Preclinical Antitumor Activity and Biodistribution of a Novel Anti-GCC Antibody-Drug Conjugate in Patient-derived Xenografts.

Guanylyl cyclase C (GCC) is a unique therapeutic target with expression restricted to the apical side of epithelial cell tight junctions thought to be only accessible by intravenously administered agents on malignant tissues where GCC expression is aberrant. In this study, we sought to evaluate the therapeutic potential of a second-generation investigational antibody-dug conjugate (ADC), TAK-164, comprised of a human anti-GCC mAb conjugated via a peptide linker to the highly cytotoxic DNA alkylator, DGN549. The in vitro binding, payload release, and in vitro activity of TAK-164 was characterized motivating in vivo evaluation. The efficacy of TAK-164 and the relationship to exposure, pharmacodynamic marker activation, and biodistribution was evaluated in xenograft models and primary human tumor xenograft (PHTX) models. We demonstrate TAK-164 selectively binds to, is internalized by, and has potent cytotoxic effects against GCC-expressing cells in vitro A single intravenous administration of TAK-164 (0.76 mg/kg) resulted in significant growth rate inhibition in PHTX models of metastatic colorectal cancer. Furthermore, imaging studies characterized TAK-164 uptake and activity and showed positive relationships between GCC expression and tumor uptake which correlated with antitumor activity. Collectively, our data suggest that TAK-164 is highly active in multiple GCC-positive tumors including those refractory to TAK-264, a GCC-targeted auristatin ADC. A strong relationship between uptake of 89Zr-labeled TAK-164, levels of GCC expression and, most notably, response to TAK-164 therapy in GCC-expressing xenografts and PHTX models. These data supported the clinical development of TAK-164 as part of a first-in-human clinical trial (NCT03449030).

KRAS Genotype Correlates with Proteasome Inhibitor Ixazomib Activity in Preclinical In Vivo Models of Colon and Non-Small Cell Lung Cancer: Potential Role of Tumor Metabolism.

In non-clinical studies, the proteasome inhibitor ixazomib inhibits cell growth in a broad panel of solid tumor cell lines in vitro. In contrast, antitumor activity in xenograft tumors is model-dependent, with some solid tumors showing no response to ixazomib. In this study we examined factors responsible for ixazomib sensitivity or resistance using mouse xenograft models. A survey of 14 non-small cell lung cancer (NSCLC) and 6 colon xenografts showed a striking relationship between ixazomib activity and KRAS genotype; tumors with wild-type (WT) KRAS were more sensitive to ixazomib than tumors harboring KRAS activating mutations. To confirm the association between KRAS genotype and ixazomib sensitivity, we used SW48 isogenic colon cancer cell lines. Either KRAS-G13D or KRAS-G12V mutations were introduced into KRAS-WT SW48 cells to generate cells that stably express activated KRAS. SW48 KRAS WT tumors, but neither SW48-KRAS-G13D tumors nor SW48-KRAS-G12V tumors, were sensitive to ixazomib in vivo. Since activated KRAS is known to be associated with metabolic reprogramming, we compared metabolite profiling of SW48-WT and SW48-KRAS-G13D tumors treated with or without ixazomib. Prior to treatment there were significant metabolic differences between SW48 WT and SW48-KRAS-G13D tumors, reflecting higher oxidative stress and glucose utilization in the KRAS-G13D tumors. Ixazomib treatment resulted in significant metabolic regulation, and some of these changes were specific to KRAS WT tumors. Depletion of free amino acid pools and activation of GCN2-eIF2α-pathways were observed both in tumor types. However, changes in lipid beta oxidation were observed in only the KRAS WT tumors. The non-clinical data presented here show a correlation between KRAS genotype and ixazomib sensitivity in NSCLC and colon xenografts and provide new evidence of regulation of key metabolic pathways by proteasome inhibition.

Antitumor activity of the investigational proteasome inhibitor MLN9708 in mouse models of B-cell and plasma cell malignancies.

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 iMyc(Cα)/Bcl-X(L) GEM model was used to assess their effects on de novo PCM and overall survival. The newly developed DP54-Luc-disseminated model of iMyc(Cα)/Bcl-X(L) 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 iMyc(Cα)/Bcl-X(L) 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.

Preclinical evaluation of the antitumor activity of bortezomib in combination with vitamin C or with epigallocatechin gallate, a component of green tea.

PURPOSE: To investigate whether clinically relevant levels of epigallocatechin gallate (EGCG, a component of green tea) or vitamin C (ascorbic acid) could antagonize bortezomib antitumor activity in CWR22 human prostate xenograft tumors. METHODS: The pharmacokinetics (PK) of EGCG and ascorbic acid were determined in immunocompromised mice and compared with concentrations measured in human PK studies of dietary supplements. Antitumor activity of bortezomib in combination with EGCG or ascorbic acid was determined using several dosing regimens to evaluate different target plasma concentrations of EGCG and ascorbic acid. RESULTS: Bortezomib dosed twice-weekly at 0.8 mg/kg IV demonstrated tumor growth inhibition (TGI) of 53.9-58.9%. However, when combined with EGCG such that the plasma concentrations of EGCG were >200 µM at the time of bortezomib dosing, all antitumor activity was abrogated (TGI = -17.7%). A lower concentration of EGCG (11-16 µM), which is severalfold higher than measured clinically in humans taking EGCG supplements (0.6-3 µM), was not antagonistic to bortezomib (TGI 63.5%). Pharmacodynamic studies of proteasome inhibition reflected these findings. Ascorbic acid (40 and 500 mg/kg PO daily) was evaluated under a similar study design and did not antagonize bortezomib antitumor activity (TGI 57.2 and 72.2%). CONCLUSIONS: No antagonism of bortezomib is seen in preclinical in vivo experiments, where EGCG or ascorbic acid plasma concentrations are commensurate with dietary or supplemental intake. The data suggest that patients receiving bortezomib treatment do not need to avoid normal dietary consumption of green tea, vitamin C-containing foods, or EGCG or vitamin C dietary supplements.

Evaluation of the proteasome inhibitor MLN9708 in preclinical models of human cancer.

The proteasome was validated as an oncology target following the clinical success of VELCADE (bortezomib) for injection for the treatment of multiple myeloma and recurring mantle cell lymphoma. Consequently, several groups are pursuing the development of additional small-molecule proteasome inhibitors for both hematologic and solid tumor indications. Here, we describe MLN9708, a selective, orally bioavailable, second-generation proteasome inhibitor that is in phase I clinical development. MLN9708 has a shorter proteasome dissociation half-life and improved pharmacokinetics, pharmacodynamics, and antitumor activity compared with bortezomib. MLN9708 has a larger blood volume distribution at steady state, and analysis of 20S proteasome inhibition and markers of the unfolded protein response confirmed that MLN9708 has greater pharmacodynamic effects in tissues than bortezomib. MLN9708 showed activity in both solid tumor and hematologic preclinical xenograft models, and we found a correlation between greater pharmacodynamic responses and improved antitumor activity. Moreover, antitumor activity was shown via multiple dosing routes, including oral gavage. Taken together, these data support the clinical development of MLN9708 for both hematologic and solid tumor indications.

The relationship among tumor architecture, pharmacokinetics, pharmacodynamics, and efficacy of bortezomib in mouse xenograft models.

Understanding a compound's preclinical pharmacokinetic, pharmacodynamic, and efficacy relationship can greatly facilitate its clinical development. Bortezomib is a first-in-class proteasome inhibitor whose pharmacokinetic/pharmacodynamic parameters are poorly understood in terms of their relationship with efficacy. Here we characterized the bortezomib pharmacokinetic/pharmacodynamic/efficacy relationship in the CWR22 and H460 xenograft models. These studies allowed us to specifically address the question of whether the lack of broad bortezomib activity in solid tumor xenografts was due to insufficient tumor penetration. In vivo studies showed that bortezomib treatment resulted in tumor growth inhibition in CWR22 xenografts, but not in H460 xenografts. Using 20S proteasome inhibition as a pharmacodynamic marker and analyzing bortezomib tumor exposures, we show that efficacy was achieved only when suitable drug exposures drove proteasome inhibition that was sustained over time. This suggested that both the magnitude and duration of proteasome inhibition were important drivers of efficacy. Using dynamic contrast-enhanced magnetic resonance imaging and high-resolution computed tomographic imaging of vascular casts, we characterized the vasculature of CWR22 and H460 xenograft tumors and identified prominent differences in vessel perfusion, permeability, and architecture that ultimately resulted in variations in bortezomib tumor exposure. Comparing and contrasting the differences between a bortezomib-responsive and a bortezomib-resistant model with these techniques allowed us to establish a relationship among tumor perfusion, drug exposure, pharmacodynamic response and efficacy, and provided an explanation for why some solid tumor models do not respond to bortezomib treatment.