Detection of Experimental and Clinical Immune Complexes by Measuring SHIP-1 Recruitment to the Inhibitory FcγRIIB.

Fc γ receptors (FcγR) are involved in multiple aspects of immune cell regulation, are central to the success of mAb therapeutics, and underpin the pathology of several autoimmune diseases. However, reliable assays capable of accurately measuring FcγR interactions with their physiological ligands, IgG immune complexes (IC), are limited. A method to study and detect IC interactions with FcγRs was therefore developed. This method, designed to model the signaling pathway of the inhibitory FcγRIIB (CD32B), used NanoLuc Binary Interaction Technology to measure recruitment of the Src homology 2 domain-containing inositol phosphatase 1 to the ITIM of this receptor. Such recruitment required prior cross-linking of an ITAM-containing activatory receptor, and evoked luciferase activity in discrete clusters at the cell surface, recapitulating the known biology of CD32B signaling. The assay detected varying forms of experimental IC, including heat-aggregated IgG, rituximab-anti-idiotype complexes, and anti-trinitrophenol-trinitrophenol complexes in a sensitive manner (≤1 µg/ml), and discriminated between complexes of varying size and isotype. Proof-of-concept for the detection of circulating ICs in autoimmune disease was provided, as responses to sera from patients with systemic lupus erythematosus and rheumatoid arthritis were detected in small pilot studies. Finally, the method was translated to a stable cell line system. In conclusion, a rapid and robust method for the detection of IC was developed, which has numerous potential applications including the monitoring of IC in autoimmune diseases and the study of underlying FcγR biology.

Antigenic modulation limits the effector cell mechanisms employed by type I anti-CD20 monoclonal antibodies.

Following the success of rituximab, 2 other anti-CD20 monoclonal antibodies (mAbs), ofatumumab and obinutuzumab, have entered clinical use. Ofatumumab has enhanced capacity for complement-dependent cytotoxicity, whereas obinutuzumab, a type II mAb, lacks the ability to redistribute into lipid rafts and is glycoengineered for augmented antibody-dependent cellular cytotoxicity (ADCC). We previously showed that type I mAbs such as rituximab have a propensity to undergo enhanced antigenic modulation compared with type II. Here we assessed the key effector mechanisms affected, comparing type I and II antibodies of various isotypes in ADCC and antibody-dependent cellular-phagocytosis (ADCP) assays. Rituximab and ofatumumab depleted both normal and leukemic human CD20-expressing B cells in the mouse less effectively than glycoengineered and wild-type forms of obinutuzumab, particularly when human immunoglobulin G1 (hIgG1) mAbs were compared. In contrast to mouse IgG2a, hIgG1 mAbs were ineffective in ADCC assays with murine natural killer cells as effectors, whereas ADCP was equivalent for mouse IgG2a and hIgG1. However, rituximab's ability to elicit both ADCC and ADCP was reduced by antigenic modulation, whereas type II antibodies remained unaffected. These data demonstrate that ADCP and ADCC are impaired by antigenic modulation and that ADCP is the main effector function employed in vivo.

Highly sensitive detection of dye-labelled DNA using nanostructured gold surfaces.

Careful control of surface chemistry results in strong surface enhanced resonance Raman scattering from dye-labelled oligonucleotides assembled on nanostructured gold surfaces, releasing their potential as reliable enhancing surfaces.

Soluble CD30 binds to CD153 with high affinity and blocks transmembrane signaling by CD30.

CD30 is an unusual member of the TNF receptor superfamily with a duplicated segment within its extracellular domain that may function as an additional ligand binding site. The extracellular domain of CD30 is shed from cells and soluble CD30 levels are elevated in certain disease states. Soluble CD30 may bind to its ligand, CD153, with high affinity and block its interaction with membrane-bound CD30. We have generated soluble recombinant forms of CD30 and CD153 in order to characterize their interaction and assess their biological activity. Soluble trimeric CD153 bound to membrane-anchored CD30 with a relatively high affinity (K(D)=23 nM) and was effective in triggering cell death and TNF-alpha production in the presence of cross-linking antibodies. The affinity and kinetics of the interaction between soluble CD30 and CD153 were determined using the BIAcore biosensor. In contrast to other members of the TNF receptor superfamily, soluble monomeric CD30 bound to its ligand with high affinity (K(D)=4.5 nM) and prevented the interaction of cellular CD153 with immobilized CD30. Furthermore, soluble CD30 blocked cell death signals generated by cell surface-expressed CD30 effectively. These data suggest that soluble CD30 may have biological functions in vivo.