Selective Depletion of Antigen-Specific Antibodies for the Treatment of Demyelinating Disease.

Current treatments for antibody-mediated autoimmunity are associated with lack of specificity, leading to immunosuppressive effects. To overcome this limitation, we have developed a class of antibody-based therapeutics for the treatment of autoimmunity involving antibodies that recognize the autoantigen, myelin oligodendrocyte glycoprotein (MOG). These agents ("Seldegs," for selective degradation) selectively eliminate antigen (MOG)-specific antibodies without affecting the levels of antibodies of other specificities. Seldeg treatment of mice during antibody-mediated exacerbation of experimental autoimmune encephalomyelitis by patient-derived MOG-specific antibodies results in disease amelioration. Consistent with their therapeutic effects, Seldegs deliver their targeted antibodies to Kupffer and liver sinusoidal endothelial cells that are known to have tolerogenic effects. Our results show that Seldegs can ameliorate disease mediated by MOG-specific antibodies and indicate that this approach also has the potential to treat other autoimmune diseases where the specific clearance of antibodies is required.

Myelin oligodendrocyte glycoprotein-specific antibodies from multiple sclerosis patients exacerbate disease in a humanized mouse model.

Myelin oligodendrocyte glycoprotein (MOG) is exposed on the outer surface of the myelin sheath, and as such, represents a possible target antigen for antibodies in multiple sclerosis (MS) and other demyelinating diseases. However, despite extensive analyses, whether MOG-specific antibodies contribute to pathogenesis in human MS remains an area of uncertainty. In the current study we demonstrate that antibodies derived from adult MS patients exacerbate experimental autoimmune encephalomyelitis (EAE) in 'humanized' mice that transgenically express human FcγRs (hFcγRs). Importantly, this exacerbation is dependent on MOG recognition by the human-derived antibodies. The use of mice that express hFcγRs has allowed us to also investigate the contribution of these receptors to disease in the absence of confounding effects of cross-species differences. Specifically, by engineering the Fc region of MOG-specific antibodies to modulate FcγR and complement (C1q) binding, we reveal that FcγRs but not complement activation contribute to EAE pathogenesis. Importantly, selective enhancement of the affinities of these antibodies for specific FcγRs reveals that FcγRIIA is more important than FcγRIIIA in mediating disease exacerbation. These studies not only provide definitive evidence for the contribution of MOG-specific antibodies to MS, but also reveal mechanistic insight that could lead to new therapeutic targets.

Antigen dynamics govern the induction of CD4(+) T cell tolerance during autoimmunity.

Antigen-specific T cell tolerance holds great promise for the treatment of autoimmune diseases. However, strategies to induce durable tolerance using high doses of soluble antigen have to date been unsuccessful, due to lack of efficacy and the risk of hypersensitivity. In the current study we have overcome these limitations by developing a platform for tolerance induction based on engineering the immunoglobulin Fc region to modulate the dynamic properties of low doses (1 µg/mouse; ∼50 µg/kg) of Fc-antigen fusions. Using this approach, we demonstrate that antigen persistence is a dominant factor governing the elicitation of tolerance in the model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE), induced by immunizing B10.PL mice with the N-terminal epitope of myelin basic protein. Unexpectedly, our analyses reveal a stringent threshold of antigen persistence for both prophylactic and therapeutic treatments, although distinct mechanisms lead to tolerance in these two settings. Importantly, the delivery of tolerogenic Fc-antigen fusions during ongoing disease results in the downregulation of T-bet and CD40L combined with amplification of Foxp3(+) T cell numbers. The generation of effective, low dose tolerogens using Fc engineering has potential for the regulation of autoreactive T cells.

FcRn: from molecular interactions to regulation of IgG pharmacokinetics and functions.

The neonatal Fc receptor, FcRn, is related to MHC class I with respect to its structure and association with ß2microglobulin (ß2m). However, by contrast with MHC class I molecules, FcRn does not bind to peptides, but interacts with the Fc portion of IgGs and belongs to the Fc receptor family. Unlike the 'classical' Fc receptors, however, the primary functions of FcRn include salvage of IgG (and albumin) from lysosomal degradation through the recycling and transcytosis of IgG within cells. The characteristic feature of FcRn is pH-dependent binding to IgG, with relatively strong binding at acidic pH (<6.5) and negligible binding at physiological pH (7.3-7.4). FcRn is expressed in many different cell types, and endothelial and hematopoietic cells are the dominant cell types involved in IgG homeostasis in vivo. FcRn also delivers IgG across cellular barriers to sites of pathogen encounter and consequently plays a role in protection against infections, in addition to regulating renal filtration and immune complex-mediated antigen presentation. Further, FcRn has been targeted to develop both IgGs with extended half-lives and FcRn inhibitors that can lower endogenous antibody levels. These approaches have implications for the development of longer lived therapeutics and the removal of pathogenic or deleterious antibodies.

The encephalitogenic, human myelin oligodendrocyte glycoprotein-induced antibody repertoire is directed toward multiple epitopes in C57BL/6-immunized mice.

Although Abs specific for myelin oligodendrocyte glycoprotein (MOG) have been detected in patients with multiple sclerosis (MS), their contribution to pathogenesis remains poorly understood. Immunization of C57BL/6 mice with recombinant human MOG (hMOG) results in experimental autoimmune encephalomyelitis involving MOG-specific, demyelinating Abs. This model is therefore informative for understanding anti-MOG humoral responses in MS. In the current study, we have characterized the hMOG-specific Ab repertoire in immunized C57BL/6 mice using both in vitro and in vivo approaches. We demonstrate that hMOG-specific mAbs are not focused on one specific region of MOG, but instead target multiple epitopes. Encephalitogenicity of the mAbs, assessed by the ability of the mAbs to exacerbate experimental autoimmune encephalomyelitis in mice, correlates with the activity of the mAbs in binding to CNS tissue sections, but not with other in vitro assays. The targeting of different MOG epitopes by encephalitogenic Abs has implications for disease pathogenesis, because it could result in MOG cross linking on oligodendrocytes and/or immune complex formation. These studies reveal several novel features concerning pathogenic, humoral responses that may have relevance to human MS.

Neonatal Fc receptor expression in macrophages is indispensable for IgG homeostasis.

The maintenance of the homeostasis of immunoglobulin G (IgG) represents a fundamental aspect of humoral immunity that has direct relevance to the successful delivery of antibody-based therapeutics. The ubiquitously expressed neonatal Fc receptor (FcRn) salvages IgG from cellular degradation following pinocytic uptake into cells, conferring prolonged in vivo persistence on IgG. However, the cellular sites of FcRn function are poorly defined. Pinocytic uptake is a prerequisite for FcRn-mediated IgG salvage, prompting us to investigate the consequences of IgG uptake and catabolism by macrophages, which represent both abundant and highly pinocytic cells in the body. Site-specific deletion of FcRn to generate mice harboring FcRn-deficient macrophages results in IgG hypercatabolism and ~threefold reductions in serum IgG levels, whereas these effects were not observed in mice that lack functional FcRn in B cells and dendritic cells. Consistent with the degradative activity of FcRn-deficient macrophages, depletion of these cells in FcRn-deficient mice leads to increased persistence and serum levels of IgG. These studies demonstrate a pivotal role for FcRn-mediated salvage in compensating for the high pinocytic and degradative activities of macrophages to maintain IgG homeostasis.

BCAP links IL-1R to the PI3K-mTOR pathway and regulates pathogenic Th17 cell differentiation.

The toll-like receptor (TLR) and interleukin (IL)-1 family of receptors share several signaling components, including the most upstream adapter, MyD88. We previously reported the discovery of B cell adapter for phosphoinositide 3-kinase (BCAP) as a novel toll-IL-1 receptor homology domain-containing adapter that regulates inflammatory responses downstream of TLR signaling. Here we find that BCAP plays a critical role downstream of both IL-1 and IL-18 receptors to regulate T helper (Th) 17 and Th1 cell differentiation, respectively. Absence of T cell intrinsic BCAP did not alter development of naturally arising Th1 and Th17 lineages but led to defects in differentiation to pathogenic Th17 lineage cells. Consequently, mice that lack BCAP in T cells had reduced susceptibility to experimental autoimmune encephalomyelitis. More importantly, we found that BCAP is critical for IL-1R-induced phosphoinositide 3-kinase-Akt-mechanistic target of rapamycin (mTOR) activation, and minimal inhibition of mTOR completely abrogated IL-1ß-induced differentiation of pathogenic Th17 cells, mimicking BCAP deficiency. This study establishes BCAP as a critical link between IL-1R and the metabolic status of activated T cells that ultimately regulates the differentiation of inflammatory Th17 cells.

Engineered clearing agents for the selective depletion of antigen-specific antibodies.

Here we have designed a novel class of engineered antibody-based reagents ('Seldegs') that induce the selective degradation of antigen-specific antibodies. We demonstrate the rapid and specific clearance of antibodies recognizing the autoantigen, myelin oligodendrocyte glycoprotein and tumour target, HER2. Seldegs have considerable potential in multiple areas, including the treatment of antibody-mediated autoimmunity and diagnostic imaging.

Macrophage-Mediated Trogocytosis Leads to Death of Antibody-Opsonized Tumor Cells.

Understanding the complex behavior of effector cells such as monocytes or macrophages in regulating cancerous growth is of central importance for cancer immunotherapy. Earlier studies using CD20-specific antibodies have demonstrated that the Fcγ receptor (FcγR)-mediated transfer of the targeted receptors from tumor cells to these effector cells through trogocytosis can enable escape from antibody therapy, leading to the viewpoint that this process is protumorigenic. In the current study, we demonstrate that persistent trogocytic attack results in the killing of HER2-overexpressing breast cancer cells. Further, antibody engineering to increase FcγR interactions enhances this tumoricidal activity. These studies extend the complex repertoire of activities of macrophages to trogocytic-mediated cell death of HER2-overexpressing target cells and have implications for the development of effective antibody-based therapies. Mol Cancer Ther; 15(8); 1879-89. ©2016 AACR.

Use of Fc-Engineered Antibodies as Clearing Agents to Increase Contrast During PET.

UNLABELLED: Despite promise for the use of antibodies as molecular imaging agents in PET, their long in vivo half-lives result in poor contrast and radiation damage to normal tissue. This study describes an approach to overcome these limitations. METHODS: Mice bearing human epidermal growth factor receptor type 2 (HER2)-overexpressing tumors were injected with radiolabeled ((124)I, (125)I) HER2-specific antibody (pertuzumab). Pertuzumab injection was followed 8 h later by the delivery of an engineered, antibody-based inhibitor of the receptor, FcRn. Biodistribution analyses and PET were performed at 24 and 48 h after pertuzumab injection. RESULTS: The delivery of the engineered, antibody-based FcRn inhibitor (or Abdeg, for antibody that enhances IgG degradation) results in improved tumor-to-blood ratios, reduced systemic exposure to radiolabel, and increased contrast during PET. CONCLUSION: Abdegs have considerable potential as agents to stringently regulate antibody dynamics in vivo, resulting in increased contrast during molecular imaging with PET.

Autoantibody depletion ameliorates disease in murine experimental autoimmune encephalomyelitis.

Much data support a role for central nervous system antigen-specific antibodies in the pathogenesis of multiple sclerosis (MS). The effects of inducing a decrease in (auto)antibody levels on MS or experimental autoimmune encephalomyelitis (EAE) through specific blockade of FcRn, however, remain unexplored. We recently developed engineered antibodies that lower endogenous IgG levels by competing for binding to FcRn. These Abdegs ("antibodies that enhance IgG degradation") can be used to directly assess the effect of decreased antibody levels in inflammatory diseases. In the current study, we show that Abdeg delivery ameliorates disease in an EAE model that is antibody dependent. Abdegs could therefore have promise as therapeutic agents for MS.