The toll-like receptor 7 and 8 (TLR7/8) agonist Resiquimod (R848) has been recognized as a promising immunostimulator for the treatment of cutaneous cancers in multiple clinical trials. However, systemic administration of R848 often results in strong immune-related toxicities while having limited therapeutic effects to the tumor. In the present study, a prodrug-based nanocarrier delivery system was developed that exhibited high therapeutic efficiency. R848 was conjugated to α-tocopherol to constitute an R848-Toco prodrug, followed by formulating with a tocopherolmodified hyaluronic acid (HA-Toco) as a polymeric nano-suspension. In vitro evaluation showed that the delivery system prolonged the release kinetics while maintaining TLR agonist activities. When administered subcutaneously, the nano-suspension formed a depot at the injection site, inducing localized immune responses without systemic expansion. This formulation also suppressed tumor growth and recruited immune cells to the tumor in a murine model of head and neck cancer. In a pilot canine clinical trial of spontaneous mast cell tumors, the treatment led to a 67% response rate (3 partial remissions and 1 complete remission).
SUMMARYThe presentation of extremely low doses of antigen to T cells is enhanced by immunoglobulin E (IgE)-dependent antigen focusing to CD23, the low-af®nity receptor for IgE, expressed on activated B cells. CD23 contains a C-type lectin domain in its extracellular sequence and a targeting signal for coated pits, required for endocytosis, in its cytoplasmic sequence. CD23 is non-covalently associated with the major histocompatibility complex class II antigen, human leucocyte antigen HLA-DR, on the surface of human B cells, but the fate of this complex following endocytosis is unknown. To answer this question we have labelled these proteins on the surface of RPMI 8866 B cells and traced their route through the cytoplasm. Endocytosis mediated by anti-CD23 antibodies (BU38 and MHM6) led to the loss of CD23 from the cells. Endocytosis mediated by an antibody to HLA-DR (CR3/43) or an antigen±IgE complex (NP-BSA±anti-NP IgE), however, led to recycling of the HLA-DR±CD23 complex to the cell surface on a time scale (3±6 hr) consistent with the recycling of HLA-DR in antigen presentation. Along the latter pathway CD23 label was observed in cytoplasmic organelles that resembled the`compartments for peptide loading' or`class II vesicles' described by previous authors. Two features of the recycling process may contribute to the ef®ciency of antigen presentation. Peptide exchange may be facilitated by the proximity of HLA-DR and antigen in peptide loading compartments of the endosomal network. The return of CD23 with HLA-DR to the cell surface may then help to stabilize speci®c B-cell±T-cell interactions, contributing to T-cell activation.
An Fcab (Fc antigen binding) is a crystallizable fragment of IgG having C-terminal structural loops of CH3 domains engineered for antigen binding. Since introduction of novel binding sites might impair the immunoglobulin fold, repairing strategies are needed for improving the biophysical properties of promising binders without decreasing affinity to the antigen. Here, a directed evolution protocol was developed and applied for stabilization of a Her2/neu-binding Fcab. Distinct loop regions of the parental binder were softly randomized by parsimonious mutagenesis, followed by heat incubation of the yeast displayed protein library and selection for retained antigen binding. Selected Fcabs were expressed solubly in Pichia pastoris and human embryonic kidney 293 cells and characterized. Fcab clones that retained their affinity to Her2/neu but exhibited a significantly increased conformational stability and resistance to aggregation could be evolved. Moreover, we demonstrate that simultaneous selection for binding to the antigen and to structurally specific ligands (FcγRI and an antibody directed against the CH2 domain) yields even more stable Fcabs. To sum up, this study presents a very potent and generally applicable method for improving the fold and stability of antibodies, antibody fragments and alternative binding scaffolds.
Modulation of the immune response using immunoglobulin fusion proteins has shown great promise for clinical immunotherapy of autoimmune diseases. Alefacept is an immunoglobulin fusion protein composed of the first extracellular domain of human LFA-3 fused to the hinge, C H 2 and C H 3 domains of human IgG 1 . Alefacept has previously been reported to inhibit T cell proliferation. Here, we analyzed the effects of alefacept on lymphocytes in vitro and characterized the role of autologous NK cells in its mechanism of action. Alefacept, but not a C H 2 binding mutant of Alefacept, inhibited CD3-induced T cell proliferation only in the presence of live NK cells, consistent with an important role for Fc + R engagement. Alefacept caused preferential depletion of CD69+ T cell subsets. Cytotoxicity assays revealed that alefacept, but not the C H 2 binding mutant, induced NK cell-mediated death of activated T cells and sorting into CD45R0 + and CD45RA + subpopulations showed that lymphocyte deletion occurred preferentially in the CD45R0 + subset. Activated CD45R0 + cells expressed higher levels of CD2 than CD45R0 -cells, providing a possible explanation for the selective targeting of this subset. Our results suggest that selective targeting of CD45R0 + T cells by NK cells represents a potential therapeutic mechanism of action of alefacept.
CD23, the low-affinity IgE receptor, is up-regulated on interleukin (IL)-4-stimulated B cells and monocytes, with a concomitant increase in the release of soluble fragments of CD23 (sCD23) into the medium by proteolytic processing of the surface-bound intact CD23. The effect of inhibition of the processing of CD23 on IgE production in human and mouse cells and in a mouse model in vivo was evaluated. CD23 processing to sCD23 from RPMI 8866 (a human Epstein-Barr virus-transformed B cell line) cell membranes was inhibited by a broad-spectrum matrix-metalloprotease inhibitor, batimastat, with an IC50 of 0.15 microM. Batimastat also inhibited CD23 processing in whole RPMI 8866 cells as well as in IL-4-stimulated purified human monocytes with similar IC50. Batimastat inhibited IgE production from IL-4/anti-CD40-stimulated human tonsil B cells as well as mouse splenic B cells in a manner consistent with inhibition of CD23 processing. Release of soluble fragments of CD23 in the cell supernatants of tonsil B cells was inhibited over the concentration range of 1-10 microM batimastat and intact cell surface CD23 was increased on mouse splenic B cells in the presence of these concentrations of batimastat. IgE production of IL-4-stimulated human peripheral blood mononuclear cells was also blocked by 1-10 microM batimastat, again with comparable inhibition of sCD23 release over the same concentration range. Finally, in a mouse model of IgE production, batimastat inhibited IgE production in response to ovalbumin challenge as determined by serum IgE levels. Taken together, the data support a role of CD23 in IgE production and point to CD23 processing to sCD23 as a therapeutically relevant control point in the regulation of IgE synthesis.
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