Linker Domain Size Does Not Impact Bivalent HER3 Targeting Affibody Efficacy

The Epidermal Growth Factor (EGF) family of receptors, also called ErbB or HER family, is a group of tyrosine kinase transmembrane proteins that have many regulatory purposes including regulating cell proliferation and survival. Members of the HER family rely on forming dimers upon ligand binding to promote downstream signaling. Gene mutations can result in the deregulation of the HER receptors, further resulting in cancer. HER3, a receptor that is deregulated in many cancers including ovarian, breast, and lung cancer, has been found to be responsible for drug resistance to therapeutics that currently exist to target other members of the HER family. This can occur through increased phosphorylation and overexpression of the HER3 receptor. There are many HER3 targeted therapeutics, including monoclonal antibodies (mAbs), that are currently in phase 1 and 2 of clinical studies; however, no HER3 targeted therapeutics have been approved by the FDA. In addition to this, previous studies have demonstrated that not every patient will respond to a specific treatment plan or therapeutic; therefore, the development of various treatment options is essential. An engineered protein known as the affibody, which in previous studies has shown to be highly soluble, thermally stable, and small in size allowing for effective tissue penetration, has emerged as a potential therapeutic agent for cancer. In this study, it was found that multivalent affibodies, which are affibodies with more than one binding domain, are more effective at inhibiting HER3 activation, also known as phosphorylation, and inducing HER3 downregulation than monovalent affibodies in multiple cell lines. Inhibiting receptor activation can be effective at reducing cell proliferation and survivability. In addition, other modifications were made to optimize the affibodies, such as altering the length of the linker that tethers the binding domains in a multivalent affibody together, and to test for their efficacy. Finally, an albumin binding domain was incorporated into the affibody design to help increase affibody half-life, which would be essential for in vivo testing

ENGINEERED MULTIVALENCY FOR ENHANCED AFFIBODY-BASED HER3 CANCER THERAPY

The receptor tyrosine kinase HER3 is well established as a compelling therapeutic target in numerous cancers, including ovarian cancer. HER3 potently activates the PI3K/Akt pro-survival pathway, mediates drug resistance, and is implicated in cancer progression and poor clinical outcomes. Yet, conventional small molecule- and monoclonal antibody-based approaches have so far failed to yield a widely used therapeutic that directly targets HER3. Here, we investigated a novel approach involving specific, multivalent engagement of HER3 with affibody molecules as an alternative to existing therapeutics. We established that multivalent HER3-targeted affibodies more effectively inhibit neuregulin 1β-mediated HER3 activation compared to monovalent affibodies; these multivalent ligands induced rapid and prolonged HER3 downregulation, indicating a potentially valuable mechanism of action to limit HER3-mediated pro-mitogenic signaling and acquired resistance. HER3-targeted affibodies also proved highly amenable to molecular engineering approaches, as modulation of linker length, valency, and albumin binding domain (ABD) fusion placement allowed for robust retention of ligand bioactivity. We further report significant mechanistic evidence supporting HER3 downregulation as a highly specific phenomenon prompted by HER3 sequestration by multivalent ligands. Most importantly, we show that both monovalent and bivalent HER3-targeted affibody-ABD fusion proteins significantly reduce tumor burden in an adriamycin-resistant ovarian cancer model in mice. Overall, these data serve as compelling evidence for HER3 multivalent ligands as promising experimental therapeutics for the treatment of ovarian cancer as single agents as well as in combination with other drugs. Further, HER3 affibodies represent a promising template for development of targeted therapies or drug conjugates for more powerful ovarian cancer therapy in the future

Engineering Saccharomyces cerevisiae for the production and secretion of Affibody molecules

BACKGROUND: Affibody molecules are synthetic peptides with a variety of therapeutic and diagnostic applications. To date, Affibody molecules have mainly been produced by the bacterial production host Escherichia coli. There is an interest in exploring alternative production hosts to identify potential improvements in terms of yield, ease of production and purification advantages. In this study, we evaluated the feasibility of Saccharomyces cerevisiae as a production chassis for this group of proteins. RESULTS: We examined the production of three different Affibody molecules in S. cerevisiae and found that these Affibody molecules were partially degraded. An albumin-binding domain, which may be attached to the Affibody molecules to increase their half-life, was identified to be a substrate for several S. cerevisiae proteases. We tested the removal of three vacuolar proteases, proteinase A, proteinase B and carboxypeptidase Y. Removal of one of these, proteinase A, resulted in intact secretion of one of the targeted Affibody molecules. Removal of either or both of the two additional proteases, carboxypeptidase Y and proteinase B, resulted in intact secretion of the two remaining Affibody molecules. The produced Affibody molecules were verified to bind their target,\ua0human HER3, as potently as the corresponding molecules produced in E. coli in an in vitro surface-plasmon resonance binding assay. Finally, we performed a fed-batch fermentation with one of the engineered protease-deficient S. cerevisiae strains and achieved a protein titer of 530\ua0mg Affibody molecule/L. CONCLUSION: This study shows that engineered S. cerevisiae has a great potential as a production host for recombinant Affibody molecules, reaching a high titer, and for proteins where endotoxin removal could be challenging, the use of S. cerevisiae obviates the need for endotoxin removal from protein produced in E. coli

Cellular Selections Aid Translational Binding in Ligand Discovery

University of Minnesota Ph.D. dissertation.March 2017. Major: Chemical Engineering. Advisor: Benjamin Hackel. 1 computer file (PDF); x, 133 pages.Engineered protein ligands have been successfully applied as molecular targeting agents in the clinic for diagnostic and therapeutic applications. Yeast surface display selections have shown effectiveness in discovery and evolution of ligands against a variety of target molecules to meet these ends. Often, these biomarkers are transmembrane proteins, which are made up with hydrophilic extracellular and intracellular domains and hydrophobic transmembrane domains. Due to these hydrophobic domains, full length transmembrane proteins are difficult to work with in aqueous systems. Thus, ligand selections are conventionally carried out against recombinant extracellular domains of these biomarkers. These molecules may be poor models of the corresponding full length, membrane associated proteins due to instability in the absence of the truncated domains, inadequacies in protein folding and post-translational modification, presence of non-natural tags required for protein purification and immobilization for selection, or denaturation during a variety of handling steps. Thus, ligand selection campaigns against these molecules often end with the evolution of ligands that bind the soluble target, but have diminished or abolished activity against the true biomarker of interest. The work presented here aims to provide a new toolkit for ligand selection that bypasses this translational hurdle by optimizing selections of yeast surface display libraries directly against target-expressing mammalian cell monolayers. Target specific binder enrichment is rigorously optimized and substantially improved through the use of a longer flexible linker in yeast surface display which enhances the recovery of even micromolar binding interactions that enable naïve library selections. Five ligand selection strategies using soluble target and cellular selection strategies are probed using a fibronectin domain and an affibody library, showing that methods partially or completely utilizing cellular selection strategies have a higher likelihood of isolating translatable binders while also developing new ligands that bind tumor vasculature targets CD276 and Thy1. Controlled valency reduction using the reducing agent dithiothreitol (DTT) to reduce yeast-displayed ligand levels to 3,000-6,000 ligands per cell enhances affinity discrimination between a 2 nM and 17 nM binder 16-fold, yielding a modular method for affinity-based cellular selection. This method is applied to discovery and affinity maturation of fibronectin domains targeting epithelial cell adhesion molecule (EpCAM) for oncological applications. Depletion of non-specific binding ligands is achieved using a pre-blocking strategy with disadhered mammalian cells, yielding a 14-fold selectivity advantage for a specific binder relative to a non-specific binder. Collectively, the results presented in this dissertation elucidate the factors that dictate cell-cell interactions within the context of yeast display ligand selections on mammalian cell targets and provide a suite of tools for ligand selections as well as a variety of targeting molecules with diagnostic and therapeutic applications.Stern, Lawrence. (2017). Cellular Selections Aid Translational Binding in Ligand Discovery. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/196527

Dual- and triple-targeting of the HER-family members using combinations of mono- and bispecific antibodies

Epidermal growth factor receptor (EGFR)-targeted cancer treatments with antibodies like Cetuximab are successfully used in the clinic for about 20 years. However, intrinsic, as well as newly developed resistance mechanisms to EGFR-targeted therapies, are the main reason for their failure. Activation of human epidermal growth factor receptor 3 (HER3)-signaling upon EGFR-targeted therapies is frequently observed and has motivated the development of combination therapies that simultaneously block EGFR and HER3. In this study, bispecific and bivalent, or tetravalent, respectively, single-chain diabody (scDb) and scDb-Fc molecules were developed comprising the antigen-binding sites of a humanized version of Cetuximab (hu225) as well as a recently developed anti-HER3 antibody (3-43). In total, eight molecules (two scDb and six scDb-Fc) with varying linkers were engineered. The scDb hu225x3 43 Fc showed the most favorable properties regarding production yield, purity, homogeneity and linker setup. Binding of the scDb-Fc to recombinant receptors, as well as to HER-family receptor expressing cell lines revealed retained binding properties, compared to parental antibodies. Furthermore, the scDb hu225x3 43 Fc showed strong and long-lasting inhibition of downstream signaling by EGF, HRG or combination of both ligands. Proliferation studies on head and neck squamous cell carcinoma (HNSCC), triple negative breast cancer (TNBC), and colorectal cancer (CRC) cell lines revealed either similar, or stronger inhibition, compared to parental antibodies as single or combination treatment, which translated into to long-lasting growth suppression in a s.c. xenograft tumor model. Treatment with the bispecific antibody inhibited in vitro HRG-stimulated oncosphere formation of two TNBC cell lines. In an orthotopic MDA-MB-468 tumor model, superior antitumor effects were observed compared to those obtained by the parental antibodies alone or in combination. Furthermore, this was associated with a reduced number of cells with stem-like properties demonstrating that the bispecific antibody not only efficiently blocks TNBC proliferation but also the survival and expansion of the cancer stem cell population. The high degree of plasticity and compensatory signaling within the HER-family not only leads to compensatory crosstalk by HER3 but also HER2 giving the rational to combine the EGFR- and HER3-targeting scDb-Fc with a HER2-targeting antibody like Trastuzumab. The triple-targeting approach with the scDb-Fc and Trastuzumab was superior in inhibition of HRG-stimulated proliferation of the CRC cell line LIM1215 compared to the combination of IgG hu225, Trastuzumab and IgG 3 43. This was also observed in primary and secondary CRC oncosphere formation assays. Finally, in CRC patient derived organoids (PDOs) grown in HRG-supplemented medium the triple-targeting of EGFR, HER2 and HER3 provided broader efficacy than dual- or mono-targeting of receptors of the HER family. In contrast to Afatinib (anti-EGFR, -HER2, -HER4), the triple-targeted antibody approach showed efficient inhibition in all tested PDOs. Thus, the bispecific scDb-Fc alone or in combination with Trastuzumab represents a superior strategy to deal with primary and acquired resistances compared to targeting a single receptor with different antibodies or any combination of antibodies targeting two receptors of the HER-family