SEMA4D Recombinant Monoclonal Antibody

What is the molecular structure of SEMA4D and how does it relate to antibody design?

SEMA4D is expressed on the cell surface as a disulfide-linked homodimer with a molecular weight of approximately 300-kDa. The protein contains a large N-terminal β-propeller "sema" domain, followed by an Ig-like domain, a lysine-rich domain, a transmembrane domain, and a cytoplasmic tail with consensus tyrosine and serine phosphorylation sites. Upon cell activation, SEMA4D can be proteolytically cleaved to generate a physiologically active 240-kDa soluble form (sSEMA4D) .

Successful antibody design requires targeting accessible epitopes within the extracellular portion of SEMA4D, particularly within the sema domain that mediates receptor interactions. For experimental applications, researchers should consider whether their antibody recognizes both membrane-bound and soluble forms, as this will significantly impact experimental outcomes when studying tissues where proteolytic shedding occurs .

How is SEMA4D expression regulated in different cell types relevant to research applications?

SEMA4D expression varies significantly across cell types and activation states. In platelets, resting cells express detectable surface SEMA4D that transiently doubles upon activation with phorbol 12-myristate 13-acetate (PMA), followed by a decline to nearly undetectable levels due to proteolytic shedding . In immune cells, SEMA4D is upregulated upon activation, particularly in T cells.

For experimental design, researchers should: (1) determine baseline expression in their target cell population through flow cytometry; (2) characterize expression kinetics following relevant stimuli; (3) quantify proportions of membrane-bound versus soluble SEMA4D using complementary approaches such as flow cytometry and ELISA; and (4) assess potential regulation by metalloprotease inhibitors if shedding is a critical experimental parameter .

How do anti-SEMA4D antibodies affect the interactions with different receptors, and what methodologies can measure this interference?

SEMA4D interacts with three main receptors: Plexin-B1 (high-affinity), Plexin-B2, and CD72 (lower-affinity). Effective anti-SEMA4D antibodies should block binding to all three receptors. Receptor blockade can be measured using flow cytometry-based binding assays where His-tagged SEMA4D is pre-incubated with anti-SEMA4D antibodies at various concentrations, then added to receptor-expressing cells. Bound SEMA4D is detected with anti-His-APC antibodies, with decreasing fluorescence indicating successful blocking .

When designing experiments to measure receptor blocking, researchers should:

• Establish receptor expression systems for each receptor (e.g., PLXNB1-transfected 293 cells)

• Determine antibody potency as EC50 values for blocking each receptor interaction

• Include appropriate isotype controls to distinguish specific from non-specific effects

• Consider downstream signaling assays to confirm functional receptor blockade

For VX15/2503, the mean EC50 for blocking SEMA4D-Plexin-B1 interaction is approximately 1.2 nM, providing a benchmark for comparative studies .

What signaling cascades are initiated by SEMA4D-receptor interactions that can be modulated by antibody treatment?

SEMA4D receptor signaling differs based on the specific receptor engaged. For CD72, SEMA4D binding disrupts CD72's association with the tyrosine phosphatase SHP-1. In resting platelets and B cells, CD72 complexes with SHP-1, maintaining its inhibitory effects. SEMA4D binding causes dissociation of this complex, releasing SHP-1-mediated inhibition and enhancing signaling through phosphotyrosine pathways .

For Plexin-B1, SEMA4D binding activates small GTPases and downstream pathways affecting cytoskeletal organization, cellular migration, and vascular permeability. In endothelial cells, this signaling can disrupt tight junctions forming the blood-brain barrier .

To experimentally assess antibody effects on these pathways, researchers can:

• Perform co-immunoprecipitation studies to evaluate CD72/SHP-1 complex integrity

• Measure phosphorylation status of downstream signaling molecules

• Employ cell migration and permeability assays to assess functional outcomes

• Use receptor-specific cell systems to distinguish effects on different signaling pathways

What strategies overcome immunological tolerance when generating high-affinity anti-SEMA4D antibodies with cross-species reactivity?

Generating antibodies with cross-species reactivity to SEMA4D presents challenges due to high protein conservation and immunological tolerance. The most successful approach involves immunizing SEMA4D-deficient (knockout) mice to bypass tolerance mechanisms. As demonstrated with the development of mAb 67-2 (the parent antibody for humanized VX15/2503), this strategy yielded antibodies that react with mouse, rat, primate, and human SEMA4D .

Methodological steps include:

• Immunization of SEMA4D knockout mice with recombinant SEMA4D

• Screening approximately 96 parental hybridomas for cross-reactivity with both mouse and human SEMA4D

• Characterization through various assays including ELISA, competition assays, flow cytometry, and immunohistochemistry

• Selection of lead candidates based on affinity, species cross-reactivity, and functional blocking activity

• Humanization of murine antibodies for potential therapeutic development

What are the critical quality attributes and analytical methods for characterizing anti-SEMA4D antibodies for research applications?

Researchers should validate each antibody lot against these parameters before experimental use to ensure reproducible results. For antibodies intended for in vivo studies, additional testing for endotoxin levels, aggregation, and formulation stability should be performed .

How can anti-SEMA4D antibodies be used to study blood-brain barrier integrity and what specific methodologies provide quantitative assessment?

Anti-SEMA4D antibodies have demonstrated efficacy in preserving blood-brain barrier (BBB) integrity in models of neuroinflammation. SEMA4D signaling through Plexin-B1 on endothelial cells disrupts tight junctions, and antibody blockade prevents this disruption .

Methodological approaches to assess BBB integrity include:

• In vivo permeability assays:

  • Intravenous injection of tracers (Evans blue, fluorescent dextrans)

  • Quantification of tracer extravasation into brain parenchyma

  • Comparison between anti-SEMA4D treated and control groups

• Ex vivo histological assessment:

  • Immunostaining for tight junction proteins (ZO-1, claudin-5, occludin)

  • Quantitative image analysis of junction continuity and expression levels

  • Co-localization studies with endothelial markers

• In vitro barrier models:

  • Transendothelial electrical resistance (TEER) measurements using brain endothelial monolayers

  • Permeability coefficients for labeled molecules of various sizes

  • Assessment of tight junction protein localization and phosphorylation status

These methodologies allow researchers to determine both the functional and molecular effects of anti-SEMA4D treatment on BBB integrity, providing insight into potential therapeutic applications in multiple sclerosis and other neuroinflammatory conditions .

What experimental approaches best demonstrate the effects of anti-SEMA4D antibodies on oligodendrocyte precursor cell (OPC) migration and differentiation?

Anti-SEMA4D antibodies can reverse the inhibitory effects of SEMA4D on OPC survival, migration, and differentiation, making them valuable tools for studying remyelination processes. Comprehensive experimental approaches include:

• In vitro OPC cultures:

  • Primary OPC isolation from neonatal rodent brains

  • Assessment of survival (TUNEL assay, MTT assay)

  • Differentiation analysis (immunostaining for myelin basic protein, proteolipid protein)

  • Migration assays (transwell, scratch assays) with/without recombinant SEMA4D and anti-SEMA4D antibodies

• Ex vivo slice cultures:

  • Organotypic brain slice cultures

  • Focal demyelination with lysolecithin

  • Treatment with anti-SEMA4D antibodies

  • Analysis of OPC recruitment and remyelination

• In vivo demyelination models:

  • Cuprizone-induced demyelination

  • Lysolecithin focal demyelination

  • Measurement of OPC migration to lesion sites

  • Quantitative assessment of remyelination (electron microscopy, myelin staining)

  • Analysis of functional recovery (electrophysiology, behavioral testing)

These approaches collectively provide robust evidence for the role of anti-SEMA4D antibodies in promoting remyelination by modulating OPC biology in developmental and pathological contexts .

How do anti-SEMA4D antibodies affect immune cell functions in the tumor microenvironment, and what assays best evaluate these effects?

SEMA4D can guide migration and trigger cytoskeletal changes in endothelial, tumor, and immune cells within the tumor microenvironment (TME). Anti-SEMA4D antibodies can modulate these effects, potentially enhancing anti-tumor immunity. Key experimental approaches include:

• Immune cell migration and infiltration:

  • Transwell migration assays for monocytes, T cells, and dendritic cells

  • In vivo tracking of labeled immune cells in tumor models

  • Immunohistochemical quantification of tumor-infiltrating lymphocytes

  • Flow cytometric analysis of immune cell populations within tumors

• Immune cell activation and function:

  • T cell proliferation assays

  • Cytokine production profile (ELISA, intracellular cytokine staining)

  • Cytotoxic T cell killing assays against tumor targets

  • Assessment of dendritic cell maturation markers

• Tumor vasculature assessment:

  • CD31 immunostaining for vessel density

  • Pericyte coverage (α-SMA co-staining)

  • Vessel perfusion and permeability

  • Analysis of hypoxic regions within tumors

These assays allow researchers to comprehensively evaluate the multifaceted effects of anti-SEMA4D antibodies on the immunological landscape of the TME, providing insights into their potential as immunomodulatory agents in cancer therapy .

What considerations are important when designing experiments to evaluate anti-SEMA4D antibodies in combination with other immunotherapeutic approaches?

When designing combination studies with anti-SEMA4D antibodies and other immunotherapeutics, researchers should consider:

• Mechanism of action complementarity:

  • Map the molecular pathways affected by each agent

  • Identify potential synergistic or antagonistic interactions

  • Determine optimal timing for each intervention based on mechanistic understanding

• Dosing and scheduling optimization:

  • Perform dose-response studies for each agent alone

  • Test sequential versus concurrent administration

  • Establish appropriate dosing intervals based on pharmacokinetics

• Comprehensive outcome assessment:

  • Analyze changes in immune cell populations by flow cytometry

  • Evaluate both direct anti-tumor effects and immune-mediated responses

  • Assess development of immunological memory

  • Monitor potential immune-related adverse events

• Control groups and experimental design:

  • Include single-agent arms for each therapy

  • Use appropriate isotype controls

  • Consider genetic approaches (e.g., SEMA4D knockout models) as complementary evidence

  • Design experiments with sufficient statistical power for subgroup analyses

These considerations ensure robust evaluation of combination approaches and facilitate translational research toward clinical applications .

How can researchers effectively study the role of SEMA4D in platelet function and thrombus formation using anti-SEMA4D antibodies?

SEMA4D is expressed on platelets and influences thrombus formation, making it a relevant target for thrombosis research. Comprehensive experimental approaches include:

• In vitro platelet function assays:

  • Aggregometry using various agonists (collagen, ADP, thrombin)

  • Flow chamber adhesion and aggregation under shear stress

  • Clot retraction and stability assessments

  • Platelet spreading on extracellular matrix proteins

• SEMA4D shedding analysis:

  • Flow cytometry to monitor surface SEMA4D expression kinetics

  • ELISA to quantify soluble SEMA4D release

  • Effects of metalloprotease inhibitors on shedding

  • Comparison between wild-type and ADAM17-deficient platelets

• In vivo thrombosis models:

  • FeCl₃-induced carotid artery thrombosis

  • Laser-induced cremaster arteriole injury

  • Intravital microscopy for real-time thrombus formation

  • Assessment of time to occlusion and stability of formed thrombi

• Signaling studies:

  • Co-immunoprecipitation of CD72/SHP-1 complexes

  • Phosphotyrosine profiling following platelet activation

  • Analysis of signaling changes with anti-SEMA4D treatment

When designing these experiments, researchers should consider that SEMA4D appears to particularly affect collagen-induced platelet responses, suggesting a specific role in GPVI-mediated signaling pathways .

What molecular mechanisms explain the dual functions of membrane-bound versus shed SEMA4D in vascular biology?

SEMA4D appears to play dual roles in vascular responses to injury through distinct mechanisms depending on its membrane-bound or soluble state:

• Membrane-bound SEMA4D on platelets:

  • Promotes initial thrombus formation through homotypic interactions with other platelets

  • Interacts with CD72 on neighboring platelets, causing dissociation of CD72-SHP-1 complexes

  • Releases inhibitory effects of SHP-1 on phosphotyrosine-based signaling downstream of collagen receptor GPVI

  • Enhances platelet activation in a contact-dependent manner

• Shed soluble SEMA4D (sSEMA4D):

  • Becomes available to interact with receptors on endothelial cells (primarily Plexin-B1)

  • Affects monocyte migration and inflammatory responses

  • Mediates angiogenic responses in endothelial cells

  • Continues to interact with platelets in a paracrine fashion

This dual functionality creates a spatiotemporal regulation mechanism: initially membrane-bound SEMA4D promotes local thrombus formation, while subsequently shed sSEMA4D influences the broader vascular environment and inflammatory response. Researchers studying these mechanisms should design experiments that can distinguish between these two phases and the transition between them .

What controls and validation steps are critical when using anti-SEMA4D antibodies to ensure experimental rigor and reproducibility?

These controls and validation steps ensure experimental rigor and facilitate reproducibility across different research groups and experimental systems .

How can researchers overcome challenges in studying species-specific SEMA4D effects when translating findings between rodent models and human systems?

Translating findings between species presents challenges due to potential differences in SEMA4D biology. Researchers can address these challenges through:

• Cross-species antibody selection:

  • Use antibodies like VX15/2503 that recognize SEMA4D across species

  • Validate comparable binding affinities for mouse, rat, primate, and human SEMA4D

  • Confirm similar receptor-blocking potencies across species

• Comparative biology approaches:

  • Directly compare SEMA4D expression patterns across species

  • Conduct side-by-side functional assays with cells from different species

  • Identify conserved versus divergent signaling pathways

• Humanized models:

  • Utilize humanized mouse models where appropriate

  • Consider xenograft approaches for specific applications

  • Complement with ex vivo human tissue studies

• Validation in multiple species:

  • Confirm key findings in at least two different species

  • Include non-human primate studies as a translational bridge

  • Correlate with available human data from clinical samples

The cross-species reactivity of well-characterized antibodies like VX15/2503 (KD values ranging from 1.5-5.1 nM across species) provides a valuable tool for such translational research, allowing direct comparison of SEMA4D biology across experimental systems .

How can researchers utilize anti-SEMA4D antibodies to investigate the complex interplay between neural, vascular, and immune systems in neurological disorders?

Anti-SEMA4D antibodies provide a powerful tool for investigating the neurovascular-immune axis in neurological disorders. Advanced research approaches include:

• Multi-parameter intravital imaging:

  • Two-photon microscopy of cranial windows

  • Simultaneous tracking of labeled immune cells, vascular integrity, and neural activity

  • Real-time assessment of anti-SEMA4D antibody effects on cellular interactions

• Cell-specific conditional approaches:

  • Compare global anti-SEMA4D treatment with cell-specific SEMA4D deletion

  • Use Cre-lox systems to delete SEMA4D receptors from specific cell populations

  • Determine the relative contribution of different cell types to observed phenotypes

• Multi-omics integration:

  • Perform transcriptomic, proteomic, and metabolomic analyses on isolated cell populations

  • Construct network models of SEMA4D-dependent signaling changes

  • Identify convergent pathways affected by anti-SEMA4D treatment

• Temporal intervention studies:

  • Administer anti-SEMA4D antibodies at different disease stages

  • Determine windows of opportunity for therapeutic intervention

  • Assess acute versus chronic effects on disease progression

These sophisticated approaches allow researchers to dissect the complex roles of SEMA4D in maintaining and disrupting the neurovascular unit, with particular relevance to multiple sclerosis, stroke, and other neuroinflammatory conditions .

What emerging technologies and methods can enhance the study of SEMA4D functions and anti-SEMA4D antibody effects at single-cell resolution?

Emerging technologies for single-cell resolution studies of SEMA4D biology include:

• Single-cell RNA sequencing:

  • Transcriptional profiling of individual cells after anti-SEMA4D treatment

  • Identification of cell-specific response signatures

  • Trajectory analysis of developmental or differentiation processes

• Mass cytometry (CyTOF):

  • High-dimensional phenotyping of immune and neural cell populations

  • Simultaneous measurement of multiple signaling pathways

  • Integration of cellular phenotypes with functional states

• Spatial transcriptomics and proteomics:

  • Mapping SEMA4D expression and signaling within tissue architecture

  • Visualization of cellular interactions at receptor interfaces

  • Correlation of molecular profiles with histopathological features

• Live-cell biosensors:

  • FRET-based reporters for real-time signaling dynamics

  • Optogenetic control of SEMA4D signaling components

  • Single-molecule tracking of SEMA4D-receptor interactions

• Engineered organoid systems:

  • Brain organoids incorporating vascular and immune components

  • Controlled perturbation of SEMA4D signaling in a human tissue context

  • Assessment of developmental and disease-relevant processes

These cutting-edge approaches will provide unprecedented insights into the cellular and molecular mechanisms of SEMA4D function and the effects of antibody-mediated intervention, advancing both basic research and therapeutic development .