EPFL3 Antibody

What is the P2G3 antibody and how does it target SARS-CoV-2?

P2G3 is a highly potent monoclonal neutralizing antibody developed by researchers at Lausanne University Hospital (CHUV) and EPFL. It was isolated from B-cells of a post-infected donor who received two doses of the mRNA-1273 vaccine and demonstrated exceptional serum antibody levels with broad effectiveness against multiple SARS-CoV-2 variants.
The P2G3 antibody functions by:

• Binding to the spike protein of SARS-CoV-2 with high affinity (IC50s of 0.006–0.010 µg/ml for spike proteins from the original 2019-nCoV and Alpha, Beta, Gamma, and Delta variants)

• Blocking the interaction between the receptor-binding domain (RBD) of the spike protein and ACE2 receptors on human cells

• Binding to a unique epitope that partially overlaps with those recognized by AZD1061 and S309/Sotrovimab antibodies

• Demonstrating strong neutralizing activity against Omicron variants, with an IC80 value of 0.035 µg/ml against Omicron BA.1, making it approximately 23-fold more potent than AZD1061/AZD8895, 60-fold more potent than Sotrovimab, and 88-fold more potent than ADG-2

What makes EphA3 a valuable target for antibody-based cancer therapies?

EphA3 has emerged as a promising target for cancer immunotherapy due to several key characteristics:

• Expression pattern: EphA3 is highly expressed in various cancers including leukemia, glioblastoma, sarcoma, and melanoma, while having very low expression in normal adult tissues, creating a therapeutic window

• Cell distribution: In glioblastoma, EphA3 is predominantly expressed on glioma stem cells (GSCs) and tumor-associated mesenchymal stromal cells

• Functional role: EphA3 has demonstrated oncogenic functions in glioblastoma, where it supports tumor cell survival, self-renewal, and tumor formation

• Disease progression: EphA3 is significantly elevated in recurrent post-treatment versus primary treatment-naïve glioblastoma, making it particularly relevant for addressing treatment-resistant disease

• Accessibility: EphA3-targeting antibodies like IIIA4 can effectively cross the blood-tumor barrier and accumulate at tumor sites with minimal normal brain reactivity

How are monoclonal antibodies isolated from COVID-19 patients for research purposes?

The isolation of monoclonal antibodies from COVID-19 patients involves a systematic approach:

• Patient selection: Researchers screen serum samples from a cohort of infected/vaccinated donors to identify those with high neutralizing antibody titers. For example, P2G3 was isolated from a post-infected donor who received two doses of the mRNA-1273 vaccine and showed exceptionally high serum antibody levels

• B-cell isolation: Peripheral blood mononuclear cells (PBMCs) are isolated from selected donors, and B-cells are purified

• Screening: B-cell clone supernatants are screened for high-affinity binding to the target antigen (e.g., SARS-CoV-2 spike protein)

• Cloning and expression: Heavy and light chain genes from promising B-cell clones are cloned and expressed in production cells (such as ExpiCHO cells for P2G3)

• Characterization: Purified antibodies undergo initial profiling for:

  • Binding affinity to target antigens

  • Cross-reactivity with variant forms

  • Competitive binding studies with existing antibodies

  • Neutralization potency in pseudovirus and live virus assays

What in vivo models are used to validate antibody efficacy prior to clinical trials?

Researchers at EPFL employ several animal models to evaluate antibody efficacy:
For anti-SARS-CoV-2 antibodies:

• Hamster infection model: Used for prophylactic protection studies with P2G3, where antibody-treated animals showed protection against viral challenge

• Non-human primate models: Used for evaluating the impact on viral replication (measured by genomic RNA and subgenomic RNA levels in tracheal, nasopharyngeal, and bronchoalveolar lavage samples)
  For anti-cancer antibodies:

• Subcutaneous xenograft models: Used to assess anti-tumor activity of EphA3 antibodies by measuring tumor volume over time

• Orthotopic xenograft models: More clinically relevant models where tumor cells are implanted directly into the organ of origin (e.g., brain for glioblastoma studies)

• Patient-derived xenografts: Models using tumor cells directly from patients to better represent tumor heterogeneity

How does antibody drug conjugate (ADC) methodology differ from traditional antibody therapeutics?

Antibody drug conjugates (ADCs) represent an advanced approach to enhancing antibody therapeutic efficacy:
Methodological components:

• Targeting antibody: Provides tumor specificity (e.g., IIIA4 for targeting EphA3)

• Linker chemistry: Connects the antibody to the payload

• Cytotoxic payload: Delivers therapeutic effect (e.g., USAN - Urea-Seco-Analogue of the Natural product duocarmycin)
  Key methodological differences from traditional antibodies:

• Mechanism of action: While traditional antibodies primarily function through antigen binding and immune system recruitment, ADCs deliver cytotoxic agents directly to target cells

• Potency: ADCs typically show significantly higher potency due to the cytotoxic payload

• Efficacy measurement: Success is measured by:

  • Tumor localization (e.g., PET/CT imaging showing antibody delivery across the blood-tumor barrier)

  • Direct tumor cell killing rather than just immune system activation

  • Survival improvement in animal models
    Research findings for EphA3-ADC:

What methodologies are used to assess antibody-mediated functional activities beyond direct neutralization?

Advanced antibodies are evaluated for multiple functional activities using specialized assays:
Antibody-Dependent Cellular Cytotoxicity (ADCC):

• Method involves co-culturing target cells (expressing the target antigen) with effector cells (NK cells or engineered reporter cells)

• Readouts include target cell lysis or reporter activation

• P2G3 and P5C3 demonstrated ADCC activity with IC80 values in the range of 0.074-0.10 μg/ml
  Antibody-Dependent Cellular Phagocytosis (ADCP):

• Utilizes fluorescently labeled target cells or beads coated with target antigen

• Measures uptake by phagocytic cells (monocytes/macrophages)

• P2G3 showed potent ADCP activity against Omicron spike-coated beads, 3-fold improved relative to P5C3
  Fc-mediated functions enhancement:

• Engineering modifications like the LS mutation (M428L/N434S) in the Fc domain extend half-life in vivo

• This provides a key advantage for prophylactic use
  Combination effects:

• P2G3/P5C3 combination showed enhanced ADCP activities compared to individual antibodies against both ancestral and Omicron spike proteins

How are switchable antibodies (SwAbs) designed and what advantages do they offer for controlled therapeutic applications?

Switchable antibodies represent an innovative approach to controlling antibody activity using small molecules:
Design methodology:

• A chemically disrupted heterodimer (CDH) is placed between the epitope-binding region and the Fc region

• The CDH consists of two proteins that associate (e.g., LD3 and Bcl-2) but can be disrupted by a small molecule (e.g., Venetoclax)

• The epitope-binding fragment (Fab) is fused to one component (LD3), while the Fc region is fused to the other (Bcl-2)
  Functional mechanism:

• In the absence of the small molecule, the antibody remains fully functional

• Addition of the small molecule disrupts the CDH, separating the Fab from the Fc

• This leads to loss of Fc-mediated benefits including:

  • Extended half-life

  • Avidity effects from dimerization

  • Effector functions
    Experimental validation:

• Size Exclusion Chromatography Multiangle Light Scattering (SEC-MALS) was used to assess complex disruption

• Engineered variant LD3_v4 showed >90% disruption upon Venetoclax treatment compared to only 3% with the original LD3

• Flow cytometry demonstrated that Venetoclax treatment reduced binding of SwAbs to target cells
  Advantages:

• On-demand control of antibody activity and half-life

• Enhanced safety profile for highly toxic therapies like immunostimulatory agents

• Potential for intermittent dosing regimens

What computational approaches are being developed for epitope-specific antibody design?

EPFL researchers are at the forefront of developing computational methods for designing epitope-specific antibodies:
Current methodological challenges:

• Traditional antibody discovery requires extensive libraries and screening campaigns

• The fundamental limitation is the need to get an initial binding signal before optimization
  Deep learning approaches under development:

• Generative models for immunoglobulin 3D structures:

  • Neural networks model diverse antibody structures with unprecedented speed

  • These models can generate completely novel structures rather than merely optimizing existing ones

• Protein-protein interface design pipeline:

  • Optimizes not only spatial orientations but fully-flexible protein structures

  • Can target specific epitopes with high precision

• Design algorithm (TopoBuilder):

  • Allows construction of proteins virtually "as if putting Lego bricks together"

  • Enables assembly of artificial proteins with novel functions
    Application examples:

• De novo proteins designed for respiratory syncytial virus (RSV) vaccine development

• These artificial proteins trigger the immune system to produce specific antibodies against viral weak spots

• Preliminary experimental results support the feasibility of this approach

How are Antibody-Peptide Inhibitor Conjugates (APICs) designed to improve target specificity?

APICs represent a novel approach to targeted enzyme inhibition:
Design methodology:

• Target identification: Select enzymes overexpressed in cancer (e.g., cathepsins)

• Peptide inhibitor design: Create non-natural peptide inhibitors (NNPIs) with:

  • Modified peptide sequences that include a Michael acceptor

  • Capability to covalently bind to and inhibit target enzymes

• Antibody conjugation: Link the peptide inhibitors to antibodies specific for cancer cell markers
  Advantages over traditional approaches:

• Enhanced specificity: Delivers inhibitors only to cancer cells, reducing systemic side effects

• Improved efficacy: Concentrates inhibitory effect where needed

• Reduced toxicity: Prevents inhibition of essential enzymes in healthy tissues

• Modular design: Platform can be adapted to different targets and cancer types
  Target example - Cathepsins:

• Family of enzymes responsible for protein degradation and tissue remodeling

• Implicated in various cancers, osteoporosis, and autoimmune diseases

• Previous clinical trials with small molecule inhibitors failed due to lack of efficacy or toxicity

• APICs overcome these limitations through targeted delivery

What approaches are used to validate antibody specificity for research applications?

Validating antibody specificity is crucial for research reliability:
Comprehensive validation approaches:

• Third-party testing:

  • Independent evaluation separate from manufacturer and end-user

  • Testing against multiple samples and conditions

  • Example: Ayoubi et al. tested 614 commercial antibodies across 65 neuroscience-related targets

• Knockout controls:

  • Using cells where the target protein has been deleted

  • Creates a true negative control to assess non-specific binding

  • Proposed comprehensive repository of knockout cells would enable widespread validation
    Validation findings:

• Only 48% of 3,313 antibodies recommended for western blotting recognized their intended protein

• Recombinant antibodies showed superior performance compared to monoclonal and polyclonal antibodies

• Citation frequency in literature is not a reliable indicator of antibody quality
  Methodological recommendations:

• Centralized third-party validation funded by grant institutions

• Raw data sharing in open repositories (e.g., ZENODO)

• Manufacturer incentivization through provision of validation data they can use in marketing

• Development of knockout cell repositories for negative controls

Table 1: Neutralization Potency of P2G3 Against SARS-CoV-2 Variants

Table 2: Survival Outcomes in EphA3 Antibody Drug Conjugate Treatment of Glioblastoma Models

Table 3: Antibody-Dependent Cellular Functions of P2G3 and P5C3

Table 4: Comparison of Different Antibody Types Used in Research Applications