iMycCα/Bcl-XL GEM model was used to assess their effects on de novo PCM and overall survival. The newly developed DP54-Luc–disseminated model of iMycCα/Bcl-XL was used to determine antitumor activity and effects on osteolytic bone disease.Results: MLN2238 has an improved pharmacodynamic profile and antitumor activity compared with bortezomib in both OCI-Ly10 and PHTX22L models. Although both MLN2238 and bortezomib prolonged overall survival, reduced splenomegaly, and attenuated IgG2a levels in the iMycCα/Bcl-XL GEM model, only MLN2238 alleviated osteolytic bone disease in the DP54-Luc model.Conclusions: Our results clearly showed the antitumor activity of MLN2238 in a variety of mouse models of B-cell lymphoma and PCM, supporting its clinical development. MLN9708 is being evaluated in multiple phase I and I/II trials. Clin Cancer Res; 17(23); 7313–23. ©2011 AACR.

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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.

Glioblastoma multiforme (GBM) is heavily reliant upon angiogenesis for growth and represents one of the most vascularised human tumors. MLN0518 (Tandutinib) is a type III receptor tyrosine kinase inhibitor (RTKi) with activity against PDGFRα/β, FLT3 and c-KIT in the submicromolar range, but exhibits no significant inhibition of a broad range of other kinases. MLN0518 is currently in phase II trials as monotherapy and in combination with Bevacizumab for recurrent glioblastoma. In this study we aimed to investigate 1) vascular haemodynamic and structural response, and 2) hypoxic changes of C6 glioma xenografts to MLN0518 using immunohistochemical and MRI methods. C6 rat glioma xenografts were grown subcutaneously in nude mice. When tumors reached 200±100mm3 they were stratified to receive either vehicle (5% dextrose, n=9) or 20mg/kg MLN0518 (n=9) subcutaneously BID for 10 days. At study end tumors were excised and the total endothelial cell concentration (CD31), degree of perfused vasculature (Hoechst 33342), and hypoxia (pimonidazole) were quantified by immunohistochemistry and fluorescence microscopy. Tumors in mice treated with MLN0518 grew significantly slower than vehicle-treated tumors (volume doubling time 5.9±0.4 days compared with 4.6±0.2 days, p There was no significant difference in either Hoechst 33342 perfused area or total endothelial cell area as assessed by CD31 staining. However, when the distribution of staining was analyzed it revealed that the percentage of the total vessels perfused at the time of Hoechst 33342 injection was 25.4% lower in MLN0518 than vehicle-treated tumors (p Finally, and most strikingly, despite having a lower perfused vessel area MLN0518-treated tumors were 28.0% less hypoxic than control tumors as assessed by pimonidazole adduct formation (p The data demonstrate that chronic administration of MLN0518 limits the growth of C6 glioma xenografts and reduces both the mean perfused vessel fraction and hypoxic area. The co-registration of Hoechst 33342 and CD31 staining also highlights the limitation of solely using a pan-endothelial marker to assess vascular response. To complement these growth and histological readouts we are currently exploring the use of susceptibility contrast enhanced magnetic resonance imaging to derive imaging biomarkers of tumor fractional blood volume (%) and vessel size index (µm). Tumor hypoxia is well known to promote a more metastatic, malignant, angiogenic and chemo-/radio-resistant lesion. MLN0518 is one of few chronically dosed RTK inhibitors that reduce hypoxia in xenograft tumors. Thus, MLN0518 may prove effective for the treatment of well vascularised tumors such as GBM. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B257.