Small Molecule Drugs That Inhibit Phagocytosis.

In our initial publication on the in vitro testing of more than 200 compounds, we demonstrated that small molecules can inhibit phagocytosis. We therefore theorized that a small molecule drug discovery-based approach to the treatment of immune cytopenias (ITP, AIHA, HTR, DHTR) is feasible. Those earlier studies showed that small molecules with anti-phagocytic groups, such as the pyrazole core, are good models for producing efficacious phagocytosis inhibitors with low toxicity. We recently screened a chemical library of 80 compounds containing pyrazole/isoxazole/pyrrole core structures and found four hit molecules for further follow-up, all having the pyrazole core structure. Subsequent evaluation via MTT viability, LDH release, and apoptosis, led to the selection of two lead compounds with negligible toxicity and high efficacy. In an in vitro assay for inhibition of phagocytosis, their IC50 values were 2-4 µM. The rational development of these discoveries from hit to lead molecule stage, viz. independent synthesis/scale up of hit molecules, and in vivo activities in mouse models of autoimmune disease, will result in the selection of a lead compound(s) for further pre-clinical evaluation.

IgG3 anti-Kell allotypic variation results in differential antigen binding and phagocytosis.

BACKGROUND: Human immunoglobulin G (hIgG) includes four different subtypes (IgG1, IgG2, IgG3, and IgG4). Due to genetic variations, each IgG subtype contains different isoallotypes. It was previously shown that a Food and Drug Administration-approved monoclonal anti-IgG failed to recognize 2 of 15 recombinant, human IgG3 anti-Kell (K1) isoallotypes (rIgG3-03 and rIgG3-13) by indirect antiglobulin test (IAT). STUDY DESIGN AND METHODS: We expressed and purified 15 recombinant human rIgG3 anti-K1 isoallotypes and investigated their antigen binding and ability to induce phagocytosis using homozygous (KK) and heterozygous (Kk) K1-positive red blood cells (RBCs) by gel IAT, flow cytometry, and a monocyte monolayer assay (MMA) with peripheral blood monocytes and cultured inflammatory (M1) and anti-inflammatory (M2) macrophages. RESULTS: MMA results showed that differences in the Fc region of rIgG3 anti-K1 led to distinctive phagocytic activity with both monocytes and M1 macrophages. rIgG3-18 and rIgG3-19 showed an enhanced ability to induce phagocytosis. Differences in Fc regions also led to variations in the number of antibodies bound to KK RBCs. Despite the differences in phagocytic activity, all 15 rIgG3 clones are predicted to induce clinically significant hemolysis if K1-positive blood was transfused into patients. CONCLUSION: These results argue that antiglobulin reagents that fail to detect isoallotype rIgG3-03 or rIgG3-13 could present a transfusion risk or lack of detection of a potentially clinically significant anti-K1 in hemolytic disease of the fetus and newborn.