Kim Rosenthal

S100A9 Induced Inflammatory Responses Are Mediated by Distinct Damage Associated Molecular Patterns (DAMP) Receptors In Vitro and In Vivo

Release of endogenous damage associated molecular patterns (DAMPs), including members of the S100 family, are associated with infection, cellular stress, tissue damage and cancer. The extracellular functions of this family of calcium binding proteins, particularly S100A8, S100A9 and S100A12, are being delineated. They appear to mediate their functions via receptor for advanced glycation endproducts (RAGE) or TLR4, but there remains considerable uncertainty over the relative physiological roles of these DAMPs and their pattern recognition receptors. In this study, we surveyed the capacity of S100 proteins to induce proinflammatory cytokines and cell migration, and the contribution RAGE and TLR4 to mediate these responses in vitro. Using adenoviral delivery of murine S100A9, we also examined the potential for S100A9 homodimers to trigger lung inflammation in vivo. S100A8, S100A9 and S100A12, but not the S100A8/A9 heterodimer, induced modest levels of TLR4-mediated cytokine production from human PBMC. In contrast, for most S100s including S100A9, RAGE blockade inhibited S100-mediated cell migration of THP1 cells and major leukocyte populations, whereas TLR4-blockade had no effect. Intranasal administration of murine S100A9 adenovirus induced a specific, time-dependent predominately macrophage infiltration that coincided with elevated S100A9 levels and proinflammatory cytokines in the BAL fluid. Inflammatory cytokines were markedly ablated in the TLR4-defective mice, but unexpectedly the loss of TLR4 signaling or RAGE-deficiency did not appreciably impact the S100A9-mediated lung pathology or the inflammatory cell infiltrate in the alveolar space. These data demonstrate that physiological levels of S100A9 homodimers can trigger an inflammatory response in vivo, and despite the capacity of RAGE and TLR4 blockade to inhibit responses in vitro, the response is predominately independent of both these receptors.

Inhibition of CD73 AMP hydrolysis by a therapeutic antibody with a dual, non-competitive mechanism of action.

ABSTRACT CD73 (ecto-5′-nucleotidase) has recently been established as a promising immuno-oncology target. Given its role in activating purinergic signaling pathways to elicit immune suppression, antagonizing CD73 (i.e., releasing the brake) offers a complimentary pathway to inducing anti-tumor immune responses. Here, we describe the mechanistic activity of a new clinical therapeutic, MEDI9447, a human monoclonal antibody that non-competitively inhibits CD73 activity. Epitope mapping, structural, and mechanistic studies revealed that MEDI9447 antagonizes CD73 through dual mechanisms of inter-CD73 dimer crosslinking and/or steric blocking that prevent CD73 from adopting a catalytically active conformation. To our knowledge, this is the first report of an antibody that inhibits an enzymes function through 2 distinct modes of action. These results provide a finely mapped epitope that can be targeted for selective, potent, and non-competitive inhibition of CD73, as well as establish a strategy for inhibiting enzymes that function in both membrane-bound and soluble states.

Development of a Novel Ectonucleotidase Assay Suitable for High-Throughput Screening

5′-Ectonucleotidase (NT5E) catalyzes the conversion of adenosine monophosphate to adenosine and free phosphate. The role of this ectonucleotidase and its production of adenosine are linked with immune function, angiogenesis, and cancer. NT5E activity is typically assayed either by chromatographic quantification of substrates and products using high-performance liquid chromatography (HPLC) or by quantification of free phosphate using malachite green. These methods are not suitable for robust screening assays of NT5E activity. HPLC is not readily suitable for the rapid and efficient assay of multiple samples and malachite green is highly sensitive to the phosphate-containing buffers common in various media and sample buffers. Here the development and validation of a novel high-throughput ectonucleotidase screening assay are described, which makes use of a luciferase-based assay reagent, the Promega CellTiter-Glo kit, to measure the catabolism of AMP by NT5E. This multiwell plate-based assay facilitates the screening of potential ectonucleotidase antagonists and is unaffected by the presence of contaminating phosphate molecules present in screening samples.

Use of a neutralizing antibody helps identify structural features critical for binding of Clostridium difficile toxin TcdA to the host cell surface

Clostridium difficile is a clinically significant pathogen that causes mild-to-severe (and often recurrent) colon infections. Disease symptoms stem from the activities of two large, multidomain toxins known as TcdA and TcdB. The toxins can bind, enter, and perturb host cell function through a multistep mechanism of receptor binding, endocytosis, pore formation, autoproteolysis, and glucosyltransferase-mediated modification of host substrates. Monoclonal antibodies that neutralize toxin activity provide a survival benefit in preclinical animal models and prevent recurrent infections in human clinical trials. However, the molecular mechanisms involved in these neutralizing activities are unclear. To this end, we performed structural studies on a neutralizing monoclonal antibody, PA50, a humanized mAb with both potent and broad-spectrum neutralizing activity, in complex with TcdA. Electron microscopy imaging and multiangle light-scattering analysis revealed that PA50 binds multiple sites on the TcdA C-terminal combined repetitive oligopeptides (CROPs) domain. A crystal structure of two PA50 Fabs bound to a segment of the TcdA CROPs helped define a conserved epitope that is distinct from previously identified carbohydrate-binding sites. Binding of TcdA to the host cell surface was directly blocked by either PA50 mAb or Fab and suggested that receptor blockade is the mechanism by which PA50 neutralizes TcdA. These findings highlight the importance of the CROPs C terminus in cell-surface binding and a role for neutralizing antibodies in defining structural features critical to a pathogens mechanism of action. We conclude that PA50 protects host cells by blocking the binding of TcdA to cell surfaces.

Tumor uptake of pegylated diabodies: Balancing systemic clearance and vascular transport

ABSTRACT The accumulation, dissemination and clearance of monoclonal antibody‐based therapeutics or imaging reagents targeting tumor associated antigens is governed by several factors including affinity, size, charge, and valency. Tumor targeting antibody fragments have distinct advantages over intact monoclonal antibodies such as enhanced penetration within the tumor and rapid accumulation but are subject to rapid clearance. Polyethylene glycol (PEG)‐modified antibody fragments can provide a way to balance tumor penetration and accumulation with improved serum persistence. In this study, we use a diabody, the dimeric antibody fragment, targeting the 5T4 antigen to assess the impact of PEGs of distinct size and shape on tumor accumulation and pharmacokinetics (PK). We show that PEG‐modified diabodies improved the PK of the parental diabody from a half‐life of 40min to over 40h for the higher molecular weight PEG conjugated diabodies. This improvement correlates with the increasing hydrodynamic size of pegylated diabodies, and can serve as a better predictor of the PK behavior of pegylated molecules than molecular weight alone. Tumor uptake profiles determined by quantitative PET imaging differed significantly based on PEG size and shape with diabody‐PEG5K showing peak accumulation early on, but with the larger diabody‐PEG20K showing better sustained tumor uptake at later time points. In addition, we demonstrate that a diabody‐PEG20K‐B with a hydrodynamic radius (Rh) of 6nm had superior tumor uptake than the larger diabody‐PEG40K‐B with Rh of 12nm, indicating that beyond 6nm, larger pegylated diabodies have a slower tumor uptake rate while having comparable clearance kinetics. Our data demonstrate that pegylated diabodies with Rh of ˜6nm have an optimal size and PK profile for tumor uptake. Understanding the impact of pegylation on PK and tumor uptake could facilitate the development of pegylated diabodies as therapeutics.

Generation and Characterization of an IgG4 Monomeric Fc Platform

The immunoglobulin Fc region is a homodimer consisted of two sets of CH2 and CH3 domains and has been exploited to generate two-arm protein fusions with high expression yields, simplified purification processes and extended serum half-life. However, attempts to generate one-arm fusion proteins with monomeric Fc, with one set of CH2 and CH3 domains, are often plagued with challenges such as weakened binding to FcRn or partial monomer formation. Here, we demonstrate the generation of a stable IgG4 Fc monomer with a unique combination of mutations at the CH3-CH3 interface using rational design combined with in vitro evolution methodologies. In addition to size-exclusion chromatography and analytical ultracentrifugation, we used multi-angle light scattering (MALS) to show that the engineered Fc monomer exhibits excellent monodispersity. Furthermore, crystal structure analysis (PDB ID: 5HVW) reveals monomeric properties supported by disrupted interactions at the CH3-CH3 interface. Monomeric Fc fusions with Fab or scFv achieved FcRn binding and serum half-life comparable to wildtype IgG. These results demonstrate that this monomeric IgG4 Fc is a promising therapeutic platform to extend the serum half-life of proteins in a monovalent format.

Abstract 4328: Phenotypic selection for identification of functional antibodies and development of high throughput screening assays.

Discovery of therapeutic antibodies requires the continued identification of tractable targets, and we have employed a phenotypic screening strategy to this end. In this study, antibodies from phage display libraries were screened for their abilities to inhibit MDA-MB-231 (‘triple negative’) breast cancer cells. The screening cascade included tests for specific cell binding, antibody internalization, and cytotoxicity as an antibody-drug conjugate. Targets of these antibodies then were identified using immunoprecipitation and mass spectrometry, and confirmed by showing diminished antibody binding after siRNA-mediated knockdown of the putative target gene. One target was identified as NT5E (also known as CD73), a 5’-ectonucleotidase. NT5E catalyzes the conversion of adenosine monophosphate to adenosine, and its function has been linked with immunity, angiogenesis, and cancer. To screen for additional inhibitors of this target, a novel, high-throughput ectonucleotidase assay was developed. NT5E activity is typically assayed either by high performance liquid chromatography or by quantification of free phosphate using malachite green, and neither of these methods is suitable for high-throughput screening. One inhibitory antibody was found to significantly slow growth of MDA-MB-231 xenograft tumors in mice. In summary, our phenotypic screening approach provides a mechanism for rapidly discovering and evaluating new antibody targets and is being used to accelerate the discovery of new cancer drugs. Citation Format: Carl Hay, Steven Rust, Erin Sult, Lori Clarke, Kim Rosenthal, Sandrine Guillard, David Lowne, Matt Flynn, Lutz Jermutus, Ralph Minter, Robert Hollingsworth, Kris Sachsenmeier. Phenotypic selection for identification of functional antibodies and development of high throughput screening assays. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4328. doi:10.1158/1538-7445.AM2013-4328