Discovery of BIIB068: A Selective, Potent, Reversible Bruton's Tyrosine Kinase Inhibitor as an Orally Efficacious Agent for Autoimmune Diseases.

Autoreactive B cell-derived antibodies form immune complexes that likely play a pathogenic role in autoimmune diseases. In systemic lupus erythematosus (SLE), these antibodies bind Fc receptors on myeloid cells and induce proinflammatory cytokine production by monocytes and NETosis by neutrophils. Bruton's tyrosine kinase (BTK) is a non-receptor tyrosine kinase that signals downstream of Fc receptors and plays a transduction role in antibody expression following B cell activation. Given the roles of BTK in both the production and sensing of autoreactive antibodies, inhibitors of BTK kinase activity may provide therapeutic value to patients suffering from autoantibody-driven immune disorders. Starting from an in-house proprietary screening hit followed by structure-based rational design, we have identified a potent, reversible BTK inhibitor, BIIB068 (1), which demonstrated good kinome selectivity with good overall drug-like properties for oral dosing, was well tolerated across preclinical species at pharmacologically relevant doses with good ADME properties, and achieved >90% inhibition of BTK phosphorylation (pBTK) in humans.

Transport of PEGylated liposomes from the splenic marginal zone to the follicle in the induction phase of the accelerated blood clearance phenomenon.

The accelerated blood clearance (ABC) phenomenon has been reported to enhance the clearance of PEGylated liposomes from the blood circulation when the liposomes are injected into the same animal repeatedly. We have shown that anti-PEG IgM production from splenic B cells is crucial in the ABC phenomenon. In this study, we describe the crucial role of marginal zone (MZ) B cells in the anti-PEG IgM production and recognition of PEGylated liposomes in the induction phase of ABC phenomenon. Suppression of the anti-PEG IgM production was correlated with the disappearance of IgM(high) cells in the MZ, particularly MZ-B cells, following cyclophosphamide (CPA)-treatment, confirming that splenic MZ-B cells are responsible for anti-PEG IgM production. The MZ-B cells stimulated by a first dose of PEGylated liposomes internalized the second dose of PEGylated liposomes in a PEG modification-dependent manner and transported the liposomes into the follicle (FO) region. To the best of our knowledge, this is the first report showing that PEGylated liposome is recognized by MZ-B cells and transported to the FO region like blood-borne antigens or immune complexes. It is likely that PEGylated liposomes are recognized as a TI-2 antigen by the first line of defense against life-threatening infections by blood-borne organisms. Our study may have implications for immunogenicity of synthesized polymer-grafted therapeutics including nanocarriers, nucleic acids and proteins.

Surface-linked liposomal antigen induces IgE selective unresponsiveness in a T-cell independent fashion.

We previously reported that surface-linked liposomal antigen induced IgE-selective unresponsiveness. The results were consistent even when different coupling procedures for antigen with liposomes, or for liposomes with different lipid components, were employed. During the course of an investigation intended to clarify the mechanism of IgE-selective unresponsiveness induced by surface-coupled liposomal antigens, we discovered an alternative approach to regulate the production of IgE, one that is independent of the activity of T-cells. Immunization of mice with OVA-liposome conjugates induced IgE- selective unresponsiveness without apparent Th1 polarization. Neither interleukin-12 (IL-12), IL-10, nor CD8(+) T-cells participated in the regulation. Further, CD4(+) T-cells of mice immunized with OVA-liposome were capable of inducing antigen-specific IgE synthesis in athymic nude mice immunized with alum-adsorbed OVA. On the other hand, immunization of the recipient mice with OVA-liposome did not induce anti-OVA IgE production, even when CD4(+) T-cells of mice immunized with alum-adsorbed OVA were transferred. In the secondary immune response, OVA-liposome enhanced anti-OVA IgG antibody production but not the ongoing IgE production, suggesting that the IgE-selective unresponsiveness induced by the liposomal antigen involved direct effects on IgE but not IgG switching in vivo. These results suggest the role of an alternative mechanism, one not involving T-cells, in the regulation of IgE synthesis, and raise the possibility that surface-linked liposomal antigen is potentially applicable for the development of a novel vaccine that induces the least IgE synthesis. Moreover, given the relatively low allergic response to and increased antigenicity of the allergen, this form of antigen preparation would be applicable to allergen immunotherapy.

T-independent IgA responses to microbial polysaccharides.

There is accumulating evidence indicating the presence in vivo of T-independent routes of IgA response in addition to the conventional T-dependent IgA response. Factors influencing these alternative pathways of IgA responses may include the structural characteristics of a stimulating antigen, the nature of responding B cells, and the microenvironment. The structural complexity of polysaccharide antigens has made it difficult to summarize a general scheme for the antibody responses they induce. Instead, one may expect that each individual polysaccharide may be able to create a unique microenvironment by activation of specific cell populations in the repertoires of non-T cell types. A specific pattern of B cell response may thus be elicited by TI stimulation. Recognition of such a unique property of a TI antigens is necessary for us to better understand the T-independent IgA response. Information obtained may have an impact on the development of vaccination strategies directed at the mucosal immunity mediated by IgA antibodies.

The consequences of the intracellular retention of pathogen-derived T-cell-independent antigens on protein presentation to T cells.

Intracellular pathogens can be considered as particulate antigens chemically composed of a complex mixture of T-cell-dependent antigens (TD) (peptides and proteins) and T-cell-independent antigens (TI) (glycolipids and complex polysaccharides). A large range of saccharides (from oligosaccharides to complex polysaccharides) derived from pathogenic microorganisms are being isolated and characterized. They are currently implicated in signaling systems and concomitant host-parasite relationships. However, there are not many structure-function relationships described for these pathogens. This is particularly true of polysaccharides. In this report we have reviewed the role of defined TI antigens in the processing and presentation of defined TD antigens to specific T cells by antigen-presenting cells (APC). We also considered the importance of some of the chemical characteristics shared by different carbohydrates implicated in the inhibition of antigen presentation. These findings are discussed in relation to the clear immunopathological consequences of long retention periods of complex carbohydrate molecules derived from intracellular parasites inside certain APC and the absence of antigen presentation impairment in physiological situations such as the removal of senescent or damaged red blood cells by splenic macrophages or intracellular accumulation of carbohydrates in colostrum and milk macrophages during lactation.

The cellular location of a foreign B cell epitope expressed by recombinant bacteria determines its T cell-independent or T cell-dependent characteristics.

We have targeted two foreign B cell antigenic determinants to different locations in the Escherichia coli cell to examine what effect this had on antibody responses elicited by the recombinant bacteria. The two epitopes were the 132-145 peptide from the PreS2 region of hepatitis B virus and the C3 neutralization epitope of poliovirus type 1. They were each expressed in two forms either on the surface, as part of the outer-membrane protein LamB, or soluble in the periplasm, as part of the periplasmic protein MalE. When live bacteria expressing the foreign epitope at the cell surface were used for immunization of mice, they induced T cell-independent antibody responses characterized by a rapid induction of IgM and IgG antibodies. In contrast, when the same foreign epitope was inserted into the MalE protein, the antibody response was only detectable after 3 wk, belonged only to the IgG class and was strictly T cell dependent. This study has therefore identified two major pathways by which epitopes expressed by bacterial cells can stimulate specific antibody responses. The first pathway is mediated by direct activation of B cells by bacterial cell-surface Ag and does not require T cell help. The second pathway is T cell dependent and concerns Ag that can be released from the bacteria in a soluble form. We have also studied the effect of the exact position of the B cell antigenic determinant within the LamB protein and with respect to the outer membrane by comparing the immunogenicity of the PreS epitope inserted at three different permissive sites of LamB. The data indicated that to obtain an antibody response with intact bacteria, the epitope must be protruding sufficiently from the outside of the outer membrane. In contrast, when semipurified hybrid proteins were used as immunogen, the exact position of the B cell antigenic determinant within solubilized LamB protein does not influence its immunogenicity.

A polymer containing a repeating peptide sequence can stimulate T-cell-independent IgG antibody production in vivo.

The immunogenicity of a CD4 peptide sequence 303-315 (V4 domain) was determined for three forms of the peptide: (a) a polymer consisting of repeating peptide units, (b) a peptide linked to a large protein carrier, chicken serum albumin, and (c) unmodified free peptide. Two in vivo systems were used: a T-cell-competent BALB/c mouse and a T-cell-deficient nude mouse. In BALB/c mice the IgG antibody responses to the peptide-CSA conjugate were at least 100-fold greater than the response to the polymer, and the free peptide gave no response. However, in contrast to the normal BALB/c mice, in the nude mice the polymer produced the only significant response. Nude mice immunized with the polymer produced a predominantly IgG response with almost equal amounts of IgG1 and IgG 2a + 2b. This IgG subclass distribution was very similar to that obtained in the BALB/c for the peptide-CSA conjugate. Positive sera from both groups were able to react with immobilized CD4 molecule on Western blots, confirming the specificity of both the T-cell-independent as well as T-cell-dependent response. These results suggest that repeating epitopes on the same peptide polymer molecule may cause cross-linking of antigen-specific immunoglobin receptors on B-cells, and thus behave as a relatively T-cell-independent immunogen which can induce isotype switching.