Autoantibody immune complex (IC) activation of Fcγ receptors (FcγRs) is a common pathogenic hallmark of multiple autoimmune diseases. Given that the IC structural features that elicit FcγR activation are poorly understood and the FcγR system is highly complex, few therapeutics can directly block these processes without inadvertently activating the FcγR system. To address these issues, the structure activity relationships of an engineered panel of multivalent Fc constructs were evaluated using sensitive FcγR binding and signaling cellular assays. These studies identified an Fc valency with avid binding to FcγRs but without activation of immune cell effector functions. These observations directed the design of a potent trivalent immunoglobulin G-Fc molecule that broadly inhibited IC-driven processes in a variety of immune cells expressing FcγRs. The Fc trimer, Fc3Y, was highly efficacious in three different animal models of autoimmune diseases. This recombinant molecule may represent an effective therapeutic candidate for FcγR-mediated autoimmune diseases.

Aptamers are short oligonucleotides that fold into well-defined three-dimensional architectures thereby enabling specific binding to molecular targets such as proteins. To be successful as a novel therapeutic modality, it is important for aptamers to not only bind their targets with high specificity and affinity, but also to exhibit favorable properties with respect to in vivo stability, cost-effective synthesis, and tolerability (i.e., safety). We describe methods for generating aptamers comprising 2 - deoxy purines and 2 -O-methyl pyrimidines (dRmY) that broadly satisfy many of these additional constraints. Conditions under which dRmY transcripts can be efficiently synthesized using mutant T7 RNA polymerases have been identified and used to generate large libraries from which dRmY aptamers to multiple target proteins, including interleukin (IL)-23 and thrombin, have been successfully discovered using the SELEX process. dRmY aptamers are shown to be highly nuclease-resistant, long-lived in vivo, efficiently synthesized, and capable of binding protein targets in a manner that inhibits their biologic activity with K(D) values in the low nM range. We believe that dRmY aptamers have considerable potential as a new class of therapeutic aptamers.

It is generally recognized that only relatively small molecular weight (typically < ∼ 500 Da) drugs can effectively permeate through intact stratum corneum. Here, we challenge this orthodoxy using a 62-nucleotide (molecular weight = 20,395 Da) RNA-based aptamer, highly specific to the human IL-23 cytokine, with picomolar activity. Results demonstrate penetration of the aptamer into freshly excised human skin using two different fluorescent labels. A dual hybridization assay quantified aptamer from the epidermis and dermis, giving levels far exceeding the cellular half maximal inhibitory concentration values (>100,000-fold), and aptamer integrity was confirmed using an oligonucleotide precipitation assay. A T helper 17 response was stimulated in freshly excised human skin resulting in significantly upregulated IL-17f, and IL-22; topical application of the IL-23 aptamer decreased both IL-17f and IL-22 by approximately 45% but did not result in significant changes to IL-23 mRNA levels, confirming that the aptamer did not globally suppress mRNA levels. This study demonstrates that very-large-molecular-weight RNA aptamers can permeate across the intact human skin barrier to therapeutically relevant levels into both the epidermis and dermis and that the skin-penetrating aptamer retains its biologically active conformational structure capable of binding to endogenous IL-23.

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γδ T cells are a unique subset of T cells found in both blood (Vδ2) and tissues (Vδ1). Unlike canonical αβ T cells that recognize peptide antigens through MHC class I/II molecules, γδ T cells recognize lipids and metabolites presented by non-MHC molecules. γδ T cells are highly cytotoxic and can rapidly kill both infected and cancer cells. Their cytotoxicity and lack of MHC restriction makes γδ T cells a promising target for allogeneic cell transfer therapy. In collaboration with MRL Oncology and ID/Vax we are investigating γδ T cells as a potential “off-the-shelf” cell transfer therapy. The ESC is currently optimizing ex vivo expansion protocols for both Vδ1 and Vδ2 T cells. Vδ2 cells rapidly expand from PBMCs after exposure to Zometa/IL-2, and are efficient cancer killers in vitro. To assess activation state, targeted metabolomics analysis was performed during Vδ2 expansion, and displayed significant differences in glutamate utilization, lactic acid, myo-inositol, and several essential and non-essential amino acids. We have also isolated and expanded Vδ1 T cells from PBMCs and human tissues (colon). These cells express high levels of NKG2D, which is suggestive of cytotoxic potential and we are currently assessing their cancer cell killing capacity in vitro. In collaboration with GpGx, we are identifying the dominant T-cell receptor (TCR) repertoires present in these populations by performing sc-TCRseq, sc-REAPseq, and comprehensive immunophenotyping. The results of these assays will be used to identify the optimum expansion protocol as well as to inform on the mechanisms underlying γδ T cell cancer targeting.

Sepsis is a life-threatening condition caused by a dysregulated host response to infection. Despite continued efforts to understand the pathophysiology of sepsis, no effective therapies are currently available. While singular components of the aberrant immune response have been investigated, comprehensive studies linking different data layers are lacking. Using an integrated systems immunology approach, we evaluated neutrophil phenotypes and concomitant changes in cytokines and metabolites in sepsis patients. Our findings identify novel differentially expressed immature neutrophil subsets in sepsis patients. These and other subsets correlate with various proteins, metabolites, and lipids, including pentraxin-3, angiopoietin-2 and lysophosphatidylcholines, in sepsis patients. These results enabled the construction of a predictive model based on weighted multi-omics linear regression analysis for patient classification. These findings could help inform earlier patient stratification and treatment options, as well as facilitate future mechanistic studies targeting the trifecta of surface marker expression, cytokines, and metabolites.

Following coronary artery damage, von Willebrand Factor (vWF) multimers adhere to exposed collagen via the vWF A3 domain. Platelets passing through damaged vessels under the conditions of high shear force associated with stenosed arteries will adhere to the vWF A1 domain via GPIb receptors. This initial platelet binding event triggers the formation of a thrombus that can limit blood flow to the myocardium and produce the symptoms of acute coronary syndrome (ACS). Through in vitro selection (SELEX) and subsequent lead optimization, we have generated a nuclease-resistant DNA aptamer that binds to the vWF A1 domain with high affinity. The anti-vWF aptamer inhibits both botrocetin-induced platelet aggregation of platelet rich plasma and shear force-induced platelet aggregation in citrated whole blood. Continuous intravenous infusion of the aptamer in cynomolgus macaques is able to effectively inhibit thrombus formation induced by electrical injury to the carotid artery. Modest effects on template bleeding are observed with the vWF-specific aptamer relative to anti-GPIIb/IIIa therapy. Based upon these results, this aptamer is an excellent candidate for treatment of ACS.