Ching Ching Leow

MEDI3617, a human anti-Angiopoietin 2 monoclonal antibody, inhibits angiogenesis and tumor growth in human tumor xenograft models

Angiopoietin 2 (Ang2) is an important regulator of angiogenesis, blood vessel maturation and integrity of the vascular endothelium. The correlation between the dynamic expression of Ang2 in tumors with regions of high angiogenic activity and a poor prognosis in many tumor types makes Ang2 an ideal drug target. We have generated MEDI3617, a human anti-Ang2 monoclonal antibody that neutralizes Ang2 by preventing its binding to the Tie2 receptor in vitro, and inhibits angiogenesis and tumor growth in vivo. Treatment of mice with MEDI3617 resulted in inhibition of angiogenesis in several mouse models including: FGF2-induced angiogenesis in a basement extract plug model, tumor and retinal angiogenesis. In xenograft tumor models, treatment with MEDI3617 resulted in a reduction in tumor angiogenesis and an increase in tumor hypoxia. The administration of MEDI3617 as a single agent to mice bearing human tumor xenografts resulted in tumor growth inhibition against a broad spectrum of tumor types. Combining MEDI3617 with chemotherapy or bevacizumab resulted in a delay in tumor growth and no body weight loss was observed in the combination groups. These results, combined with pharmacodynamic studies, demonstrate that treatment of tumor-bearing mice with MEDI3617 significantly inhibited tumor growth as a single agent by blocking tumor angiogenesis. Together, these data show that MEDI3617 is a robust antiangiogenic agent and support the clinical evaluation and biomarker development of MEDI3617 in cancer patients.

Antitumor efficacy of IPI-504, a selective heat shock protein 90 inhibitor against human epidermal growth factor receptor 2–positive human xenograft models as a single agent and in combination with trastuzumab or lapatinib

IPI-504 is a novel, highly soluble small-molecule inhibitor of heat shock protein 90 (Hsp90), a protein chaperone essential for regulating homeostasis of oncoproteins and cell signaling proteins. Human epidermal growth factor receptor 2 (HER2; ErbB2) oncoprotein, expressed in a subset of metastatic breast cancers, is a Hsp90 client protein. In this study, we investigated the antitumor activity and the mechanism of action of IPI-504 in HER2+, trastuzumab-sensitive and trastuzumab-refractory cell lines in vitro and in vivo. IPI-504 exhibited potent antiproliferative activities (range of IC50, 10-40 nmol/L) against several tumor cell lines examined, whereby mechanism of action was mediated through HER2 and Akt degradation. Both intravenous and oral administration of IPI-504 assessed in multiple schedules showed potent tumor growth inhibition in vivo with corresponding degradation of HER2. The tolerability and efficacy of IPI-504 combined with either trastuzumab or lapatinib were also investigated in HER2+ tumor xenograft models. Combination of IPI-504 with trastuzumab significantly enhanced tumor growth delay and induced greater responses when compared with either agent alone. Although, as expected, trastuzumab alone did not exhibit any significant antitumor activity in the trastuzumab-resistant JIMT-1 model, IPI-504 administered in combination with trastuzumab yielded greater antitumor efficacy than either agent alone. Finally, combination of IPI-504 and lapatinib was well tolerated up to 50 mg/kg IPI-504 and 100 mg/kg lapatinib and resulted in significant delay in tumor growth, including partial and complete tumor responses. These lines of evidence support the development of IPI-504 in HER2-positive breast cancers as a single agent and in combination with either trastuzumab or lapatinib.[Mol Cancer Ther 2009;8(8):2131–41]

Doxil Synergizes with Cancer Immunotherapies to Enhance Antitumor Responses in Syngeneic Mouse Models

Based on the previously described roles of doxorubicin in immunogenic cell death, both doxorubicin and liposomal doxorubicin (Doxil) were evaluated for their ability to boost the antitumor response of different cancer immunotherapies including checkpoint blockers (anti–PD-L1, PD-1, and CTLA-4 mAbs) and TNF receptor agonists (OX40 and GITR ligand fusion proteins) in syngeneic mouse models. In a preventative CT26 mouse tumor model, both doxorubicin and Doxil synergized with anti–PD-1 and CTLA-4 mAbs. Doxil was active when CT26 tumors were grown in immunocompetent mice but not immunocompromised mice, demonstrating that Doxil activity is increased in the presence of a functional immune system. Using established tumors and maximally efficacious doses of Doxil and cancer immunotherapies in either CT26 or MCA205 tumor models, combination groups produced strong synergistic antitumor effects, a larger percentage of complete responders, and increased survival. In vivo pharmacodynamic studies showed that Doxil treatment decreased the percentage of tumor-infiltrating regulatory T cells and, in combination with anti–PD-L1, increased the percentage of tumor-infiltrating CD8+ T cells. In the tumor, Doxil administration increased CD80 expression on mature dendritic cells. CD80 expression was also increased on both monocytic and granulocytic myeloid cells, suggesting that Doxil may induce these tumor-infiltrating cells to elicit a costimulatory phenotype capable of activating an antitumor T-cell response. These results uncover a novel role for Doxil in immunomodulation and support the use of Doxil in combination with checkpoint blockade or TNFR agonists to increase response rates and antitumor activity.

Multivalent Scaffold Proteins as Superagonists of TRAIL Receptor 2–Induced Apoptosis

Activation of TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2) can induce apoptosis in a variety of human cancer cell lines and xenografts, while lacking toxicity in normal cells. The natural ligand and agonistic antibodies show antitumor activity in preclinical models of cancer, and this had led to significant excitement in the clinical potential of these agents. Unfortunately, this optimism has been tempered by trial data that, thus far, are not showing clear signs of efficacy in cancer patients. The reasons for discrepant preclinical and clinical observations are not understood, but one possibility is that the current TRAILR2 agonists lack sufficient potency to achieve a meaningful response in patients. Toward addressing that possibility, we have developed multivalent forms of a new binding scaffold (Tn3) that are superagonists of TRAILR2 and can induce apoptosis in tumor cell lines at subpicomolar concentrations. The monomer Tn3 unit was a fibronectin type III domain engineered for high-affinity TRAILR2 binding. Multivalent presentation of this basic unit induced cell death in TRAILR2-expressing cell lines. Optimization of binding affinity, molecular format, and valency contributed to cumulative enhancements of agonistic activity. An optimized multivalent agonist consisting of 8 tandem Tn3 repeats was highly potent in triggering cell death in TRAIL-sensitive cell lines and was 1 to 2 orders of magnitude more potent than TRAIL. Enhanced potency was also observed in vivo in a tumor xenograft setting. The TRAILR2 superagonists described here have the potential for superior clinical activity in settings insensitive to the current therapeutic agonists that target this pathway. Mol Cancer Ther; 12(7); 1235–44. ©2013 AACR.

A Novel Murine GITR Ligand Fusion Protein Induces Antitumor Activity as a Monotherapy That Is Further Enhanced in Combination with an OX40 Agonist

Purpose: To generate and characterize a murine GITR ligand fusion protein (mGITRL-FP) designed to maximize valency and the potential to agonize the GITR receptor for cancer immunotherapy. Experimental Design: The EC50 value of the mGITRL-FP was compared with an anti-GITR antibody in an in vitro agonistic cell–based reporter assay. We assessed the impact of dose, schedule, and Fc isotype on antitumor activity and T-cell modulation in the CT26 tumor model. The activity of the mGITRL-FP was compared with an agonistic murine OX40L-FP targeting OX40, in CT26 and B16F10-Luc2 tumor models. Combination of the mGITRL-FP with antibodies targeting PD-L1, PD-1, or CTLA-4 was analyzed in mice bearing CT26 tumors. Results: The mGITRL-FP had an almost 50-fold higher EC50 value compared with an anti-murine GITR antibody. Treatment of CT26 tumor-bearing mice with mGITRL-FP–mediated significant antitumor activity that was dependent on isotype, dose, and duration of exposure. The antitumor activity could be correlated with the increased proliferation of peripheral CD8+ and CD4+ T cells and a significant decrease in the frequency of intratumoral Tregs. The combination of mGITRL-FP with mOX40L-FP or checkpoint inhibitor antagonists enhanced antitumor immunity above that of monotherapy treatment. Conclusions: These results suggest that therapeutically targeting GITR represents a unique approach to cancer immunotherapy and suggests that a multimeric fusion protein may provide increased agonistic potential versus an antibody. In addition, these data provide, for the first time, early proof of concept for the potential combination of GITR targeting agents with OX40 agonists and PD-L1 antagonists. Clin Cancer Res; 23(13); 3416–27. ©2017 AACR.

A Monoclonal Antibody to ADAM17 Inhibits Tumor Growth by Inhibiting EGFR and Non–EGFR-Mediated Pathways

ADAM17 is the primary sheddase for HER pathway ligands. We report the discovery of a potent and specific ADAM17 inhibitory antibody, MEDI3622, which induces tumor regression or stasis in many EGFR-dependent tumor models. The inhibitory activity of MEDI3622 correlated with EGFR activity both in a series of tumor models across several indications as well in as a focused set of head and neck patient–derived xenograft models. The antitumor activity of MEDI3622 was superior to that of EGFR/HER pathway inhibitors in the OE21 esophageal model and the COLO205 colorectal model suggesting additional activity outside of the EGFR pathway. Combination of MEDI3622 and cetuximab in the OE21 model was additive and eradicated tumors. Proteomics analysis revealed novel ADAM17 substrates that function outside of the HER pathways and may contribute toward the antitumor activity of the monoclonal antibody. Mol Cancer Ther; 14(7); 1637–49. ©2015 AACR.

GITR ligand fusion protein agonist enhances the tumor antigen–specific CD8 T-cell response and leads to long-lasting memory

BackgroundThe expansion of antigen-specific CD8 T cells is important in generating an effective and long-lasting immune response to tumors and viruses. Glucocorticoid-induced tumor necrosis factor receptor family-related receptor (GITR) is a co-stimulatory receptor that binds the GITR ligand (GITRL). Agonism of GITR can produce important signals that drive expansion of effector T cell populations.MethodsWe explored two separate murine tumor models, CT26 and TC-1, for responsiveness to GITR Ligand Fusion Protein(GITRL-FP) monotherapy. In TC-1, GITRL-FP was also combined with concurrent administration of an E7-SLP vaccine. We evaluated tumor growth inhibition by tumor volume measurements as well as changes in CD8 T cell populations and function including cytokine production using flow cytometry. Additionally, we interrogated how these therapies resulted in tumor antigen-specific responses using MHC-I dextramer staining and antigen-specific restimulations.ResultsIn this study, we demonstrate that a GITR ligand fusion protein (GITRL-FP) is an effective modulator of antigen-specific CD8 T cells. In a CT26 mouse tumor model, GITRL-FP promoted expansion of antigen-specific T cells, depletion of regulatory T cells (Tregs), and generation of long-lasting CD8 T cell memory. This memory expansion was dependent on the dose of GITRL-FP and resulted in complete tumor clearance and protection from tumor rechallenge. In contrast, in TC-1 tumor–bearing mice, GITRL-FP monotherapy could not prime an antigen-specific CD8 T cell response and was unable to deplete Tregs. However, when combined with a vaccine targeting E7, treatment with GITRL-FP resulted in an augmentation of the vaccine-induced antigen-specific CD8 T cells, the depletion of Tregs, and a potent antitumor immune response. In both model systems, GITR levels on antigen-specific CD8 T cells were higher than on all other CD8 T cells, and GITRL-FP interacted directly with primed antigen-specific CD8 T cells.ConclusionsWhen taken together, our results demonstrate that the delivery of GITRL-FP as a therapeutic can promote anti-tumor responses in the presence of tumor-specific CD8 T cells. These findings support further study into combination partners for GITRL-FP that may augment CD8 T-cell priming as well as provide hypotheses that can be tested in human clinical trials exploring GITR agonists including GITRL-FP.

Insertion of scFv into the hinge domain of full-length IgG1 monoclonal antibody results in tetravalent bispecific molecule with robust properties.

ABSTRACT By simultaneous binding two disease mediators, bispecific antibodies offer the opportunity to broaden the utility of antibody-based therapies. Herein, we describe the design and characterization of Bs4Ab, an innovative and generic bispecific tetravalent antibody platform. The Bs4Ab format comprises a full-length IgG1 monoclonal antibody with a scFv inserted into the hinge domain. The Bs4Ab design demonstrates robust manufacturability as evidenced by MEDI3902, which is currently in clinical development. To further demonstrate the applicability of the Bs4Ab technology, we describe the molecular engineering, biochemical, biophysical, and in vivo characterization of a bispecific tetravalent Bs4Ab that, by simultaneously binding vascular endothelial growth factor and angiopoietin-2, inhibits their function. We also demonstrate that the Bs4Ab platform allows Fc-engineering similar to that achieved with IgG1 antibodies, such as mutations to extend half-life or modulate effector functions.

Abstract 1538: Levels and enzyme activity of CD73 in primary samples from cancer patients

CD73 is an ectonucleotidase that converts extracellular AMP to Adenosine. Within the tumor microenvironment, adenosine produced by CD73 is believed to have an immunosuppressive effect upon anti-tumor immunity. CD73 is found on lymphoid and myeloid cells, and blockade of this enzyme has been shown to alter the suppressive capacity of cells. Expression of surface CD73 as well as soluble CD73(sCD73) levels were measured in serum, peripheral blood mononuclear cells, and tumor samples from healthy and cancer patient donors. Enzymatic activity of both surface CD73 and sCD73 was determined and differences in levels across varying tumor types and disease status were quantified. In addition, CD73 levels and activity were measured after incubation or treatment with anti-CD73 antibody, MEDI9447. These studies suggest a potential role of surface and sCD73 as a pharmacodynamic and/ or predictive biomarker of anti-CD73 immunotherapy. Citation Format: Qihui Huang, Nicholas M. Durham, Erin Sult, Yuling Wu, Jenny Liu, Nicholas Holoweckyj, Laurie Iciek, Robert Hollingsworth, Brett Hall, Ronald Herbst, Ching Ching Leow, Kris Sachsenmeier. Levels and enzyme activity of CD73 in primary samples from cancer patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1538. doi:10.1158/1538-7445.AM2015-1538

Abstract A49: Anti-DLL4 antibodies inhibit cancer stem cells in small cell lung cancer.

Small cell lung cancer (SCLC) is a very aggressive lung cancer with features suggesting enrichment in cancer stem cells (CSCs). Delta-like ligand 4 (DLL4) is a membrane bound ligand for Notch receptors critical for functional angiogenesis. Blocking DLL4 signaling increases the density of nonfunctional blood vessels and hypoxia of tumors, and thereby inhibits growth of tumor xenografts in mice. In addition, growing evidence implicates DLL4 Notch signaling pathway in the maintenance of CSCs. Here we investigated the anti-CSC activity of anti-DLL4 mAbs using in vitro and in vivo models of SCLC. Biochemical and flow cytometry analyses revealed that multiple SCLC cell lines express DLL4, and notably, SCLC spheres cultured under CSC-enriching conditions express higher levels of DLL4. Prior to determining the ability of anti-DLL4 mAb in inhibiting CSC in vivo, we first characterized the phenotype of CSCs in the SCLC cell line NCI-H69. NCI-H69 cells enriched for high CD133 expression were more tumorigenic and expressed higher levels of Nanog, Oct 3/4, and EZH2, which are genes crucial for maintenance of CSCs, than cells with low or negative levels of CD133. Moreover, RNA level of DLL4 was found to be three fold higher in CD133 high cells compared to CD133 low cells, suggesting that expression of DLL4 correlates with the CSC phenotype. In vivo, an anti-DLL4 antibody leads to inhibition of NCI-H69 tumor growth when the antibody is administered as a single agent, or in combination with cisplatin+etoposide or topotecan, chemotherapy agents commonly used in treatment of SCLC. To determine if blockade of DLL4 inhibits CSCs in vivo, NCI-H69 tumor xenografts treated with anti-DLL4 mAb were analyzed by flow cytometry. A subset of dissociated tumor cells expressed CD133 and DLL4, and a reduction of these populations of cells was observed in the anti-DLL4 antibody treated groups. In summary, these studies highlight that, in addition to vascular expression, (1) DLL4 is frequently expressed in SCLC cells, (2) DLL4 expression correlates with a CSC phenotype, and (3) that DLL4 blockade using an anti-DLL4 mAb results in inhibition of CSCs. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A49. Citation Format: Patrick Strout, Martin Korade, Ching Ching Leow, Ivan Inigo, Suneetha Thomas, Elaine Hurt, Jon Chesebrough, Adeela Kamal, Song Cho. Anti-DLL4 antibodies inhibit cancer stem cells in small cell lung cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A49.