Therapeutic antibody glycosylation impacts antigen recognition and immunogenicity.

In this study we show that glycosylation is relevant for immune recognition of therapeutic antibodies, and that defined glycan structures can modulate immunogenicity. Concerns regarding immunogenicity arise from the high heterogeneity in glycosylation that is difficult to control and can deviate from human glycosylation if produced in non-human cell lines. While non-human glycosylation is thought to cause hypersensitivity reactions and immunogenicity, less is known about effects of Fc-associated glycan structures on immune cell responses. We postulated that glycosylation influences antigen recognition and subsequently humoral responses to therapeutic antibodies by modulating 1) recognition and uptake by dendritic cells (DCs), and 2) antigen routing, processing and presentation. Here, we compared different glycosylation variants of the antibody rituximab (RTX) in in vitro assays using human DCs and T cells as well as in in vivo studies. We found that human DCs bind and internalize unmodified RTX stronger compared to its aglycosylated form suggesting that glycosylation mediates uptake after recognition by glycan-specific receptors. Furthermore, we show that DC-uptake of RTX increases or decreases if glycosylation is selectively modified to recognize activating (by mannosylation) or inhibitory lectin receptors (by sialylation). Moreover, glycosylation seems to influence antigen presentation by DCs because specific glycovariants tend to induce either stronger or weaker T cell activation. Finally, we demonstrate that antibody glycosylation impacts anti-drug antibody (ADA) responses to RTX in vivo. Hence, defined glycan structures can modulate immune recognition and alter ADA responses. Glyco-engineering may help to decrease clinical immunogenicity and ADA-associated adverse events such as hypersensitivity reactions.

Small-molecule factor B inhibitor for the treatment of complement-mediated diseases.

Dysregulation of the alternative complement pathway (AP) predisposes individuals to a number of diseases including paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, and C3 glomerulopathy. Moreover, glomerular Ig deposits can lead to complement-driven nephropathies. Here we describe the discovery of a highly potent, reversible, and selective small-molecule inhibitor of factor B, a serine protease that drives the central amplification loop of the AP. Oral administration of the inhibitor prevents KRN-induced arthritis in mice and is effective upon prophylactic and therapeutic dosing in an experimental model of membranous nephropathy in rats. In addition, inhibition of factor B prevents complement activation in sera from C3 glomerulopathy patients and the hemolysis of human PNH erythrocytes. These data demonstrate the potential therapeutic value of using a factor B inhibitor for systemic treatment of complement-mediated diseases and provide a basis for its clinical development.

Modulation of Microglia by Voluntary Exercise or CSF1R Inhibition Prevents Age-Related Loss of Functional Motor Units.

Age-related loss of skeletal muscle innervation by motor neurons leads to impaired neuromuscular function and is a well-established clinical phenomenon. However, the underlying pathogenesis remains unclear. Studying mice, we find that the number of motor units (MUs) can be maintained by counteracting neurotoxic microglia in the aged spinal cord. We observe that marked innervation changes, detected by motor unit number estimation (MUNE), occur prior to loss of muscle function in aged mice. This coincides with gene expression changes indicative of neuronal remodeling and microglial activation in aged spinal cord. Voluntary exercise prevents loss of MUs and reverses microglia activation. Depleting microglia by CSF1R inhibition also prevents the age-related decline in MUNE and neuromuscular junction disruption, implying a causal link. Our results suggest that age-related changes in spinal cord microglia contribute to neuromuscular decline in aged mice and demonstrate that removal of aged neurotoxic microglia can prevent or reverse MU loss.

Brain region-specific enhancement of remyelination and prevention of demyelination by the CSF1R kinase inhibitor BLZ945.

Multiple sclerosis (MS) is a chronic inflammatory disease affecting the central nervous system (CNS). While multiple effective immunomodulatory therapies for MS exist today, they lack the scope of promoting CNS repair, in particular remyelination. Microglia play a pivotal role in regulating myelination processes, and the colony-stimulating factor 1 (CSF-1) pathway is a key regulator for microglia differentiation and survival. Here, we investigated the effects of the CSF-1 receptor kinase inhibitor, BLZ945, on central myelination processes in the 5-week murine cuprizone model by non-invasive and longitudinal magnetic resonance imaging (MRI) and histology. Therapeutic 2-week BLZ945 treatment caused a brain region-specific enhancement of remyelination in the striatum/cortex, which was absent in the corpus callosum/external capsule. This beneficial effect correlated positively with microglia reduction, increased oligodendrocytes and astrogliosis. Prophylactic BLZ945 treatment prevented excessive demyelination in the corpus callosum by reducing microglia and increasing oligondendrocytes. In the external capsule oligodendrocytes were depleted but not microglia and a buildup of myelin debris and axonal damage was observed. A similar microglial dysfunction in the external capsule with an increase of myelin debris was obvious in triggering receptor expressed on myeloid cells 2 (TREM2) knock-out mice treated with cuprizone. Finally, therapeutic BLZ945 treatment did not change the disease course in experimental autoimmune encephalomyelitis mice, a peripherally driven neuroinflammation model. Taken together, our data suggest that a short-term therapeutic inhibition of the CSF-1 receptor pathway by BLZ945 in the murine cuprizone model enhances central remyelination by modulating neuroinflammation. Thus, microglia-modulating therapies could be considered clinically for promoting myelination in combination with standard-of-care treatments in MS patients.

The BACE-1 inhibitor CNP520 for prevention trials in Alzheimer's disease.

The beta-site amyloid precursor protein cleaving enzyme-1 (BACE-1) initiates the generation of amyloid-ß (Aß), and the amyloid cascade leading to amyloid plaque deposition, neurodegeneration, and dementia in Alzheimer's disease (AD). Clinical failures of anti-Aß therapies in dementia stages suggest that treatment has to start in the early, asymptomatic disease states. The BACE-1 inhibitor CNP520 has a selectivity, pharmacodynamics, and distribution profile suitable for AD prevention studies. CNP520 reduced brain and cerebrospinal fluid (CSF) Aß in rats and dogs, and Aß plaque deposition in APP-transgenic mice. Animal toxicology studies of CNP520 demonstrated sufficient safety margins, with no signs of hair depigmentation, retina degeneration, liver toxicity, or cardiovascular effects. In healthy adults ≥ 60 years old, treatment with CNP520 was safe and well tolerated and resulted in robust and dose-dependent Aß reduction in the cerebrospinal fluid. Thus, long-term, pivotal studies with CNP520 have been initiated in the Generation Program.

Longitudinal noninvasive magnetic resonance imaging of brain microhemorrhages in BACE inhibitor-treated APP transgenic mice.

Currently, several immunotherapies and BACE (Beta Site APP Cleaving Enzyme) inhibitor approaches are being tested in the clinic for the treatment of Alzheimer's disease. A crucial mechanism-related safety concern is the exacerbation of microhemorrhages, which are already present in the majority of Alzheimer patients. To investigate potential safety liabilities of long-term BACE inhibitor therapy, we used aged amyloid precursor protein (APP) transgenic mice (APP23), which robustly develop cerebral amyloid angiopathy. T2*-weighted magnetic resonance imaging (MRI), a translational method applicable in preclinical and clinical studies, was used for the detection of microhemorrhages throughout the entire brain, with subsequent histological validation. Three-dimensional reconstruction based on in vivo MRI and serial Perls' stained sections demonstrated a one-to-one matching of the lesions thus allowing for their histopathological characterization. MRI detected small Perls' positive areas with a high spatial resolution. Our data demonstrate that volumetric assessment by noninvasive MRI is well suited to monitor cerebral microhemorrhages in vivo. Furthermore, 3 months treatment of aged APP23 with the potent BACE-inhibitor NB-360 did not exacerbate microhemorrhages in contrast to Aß-antibody ß1. These results substantiate the safe use of BACE inhibitors regarding microhemorrhages in long-term clinical studies for the treatment of Alzheimer's disease.

Pharmacological BACE1 and BACE2 inhibition induces hair depigmentation by inhibiting PMEL17 processing in mice.

Melanocytes of the hair follicle produce melanin and are essential in determining the differences in hair color. Pigment cell-specific MELanocyte Protein (PMEL17) plays a crucial role in melanogenesis. One of the critical steps is the amyloid-like functional oligomerization of PMEL17. Beta Site APP Cleaving Enzyme-2 (BACE2) and γ-secretase have been shown to be key players in generating the proteolytic fragments of PMEL17. The ß-secretase (BACE1) is responsible for the generation of amyloid-ß (Aß) fragments in the brain and is therefore proposed as a therapeutic target for Alzheimer's disease (AD). Currently BACE1 inhibitors, most of which lack selectivity over BACE2, have demonstrated efficacious reduction of amyloid-ß peptides in animals and the CSF of humans. BACE2 knock-out mice have a deficiency in PMEL17 proteolytic processing leading to impaired melanin storage and hair depigmentation. Here, we confirm BACE2-mediated inhibition of PMEL17 proteolytic processing in vitro in mouse and human melanocytes. Furthermore, we show that wildtype as well as bace2(+/-) and bace2(-/-) mice treated with a potent dual BACE1/BACE2 inhibitor NB-360 display dose-dependent appearance of irreversibly depigmented hair. Retinal pigmented epithelium showed no morphological changes. Our data demonstrates that BACE2 as well as additional BACE1 inhibition affects melanosome maturation and induces hair depigmentation in mice.

BACE1 inhibition induces a specific cerebrospinal fluid ß-amyloid pattern that identifies drug effects in the central nervous system.

BACE1 is a key enzyme for amyloid-ß (Aß) production, and an attractive therapeutic target in Alzheimer's disease (AD). Here we report that BACE1 inhibitors have distinct effects on neuronal Aß metabolism, inducing a unique pattern of secreted Aß peptides, analyzed in cell media from amyloid precursor protein (APP) transfected cells and in cerebrospinal fluid (CSF) from dogs by immunoprecipitation-mass spectrometry, using several different BACE1 inhibitors. Besides the expected reductions in Aß1-40 and Aß1-42, treatment also changed the relative levels of several other Aß isoforms. In particular Aß1-34 decreased, while Aß5-40 increased, and these changes were more sensitive to BACE1 inhibition than the changes in Aß1-40 and Aß1-42. The effects on Aß5-40 indicate the presence of a BACE1 independent pathway of APP degradation. The described CSF Aß pattern may be used as a pharmacodynamic fingerprint to detect biochemical effects of BACE1-therapies in clinical trials, which might accelerate development of novel therapies.