Semaphorin 4D is upregulated in neurons of diseased brains and triggers astrocyte reactivity.

BACKGROUND: The close interaction and interdependence of astrocytes and neurons allows for the possibility that astrocyte dysfunction contributes to and amplifies neurodegenerative pathology. Molecular pathways that trigger reactive astrocytes may represent important targets to preserve normal homeostatic maintenance and modify disease progression. METHODS: Semaphorin 4D (SEMA4D) expression in the context of disease-associated neuropathology was assessed in postmortem brain sections of patients with Huntington's (HD) and Alzheimer's disease (AD), as well as in mouse models of HD (zQ175) and AD (CVN; APPSwDI/NOS2-/-) by immunohistochemistry. Effects of SEMA4D antibody blockade were assessed in purified astrocyte cultures and in the CVN mouse AD model. CVN mice were treated weekly from 26 to 38 weeks of age; thereafter mice underwent cognitive assessment and brains were collected for histopathology. RESULTS: We report here that SEMA4D is upregulated in neurons during progression of neurodegenerative diseases and is a trigger of reactive astrocytes. Evidence of reactive astrocytes in close proximity to neurons expressing SEMA4D is detected in brain sections of patients and mouse models of HD and AD. We further report that SEMA4D-blockade prevents characteristic loss of GABAergic synapses and restores spatial memory and learning in CVN mice, a disease model that appears to reproduce many features of AD-like pathology including neuroinflammation. In vitro mechanistic studies demonstrate that astrocytes express cognate receptors for SEMA4D and that ligand binding triggers morphological variations, and changes in expression of key membrane receptors and enzymes characteristic of reactive astrocytes. These changes include reductions in EAAT-2 glutamate transporter and glutamine synthetase, key enzymes in neurotransmitter recycling, as well as reduced GLUT-1 glucose and MCT-4 lactate transporters, that allow astrocytes to couple energy metabolism with synaptic activity. Antibody blockade of SEMA4D prevented these changes and reversed functional deficits in glucose uptake. CONCLUSIONS: Collectively, these results suggest that SEMA4D blockade may ameliorate disease pathology by preserving normal astrocyte function and reducing the negative consequences of reactive astrogliosis.

Anti-Semaphorin 4D Rescues Motor, Cognitive, and Respiratory Phenotypes in a Rett Syndrome Mouse Model.

Rett syndrome is a neurodevelopmental disorder caused by mutations of the methyl-CpG binding protein 2 gene. Abnormal physiological functions of glial cells contribute to pathogenesis of Rett syndrome. Semaphorin 4D (SEMA4D) regulates processes central to neuroinflammation and neurodegeneration including cytoskeletal structures required for process extension, communication, and migration of glial cells. Blocking SEMA4D-induced gliosis may preserve normal glial and neuronal function and rescue neurological dysfunction in Rett syndrome. We evaluated the pre-clinical therapeutic efficacy of an anti-SEMA4D monoclonal antibody in the Rett syndrome Mecp2T158A transgenic mouse model and investigated the contribution of glial cells as a proposed mechanism of action in treated mice and in primary glial cultures isolated from Mecp2T158A/y mutant mice. SEMA4D is upregulated in neurons while glial fibrillary acidic protein and ionized calcium binding adaptor molecule 1-positive cells are upregulated in Mecp2T158A/y mice. Anti-SEMA4D treatment ameliorates Rett syndrome-specific symptoms and improves behavioural functions in both pre-symptomatic and symptomatic cohorts of hemizygous Mecp2T158A/y male mice. Anti-SEMA4D also reduces astrocyte and microglia activation in vivo. In vitro experiments demonstrate an abnormal cytoskeletal structure in mutant astrocytes in the presence of SEMA4D, while anti-SEMA4D antibody treatment blocks SEMA4D-Plexin B1 signaling and mitigates these abnormalities. These results suggest that anti-SEMA4D immunotherapy may be an effective treatment option to alleviate symptoms and improve cognitive and motor function in Rett syndrome.

Use of poxvirus display to select antibodies specific for complex membrane antigens.

Antibody discovery against complex antigens is limited by the availability of a reproducible pure source of concentrated properly folded antigen. We have developed a technology to enable direct incorporation of membrane proteins such as GPCRs and into the membrane of poxvirus. The protein of interest is correctly folded and expressed in the cell-derived viral membrane and does not require any detergents or refolding before downstream use. The poxvirus is selective in which proteins are incorporated into the viral membrane, making the antigen poxvirus an antigenically cleaner target for in vitro panning. Antigen-expressing virus can be readily purified at scale and used for antibody selection using any in vitro display platform.

Generation and preclinical characterization of an antibody specific for SEMA4D.

Semaphorin 4D (SEMA4D or CD100) is a member of the semaphorin family of proteins and an important mediator of the movement and differentiation of multiple cell types, including those of the immune, vascular, and nervous systems. Blocking the binding of SEMA4D to its receptors can result in physiologic changes that may have implications in cancer, autoimmune, and neurological disease. To study the effects of blocking SEMA4D, we generated, in SEMA4D-deficient mice, a panel of SEMA4D-specific hybridomas that react with murine, primate, and human SEMA4D. Utilizing the complementarity-determining regions from one of these hybridomas (mAb 67-2), we generated VX15/2503, a humanized IgG4 monoclonal antibody that is currently in clinical development for the potential treatment of various malignancies and neurodegenerative disorders, including multiple sclerosis and Huntington's disease. This work describes the generation and characterization of VX15/2503, including in vitro functional testing, epitope mapping, and an in vivo demonstration of efficacy in an animal model of rheumatoid arthritis.