Lihui Xu

Therapeutically Targeting ErbB3: A Key Node in Ligand-Induced Activation of the ErbB Receptor–PI3K Axis

Computational modeling of the ErbB signaling network affirms ErbB3 as a therapeutic target. Zooming In on ErbB3 Aberrant signaling involving the ErbB family of receptors, which can signal as homo- or heterodimers to activate the phosphatidylinositol 3-kinase (PI3K) signaling pathway, has been implicated as contributing to various cancers. Using a systems approach, Schoeberl et al. implicated ErbB3—a member of the ErbB family that is catalytically inactive—as critical to signaling stimulated by ligands that bind either ErbB1 or ErbB3. Computational analysis suggested that inhibiting ligand binding to ErbB3 might represent a more successful approach to treating cancers associated with ligand-induced stimulation of ErbB-PI3K signaling mediated by combinatorial receptor activation than do current therapies that target overexpressed or mutationally activated ErbB-family receptors. Moreover, experimental analysis revealed that a monoclonal antibody developed on the basis of this strategy could stop the growth of tumors grafted into immunodeficient mice. The signaling network downstream of the ErbB family of receptors has been extensively targeted by cancer therapeutics; however, understanding the relative importance of the different components of the ErbB network is nontrivial. To explore the optimal way to therapeutically inhibit combinatorial, ligand-induced activation of the ErbB–phosphatidylinositol 3-kinase (PI3K) axis, we built a computational model of the ErbB signaling network that describes the most effective ErbB ligands, as well as known and previously unidentified ErbB inhibitors. Sensitivity analysis identified ErbB3 as the key node in response to ligands that can bind either ErbB3 or EGFR (epidermal growth factor receptor). We describe MM-121, a human monoclonal antibody that halts the growth of tumor xenografts in mice and, consistent with model-simulated inhibitor data, potently inhibits ErbB3 phosphorylation in a manner distinct from that of other ErbB-targeted therapies. MM-121, a previously unidentified anticancer therapeutic designed using a systems approach, promises to benefit patients with combinatorial, ligand-induced activation of the ErbB signaling network that are not effectively treated by current therapies targeting overexpressed or mutated oncogenes.

MM-141, an IGF-IR– and ErbB3-Directed Bispecific Antibody, Overcomes Network Adaptations That Limit Activity of IGF-IR Inhibitors

Although inhibition of the insulin-like growth factor (IGF) signaling pathway was expected to eliminate a key resistance mechanism for EGF receptor (EGFR)-driven cancers, the effectiveness of IGF-I receptor (IGF-IR) inhibitors in clinical trials has been limited. A multiplicity of survival mechanisms are available to cancer cells. Both IGF-IR and the ErbB3 receptor activate the PI3K/AKT/mTOR axis, but ErbB3 has only recently been pursued as a therapeutic target. We show that coactivation of the ErbB3 pathway is prevalent in a majority of cell lines responsive to IGF ligands and antagonizes IGF-IR–mediated growth inhibition. Blockade of the redundant IGF-IR and ErbB3 survival pathways and downstream resistance mechanisms was achieved with MM-141, a tetravalent bispecific antibody antagonist of IGF-IR and ErbB3. MM-141 potency was superior to monospecific and combination antibody therapies and was insensitive to variation in the ratio of IGF-IR and ErbB3 receptors. MM-141 enhanced the biologic impact of receptor inhibition in vivo as a monotherapy and in combination with the mTOR inhibitor everolimus, gemcitabine, or docetaxel, through blockade of IGF-IR and ErbB3 signaling and prevention of PI3K/AKT/mTOR network adaptation. Mol Cancer Ther; 13(2); 410–25. ©2013 AACR.

Rapid optimization and prototyping for therapeutic antibody-like molecules

Multispecific antibody-like molecules have the potential to advance the standard-of-care in many human diseases. The design of therapeutic molecules in this class, however, has proven to be difficult and, despite significant successes in preclinical research, only one trivalent antibody, catumaxomab, has demonstrated clinical utility. The challenge originates from the complexity of the design space where multiple parameters such as affinity, avidity, effector functions, and pharmaceutical properties need to be engineered in concurrent fashion to achieve the desired therapeutic efficacy. Here, we present a rapid prototyping approach that allows us to successfully optimize these parameters within one campaign cycle that includes modular design, yeast display of structure focused antibody libraries and high throughput biophysical profiling. We delineate this approach by presenting a design case study of MM-141, a tetravalent bispecific antibody targeting two compensatory signaling growth factor receptors: insulin-like growth factor 1 receptor (IGF-1R) and v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (ErbB3). A MM-141 proof-of-concept (POC) parent molecule did not meet initial design criteria due to modest bioactivity and poor stability properties. Using a combination of yeast display, structured-guided antibody design and library-scale thermal challenge assay, we discovered a diverse set of stable and active anti-IGF-1R and anti-ErbB3 single-chain variable fragments (scFvs). These optimized modules were reformatted to create a diverse set of full-length tetravalent bispecific antibodies. These re-engineered molecules achieved complete blockade of growth factor induced pro-survival signaling, were stable in serum, and had adequate activity and pharmaceutical properties for clinical development. We believe this approach can be readily applied to the optimization of other classes of bispecific or even multispecific antibody-like molecules.

A novel screening method to assess developability of antibody-like molecules

Monoclonal antibodies and antibody-like molecules represent a fast-growing class of bio-therapeutics that has rapidly transformed patient care in a variety of disease indications. The discovery of antibodies that bind to particular targets with high affinity is now a routine exercise and a variety of in vitro and in vivo techniques are available for this purpose. However, it is still challenging to identify antibodies that, in addition to having the desired biological effect, also express well, remain soluble at different pH levels, remain stable at high concentrations, can withstand high shear stress, and have minimal non-specific interactions. Many promising antibody programs have ultimately failed in development due to the problems associated with one of these factors. Here, we present a simple high-performance liquid chromatography (HPLC)-based screening method to assess these developability factors earlier in discovery process. This method is robust and requires only microgram quantities of proteins. Briefly, we show that for antibodies injected on a commercially available pre-packed Zenix HPLC column, the retention times are inversely related to their colloidal stability with antibodies prone to precipitation or aggregation retained longer on the column with broader peaks. By simply varying the salt content of running buffer, we were also able to estimate the nature of interactions between the antibodies and the column. We believe this approach should generally be applicable to assessment of the developability of other classes of bio-therapeutic molecules, and that the addition of this simple tool early in the discovery process will lead to selection of molecules with improved developability characteristics.

Comprehensive optimization of a single-chain variable domain antibody fragment as a targeting ligand for a cytotoxic nanoparticle

Antibody-targeted nanoparticles have the potential to significantly increase the therapeutic index of cytotoxic anti-cancer therapies by directing them to tumor cells. Using antibodies or their fragments requires careful engineering because multiple parameters, including affinity, internalization rate and stability, all need to be optimized. Here, we present a case study of the iterative engineering of a single chain variable fragment (scFv) for use as a targeting arm of a liposomal cytotoxic nanoparticle. We describe the effect of the orientation of variable domains, the length and composition of the interdomain protein linker that connects VH and VL, and stabilizing mutations in both the framework and complementarity-determining regions (CDRs) on the molecular properties of the scFv. We show that variable domain orientation can alter cross-reactivity to murine antigen while maintaining affinity to the human antigen. We demonstrate that tyrosine residues in the CDRs make diverse contributions to the binding affinity and biophysical properties, and that replacement of non-essential tyrosines can improve the stability and bioactivity of the scFv. Our studies demonstrate that a comprehensive engineering strategy may be required to identify a scFv with optimal characteristics for nanoparticle targeting.

Improving the developability of an anti-EphA2 single-chain variable fragment for nanoparticle targeting

ABSTRACT Antibody-targeted nanoparticles have great promise as anti-cancer drugs; however, substantial developmental challenges of antibody modules prevent many candidates from reaching the clinic. Here, we describe a robust strategy for developing an EphA2-targeting antibody fragment for immunoliposomal drug delivery. A highly bioactive single-chain variable fragment (scFv) was engineered to overcome developmental liabilities, including low thermostability and weak binding to affinity purification resins. Improved thermostability was achieved by modifying the framework of the scFv, and complementarity-determining region (CDR)-H2 was modified to increase binding to protein A resins. The results of our engineering campaigns demonstrate that it is possible, using focused design strategies, to rapidly improve the stability and manufacturing characteristics of an antibody fragment for use as a component of a novel therapeutic construct.

Abstract 871: Nanoliposomal targeting of ephrin receptor A2 (EphA2): Preclinical in vitro and in vivo rationale

Ephrins receptors are cell to cell adhesion molecules that mediate signaling and are implicated in neuronal repulsion, cell migration and angiogenesis. Ephrin receptor A2 (EphA2) is part of the ephrin family and was shown to be overexpressed in several solid tumors including ovarian, gastric and lung cancer and is associated with poor prognosis. MM-310 is a novel EphA2 targeted docetaxel nanoliposome. Similar to other nanoparticles, MM-310 leverages organ specificity through enhanced permeability and retention, but can also leverage cellular specificity through its EphA2 targeting arm. Binding of targeted liposomes to cells in vitro was assessed using flow cytometry in a large panel cell lines. 3D spheroids were used to assess targeting as well as liposome penetration. In order to test the effect of targeting on liposome microdistribution in vivo, primary and metastatic tumor bearing animals were injected with a mixture of EphA2-targeted liposome (EphA2-Ls) and non-targeted liposome (NT-Ls) labeled with two different lipophilic fluorescent dyes. Tissues were assessed using fluorescent microscopy. In order to evaluate the contribution of EphA2-targeting to efficacy, four gastric xenograft models were treated with either MM-310 or a non-targeted version of the drug NT-310, and compared to free docetaxel. In cell suspension models, we observed a high level of specificity for EphA2-Ls, with more than one hundred-fold increase in liposome cell association. 3D spheroid assays showed that EphA2-Ls binds and penetrates EphA2+ spheroids, while non-targeted liposomes show minimal penetration. Tissue microdistribution analysis in triple negative breast and esophageal tumor models following injection of the EphA2-Ls/NT-Ls mixtures showed a target mediated shift in the microdistribution of liposomes. EphA2-Ls penetrated deeper within the lesions while the NT-Ls deposited at high levels in areas close to the microvasculature. The target mediated shift in microdistribution was also observed in lung metastasis model, with a pattern of distribution that potentially matches disseminated tumor cells. In the same animals, targeting did not affect microdistribution in normal organs such as liver, spleen and skin. Four models of gastric and esophageal cancers were used to test the potential link between cell targeting and tumor growth control. While NT-310 and MM-310 were both able to control tumor growth leading to regression in most models, in three out of four models there was a statistically significant difference between MM-310 and NT-310 at 25 mpk. Biomarker analysis is underway to evaluate the key parameters necessary to mediate targeting. In conclusion, the data suggests clear evidence of targeting in 2D cell suspension, 3D spheroids, and in primary as well as metastatic tumor models in vivo. In vivo efficacy data showed evidence of the contribution of EphA2 targeting to tumor growth control and regression in several gastric cancer models. Citation Format: Walid S. Kamoun, Lia Luus, Christine Pien, Tad Kornaga, Shinji Oyama, Zhaohua Richard Huang, Suresh Tipparaju, Dmitri B. Kirpotin, James D. Marks, Alexander Koshkaryev, Melissa Geddie, Lihui Xu, Alexey Lugovosky, Daryl C. Drummond. Nanoliposomal targeting of ephrin receptor A2 (EphA2): Preclinical in vitro and in vivo rationale. [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 871.

Abstract 22: MM-161, a first-in-class pan-FGFR antibody

Aberrant signaling of the FGFR pathway has long been known to promote tumorigenesis and angiogenesis across multiple cancer indications. However, the development of an effective and well-tolerated FGFR targeted inhibitor has been hindered by the need to block the activation of multiple mitogenic receptors while avoiding significant toxicities associated with blocking endocrine FGF ligands. Here we disclose for the first time a novel FGFR targeted antibody, MM-161, designed to block ligand-dependent signaling driven by all four FGF receptors, specifically the IIIc-isoforms. MM-161 is well tolerated in mice and cynomolgus monkeys with no significant weight loss observed in either species. Efficacy studies demonstrated that MM-161 monotherapy leads to significant tumor growth inhibition or tumor regression of xenografts of human lung, renal and endometrial cancer amongst others. Importantly, MM-161 has a dual mechanism of action by inhibiting both proliferation and angiogenesis. We will present data illustrating that inhibition of multiple FGFRs is desirable to achieve tumor regression. Furthermore, we will show combination studies with relevant standard of care therapies in models of lung and renal cancer. Taken together, our preclinical data strongly supports the clinical evaluation of MM-161 in cancer patients. Citation Format: Tamara Dake, Greg Finn, Melissa Geddie, Neeraj Kohli, Maja Razlog, Lihui Xu, Violette Paragas, Haluk Yuzugullu, Sara Ghassemifar, Yasmin Hashambhoy-Ramsay, Charlotte McDonagh, Marco Muda, Birgit Schoeberl. MM-161, a first-in-class pan-FGFR antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 22. doi:10.1158/1538-7445.AM2017-22

Abstract 3842: Design and engineering of TRAIL fusion proteins for cancer therapy

Protein-based agonists of apoptotic death receptors have shown remarkable preclinical efficacy but limited clinical response. The short circulating half-life of recombinant human TRAIL and the necessity of Fc-mediated clustering for potentiating agonistic antibodies against DR4 and DR5 have been proposed to be major impediments to the clinical success of this class. To address these limitations we have created Fc-scTRAIL, a single fusion polypeptide consisting of an IgG1 Fc region followed by three successive TRAIL monomers connected by two fifteen-amino acid linkers. While Fc-scTRAIL showed potent activity in vitro, we observed a low TM (48 °C) and rapid inactivation in serum indicating protein instability. Subsequently, we applied a directed evolution approach using yeast surface display to identify mutations that would stabilize the TRAIL trimer. When individual mutations were transferred to the Fc-scTRAIL format, we observed a dramatic increase in the TM (66-70 °C) while the combination of three mutations improved serum stability by ten-fold. Stabilized Fc-scTRAIL shows greater pro-apoptotic activity across a panel of cancer cell lines when compared to mapatumumab (anti-DR4) and drozitumab (anti-DR5), or the combination of antibodies even in the presence of anti-Fc cross-linking. Moreover, anti-Fc did not improve Fc-scTRAIL activity suggesting that the hexavalent design of the molecule maximizes death receptor activation. Currently, in vivo evaluation of Fc-scTRAIL for pharmacokinetic properties and activity is underway. We believe this format, when combined with an appropriate patient selection strategy, will result in improved clinical outcomes. Citation Format: Eric M. Tam, Hannah Hudson, Tamara Dake, Sara Ghassemifar, Andreas Raue, Yasmin Hashambhoy-Ramsay, Stephen L. Sazinsky, Anahita Daruwalla, Neeraj Kohli, Lihui Xu, Charlotte F. Mc Donagh, Birgit Schoeberl, Diana H. Chai. Design and engineering of TRAIL fusion proteins for cancer therapy. [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 3842.

Abstract 750: Nanoliposomal targeting of Ephrin receptor A2 (EphA2): Clinical translation

Ephrin receptor A2 (EphA2) is part of the Ephrin family of cell-cell junction proteins highly overexpressed in several solid tumors, and is associated with poor prognosis. We developed a novel EphA2-targeted docetaxel nanoliposome, leveraging organ specificity through enhanced permeability effect and cellular specificity through EphA2 targeting. The goal of the study was to develop the diagnostic framework enabling the clinical implementation of EphA2-based exclusion criteria in future MM-310 trials. We used qFACS and an in vitro assay for liposome (Ls)-cell interaction to identify the minimum number of EphA2 receptors to enable antibody-mediated internalization of Ls. We developed an IHC assay able to differentiate EphA2 - vs + cell lines. We characterized EphA2 staining pattern in tumor samples of various indications and developed a scoring algorithm that allows selection of patients in early clinical trials. While non targeted Ls do not associate with cells in vitro, there is a strong correlation between EphA2 expression and EphA2-Ls cell association independent of the cell line origin. We used the non-targeted Ls to determine the extent of non-specific binding that can be achieved (∼340 Ls/cell) and used partitioning to determine the minimum number of EphA2 receptors necessary to mediate targeting (∼3000 receptors/cell). We have developed and validated a qIHC assay for EphA2 (precision ∼90%, linearity 0.8 and reproducibility ∼5%). We stained a set of ∼200 tumor samples from various indications. EphA2 was found to be expressed in tumor cells, tumor-associated myofibroblasts, and tumor-associated blood vessels. Using an inclusive cutoff of 10%, EphA2 prevalence was found to range from 50% to 100% in the tumor types evaluated. No significant difference in staining was seen between metastasis and primary tumors in matched samples. In summary, we developed a diagnostic framework for prospective selection of EphA2+ patients for MM-310 trials based on a mechanistic single cell cut-off, and a clinical-grade IHC assay. Citation Format: Walid S. Kamoun, Shinji Oyama, Tad Kornaga, Theresa Feng, Lia Luus, Minh T. Pham, Dmitri B. Kirpotin, James D. Marks, Melissa Geddie, Lihui Xu, Alexey A. Lugovskoy, Monica Murphy, Carl Morrisson, Daryl C. Drummond. Nanoliposomal targeting of Ephrin receptor A2 (EphA2): Clinical translation. [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 750.