Because the variable ability of the antibody constant (Fc) domain name

Because the variable ability of the antibody constant (Fc) domain name to recruit innate immune effector cells and complement is a major factor in antibody activity in vivo convenient means of assessing these binding interactions is of high relevance to the development of enhanced antibody therapeutics and to understanding the protective or pathogenic antibody response to infection vaccination and self. with these receptors was quantified. We demonstrate qualitative and quantitative assessment of binding preferences and affinities across IgG subclasses Fc domain name point mutants and antibodies with variant glycosylation. This method can serve as a rapid proxy for biophysical methods that require substantial sample quantities high-end instrumentation and serial analysis across multiple binding interactions thereby offering a useful means to characterize monoclonal antibodies clinical antibody samples and antibody mimics or alternatively to investigate the binding preferences of candidate Fc receptors. Keywords: Fc domain name Fcγ receptor IgG antibody glycosylation lectin luminex multiplex Introduction Research and development of clinically relevant antibody therapeutics as well as an increasingly refined understanding of the humoral response to contamination and vaccination has demonstrated the crucial importance of antibodies across a range of disease says. In vivo effector function that is the ability of an antibody to interact BCL2L5 with antibody receptors expressed solubly in plasma on the surface of innate immune effector cells or even intracellularly following internalization of immune complexes is an important aspect of antibody activity. As such mechanistic understanding of how antibodies can link antigen acknowledgement to potent biological effect through the spectrum of Ig receptors is usually of critical therapeutic relevance. The binding affinity of an IgG for Fc receptors (FcR) can be modulated by IgG subclass 1 Fc domain name glycosylation 2 avidity driven by immune complex formation 3 4 IgG multimerization 5 variant disulfide bond formation 6 or via 4SC-202 amino acid point mutations recognized by recombinant protein engineering methods7 or those present naturally among GM allotypes.8 9 The producing combinatorial diversity in antibody characteristics is complemented by diversity among antibody receptors which even among classical FcγR vary in subclass binding preferences glycan sensitivity cellular distribution and expression level and can lead to outcomes ranging from immunosuppression to secretion 4SC-202 of lytic factors. For protein therapeutics rational modulation of these collective effector functions via subclass and isotype choice glycoengineering amino acid point mutations or via entirely novel binding domains promises to allow specific effector functions to be alternatively enhanced or ablated as desired.10 11 Likewise some of these modifications are available to B cells with longstanding evidence that IgG subclass selection is highly regulated and increasing evidence that this immune system is able to actively tune antibody activity based on variant glycosylation.12-15 Collectively these natural mechanisms offer a path for similar rational induction of antibody responses with specific functional profiles via vaccination.16 Furthermore beyond relatively well-characterized FcγR and match proteins a growing number of diverse and structurally unrelated Fc-binding proteins have been identified ranging from the pH-sensitive neonatal Fc receptor17 to C-type lectins such as dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) 18 FcR-Like receptors 19 4SC-202 20 mannose-binding lectin 2 (MBL2) 21 TRIM21 22 macrophage mannose receptor (MMR) 23 and Dectin-1.24 Probing the acknowledgement properties of these and other FcR for engineered and naturally-produced IgG represents an important avenue to enhance our understanding of their potential role in antibody activity in vivo. Lastly understanding the FcγR binding dynamics of other ligands of interest such as pentraxins (pattern recognition molecules that are considered innate antibodies) 4SC-202 25 or pathogen-secreted molecules that can interfere with FcγR function 26 or the development of therapeutic inhibitors of FcγR may also be crucial to providing high-resolution understanding of the role of antibodies and antibody receptors in immunity and recombinant antibody therapies. Thus high-throughput means to characterize either the ability of therapeutic proteins of interest to interact with these receptors or the ability of candidate Fc receptors to interact with different antibody species could be of high value. To this end we statement the development of a multiplexed coded.