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RNA interference (RNAi) offers the potential to treat disease at the earliest onset by selectively turning off the expression of target genes, such as intracellular oncogenes that drive cancer growth. However, the development of RNAi therapeutics as anti-cancer drugs has been limited by both a lack of efficient and target cell-specific delivery systems and the necessity to overcome numerous intracellular barriers, including serum/lysosomal instability, cell membrane impermeability, and limited endosomal escape. Here, we combine two technologies to achieve posttranscriptional gene silencing in tumor cells: Centyrins, alternative scaffold proteins binding plasma membrane receptors for targeted delivery, and small interfering RNAs (siRNAs), chemically modified for high metabolic stability and potency. An EGFR Centyrin known to internalize in EGFR-positive tumor cells was site-specifically conjugated to a beta-catenin (CTNNb1) siRNA and found to drive potent and specific target knockdown by free uptake in cell culture and in mice inoculated with A431 tumor xenografts (EGFR amplified). The generalizability of this approach was further demonstrated with Centyrins targeting multiple receptors (e.g., BCMA, PSMA, and EpCAM) and siRNAs targeting multiple genes (e.g., CD68, KLKb1, and SSB1). Moreover, by installing multiple conjugation handles, two different siRNAs were fused to a single Centyrin, and the conjugate was shown to simultaneously silence two different targets. Finally, by specifically pairing EpCAM-binding Centyrins that exhibited optimized internalization profiles, we present data showing that an EpCAM Centyrin CTNNb1 siRNA conjugate suppressed tumor cell growth of a colorectal cancer cell line containing an APC mutation but not cells with normal CTNNb1 signaling. Overall, these data demonstrate the potential of Centyrin-siRNA conjugates to target cancer cells and silence oncogenes, paving the way to a new class of anticancer drugs.

Tumor-targeted near-infrared fluorescent dyes have the potential to improve cancer surgery by enabling surgeons to locate and resect more malignant lesions where good visualization tools are required to ensure complete removal of malignant tissue. Although the tumor-targeted fluorescent dyes used in humans to date have been either small organic molecules or high molecular weight antibodies, low molecular weight protein scaffolds have attracted significant attention because they penetrate solid tumors almost as efficiently as small molecules, but can be infinitely mutated to bind almost any antigen. Here we describe the use of a 10 kDa protein scaffold, a Centyrin, to target a near-infrared fluorescent dye to tumors that overexpress the epidermal growth factor receptor (EGFR) for fluorescence-guided surgery (FGS). We have developed and optimized the dose and time required for imaging small tumor burdens with minimal background fluorescence in real-time fluorescence-guided surgery of EGFR-expressing tumor xenografts in murine models. We demonstrate that the Centyrin-near-infrared dye conjugate (CNDC) binds selectively to human EGFR

Targeted delivery of therapeutic payloads to specific tissues and cell types is an important component of modern pharmaceutical development. Antibodies or other scaffold proteins can provide the cellular address for delivering a covalently linked therapeutic via specific binding to cell-surface receptors. Optimization of the conjugation site on the targeting protein, linker chemistry and intracellular trafficking pathways can all influence the efficiency of delivery and potency of the drug candidate. In this study, we describe a comprehensive engineering experiment for an EGFR binding Centyrin, a highly stable fibronectin type III (FN3) domain, wherein all possible single-cysteine replacements were evaluated for expression, purification, conjugation efficiency, retention of target binding, biophysical properties and delivery of a cytotoxic small molecule payload. Overall, 26 of the 94 positions were identified as ideal for cysteine modification, conjugation and drug delivery. Conjugation-tolerant positions were mapped onto a crystal structure of the Centyrin, providing a structural context for interpretation of the mutagenesis experiment and providing a foundation for a Centyrin-targeted delivery platform.

A confluence of social, economic, and demographic trends has left a generation of young Americans facing an uncertain future in the workforce. If we are to improve their prospects and prepare them for rewarding careers, disparate stakeholders-employers, educators, youth advocates, and others-must work in common purpose. This article suggests ways to build community partnerships, with a special focus on engaging private employers in the effort.

Prostate specific membrane antigen (PSMA), a transmembrane glycoprotein, is a clinically validated marker of prostate cancer. PSMA is overexpressed in primary and metastatic prostate cancer when compared to normal prostate tissue which makes it an attractive target for immunotherapy. Several antibodies are being considered to target PSMA for the development of an anticancer drug. The expression levels of PSMA on tumor cells may play a role in response to treatment with anti PSMA drugs. Therefore, it is important to monitor PSMA expression on tumor cells before and during treatment. We used CellSearch® CTC assay to determine the presence of PSMA on circulating tumor cells (CTCs) using anti PSMA Centyrin as a biomarker. Centyrins are a class of alternative scaffold molecules which are proteins of small size (∼ 10 kDa). They are engineered to bind to target molecules with high specificity and sensitivity. The anti PSMA Centyrin was tested for specificity and sensitivity using tissue cultured tumor cell lines spiked into normal healthy blood and then compared to anti PSMA antibody. The anti PSMA Centyrin showed 100% positivity with the high PSMA expressing cell line (LNCap), 25% positivity with the low PSMA expressing cell line (22Rv1) and 0% positivity with the PSMA negative cell line (PC3-9). The anti PSMA Centyrin was further tested with prostate patient blood samples to determine the expression levels of PSMA on CTCs. The patient samples which contain CTCs are positive for anti PSMA Centyrin and the percentage of CTCs positive for PSMA ranged from 25-100%. Thus, PSMA expression on CTCs can be determined using CellSearch® system and anti PSMA Centyrin as a biomarker in a clinical trial. Citation Format: Bhavesh Vaidya, Peter Vulfson, Renouard Sanders, Donna Klein, Shalom Goldberg, Steve Jacobs, Chandra Rao. Detection of PSMA on circulating tumor cells from blood of prostate cancer patients using anti-PSMA Centyrin. [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 4972.

Centyrins are engineered alternative scaffold proteins that combine the affinity and specificity of antibodies with increased tissue penetration and simplified manufacturing. To identify optimal positions for chemical conjugation within the Centyrin scaffold, the crystal structure of an anti-EGFR Centyrin was solved and a library consisting of all possible single-cysteine variants was constructed and assessed for expression level in E. coli, efficiency of conjugation to maleimide derivatives, protein aggregation, thermostability, and EGFR binding. The best variants were conjugated to cytotoxic drugs and compared in a cell-killing assay. Three optimal positions were identified at which the Centyrin conjugated efficiently to maleimides; retained high expression, antigen binding, and biophysical properties; and showed increased cell-killing potency relative to the parent molecule. Citation Format: Rosa Cardoso, Shalom Goldberg, Tricia Lin, Tracy Spinka-Doms, Donna Klein, Steven Jacobs, Gary Gilliland, Karyn O’Neil. Evaluation of drug conjugation sites in an anti-EGFR Centyrin. [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 LB-062.

Many tumors compensate with a resistance mechanism once a signaling pathway is inhibited, as evidenced in patients treated with small molecule inhibitors of EGFR which frequently show increased expression of c-Met or HGF. Since both EGFR and c-Met signal through the same survival and anti-apoptotic pathways, inhibition of the pair of receptors could improve overall efficacy. This was demonstrated clinically, where the combination of onartuzumab and erlotinib showed promising Phase II trials in lung cancer patients. Centyrins are small proteins (~10 kDa) of an emerging class of proteins termed alternative scaffolds. They are engineered to bind to target molecules with high specificity and affinity, but are structurally simpler molecules than antibodies in that that they are single chain, unglycosylated proteins that lack disulfide bonds. In addition, monomeric Centyrins can be linked together such that one molecule can bind to and inhibit multiple targets. Anti-EGFR and anti-c-Met Centyrins were selected for their ability to inhibit ligand-induced phosphorylation. Molecules with a wide range of affinities were identified and bispecific molecules were generated. Phosphorylation of EGFR, c-Met, and ERK were monitored in cells treated with individual monomeric Centyrins, a mixture of monomeric Centyrins, or linked bispecific Centyrins. One bispecific EGFR/c-Met Centyrin provided a 134-fold increase in potency in inhibition of phosphorylation of c-Met compared to the mixture of the two Centyrins. The increase in potency observed in cell signaling translated to enhanced anti-proliferative activity with a potency >100-fold compared to that of the mixture of monospecific Centyrins. The bispecific EGFR/c-Met Centyrins were produced as fusion proteins linked to an albumin binding domain in order to reduce kidney filtration and evaluated in SCID-beige mice implanted with tumor cells engineered to express human HGF. Interestingly, all of the Centyrins tested significantly reduced the size of the tumors compared to the control and the degree of tumor growth inhibition correlated with the affinity to both EGFR and c-Met. In a second tumor model, complete tumor regressions were observed in all mice treated with the bispecific EGFR/c-Met Centyrin. The Centyrin platform could offer benefits over currently available treatment options. Our data demonstrate that in addition to inhibiting two targets simultaneously, a single bispecific molecule allows for significant avidity at the cellular level. This could translate into a lower dosing regimen that allows for improved efficacy through avidity and decreased toxicity. We anticipate that avidity will allow for improved specificity for tumor compared to normal tissue. In addition, the small size of Centyrins may provide an advantage for tumor targeting and accumulation. Citation Format: Donna Klein, Steve Jacobs, Moores Sheri, Mark Anderson, Ricardo Attar, Alexander Barnakov, Kerry Brosnan, Barbara Bushey, Kristen Chevalier, Diana Chin, Carla Cornejo, Mike Diem, Linus Hyun, Elise Kuhar, Francis McCabe, Kristen Picha, Tracy Spinka-Doms, Edward Swift, Karyn O'Neil. Bispecific Centyrin simultaneously targeting EGFR and c-Met demonstrates improved activity compared to the mixture of single agents. [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 LB-312. doi:10.1158/1538-7445.AM2013-LB-312

Hypersensitivity pneumonitis (HP) or extrinsic allergic alveolitis (British terminology) is defined as a pulmonary illness caused by an immunologic reaction to a variety of inhaled environmental antigens that involves the periphery of the lung.1 The affected subject acquires an abnormal sensitivity or heightened reactivity to the inciting agent, resulting in an inflammatory host response located primarily in the alveolar-air exchange portion of the lung rather than in the conducting airways seen in asthma. The distinction is important, for certain types of asthma, notably extrinsic, IgE-mediated allergic asthma, is also a form of hypersensitivity lung disease often associated with airborne organic antigens.2