CD28 Antibody

What is CD28 and what role does it play in T-cell biology?

CD28 is a critical costimulatory receptor that provides the essential "signal 2" required for complete T-cell activation. It functions alongside the T-cell receptor (TCR)/CD3 complex stimulation (signal 1) to enhance T-cell proliferation, cytokine production, and survival. CD28 and CTLA-4, together with their ligands B7-1 and B7-2, constitute one of the dominant costimulatory pathways regulating T and B cell responses .

Functionally, CD28 costimulation significantly enhances anti-tumor activity when signal 1 from TCR/CD3 is present. Without CD28 costimulation, T-cells typically experience insufficient activation and early exhaustion, limiting their effectiveness in cancer immunotherapy applications .

How do different types of CD28 antibodies function in immune response modulation?

CD28 antibodies can be categorized into two main functional types based on their ability to activate T-cells:

Non-superagonistic CD28 antibodies:

• Require TCR/CD3 stimulation to activate T-cells

• Often bind to epitopes closer to the apex of CD28, similar to natural ligands

• Show costimulatory effects only when combined with TCR/CD3 stimulation

• Demonstrate better safety profiles in preclinical models

• Example: E1P2, which mimics the function of endogenous CD80/CD86

Superagonistic CD28 antibodies:

• Can activate T-cells without concurrent TCR/CD3 stimulation

• Typically bind to lateral epitopes of CD28, away from the natural ligand binding site

• Associated with severe cytokine release syndrome, as demonstrated in the 2006 TeGenero clinical trial

• Induce polyclonal T-cell activation that can lead to systemic inflammation

• Example: TGN1412, which caused severe adverse effects in clinical trials

To experimentally differentiate between these types, researchers perform in vitro superagonistic assays where antibodies are plate-bound and T-cell activation is measured in the absence of TCR/CD3 stimulation .

What are the optimal methods for evaluating CD28 antibody binding specificity?

Comprehensive evaluation of CD28 antibody binding specificity requires multiple complementary techniques:

Flow Cytometry Analysis:

• Assess binding to primary human and mouse T-cells expressing native CD28

• Determine EC50 values through serial dilution of antibodies

• Test binding to CD28-negative cell lines to confirm specificity

• Use fluorescently-labeled secondary antibodies for detection

ELISA-Based Characterization:

• Test binding to recombinant CD28 proteins

• Determine apparent affinity through EC50 values

• Compare binding to human versus mouse CD28 to assess cross-reactivity

• Include appropriate controls (e.g., isotype control antibodies)

Protein Quality Assessment:

• Confirm purity and homogeneity of both antibody and recombinant CD28 proteins using SEC and SDS-PAGE

• Verify proper folding and oligomeric state

For CD28 antibodies specifically, testing binding to both human and mouse CD28 is crucial to facilitate preclinical safety testing. For example, E1P2 binds to both species with different affinities (EC50 values of 4.9 nM for human and 88 nM for mouse T-cells) .

How should researchers design in vitro assays to assess CD28 antibody costimulatory effects?

Assessment of CD28 antibody costimulatory effects requires several complementary approaches:

T-Cell Activation Assay Protocol:

• Coat plates with anti-CD3 antibody (e.g., OKT3) or use CD3 bispecific antibodies

• Add freshly isolated human PBMCs as effector cells

• Include CD28 antibody at various concentrations

• Incubate for 3-5 days depending on the readout

• Analyze activation markers (CD25, CD69) by flow cytometry

• Quantify cytokine production (IL-2, IFN-γ, TNF-α) by ELISA

Proliferation Assessment Methods:

• MTS colorimetric assay to measure metabolic activity

• Flow cytometric determination of absolute CD3+ T-cell counts

• CFSE or CellTrace dye dilution to track cell divisions

Tumor Cell Killing Efficiency Evaluation:

• Co-culture PBMCs with target cells (e.g., tumor cells expressing specific markers)

• Measure target cell killing when combining CD28 antibodies with CD3 bispecific antibodies

• Use Zombie Violet or similar dyes for live/dead discrimination

When performing these assays, include appropriate controls: isotype control antibodies, conditions without CD3 stimulation to check for superagonistic activity, and conditions with CD3 stimulation alone to assess the costimulatory effect .

What parameters should be monitored in safety studies for CD28-targeting therapeutics?

After the TGN1412 incident in 2006, comprehensive safety monitoring for CD28 antibodies has become essential:

Cytokine Release Monitoring:

• Measure pro-inflammatory cytokines (IL-2, IFN-γ, TNF-α, IL-6)

• Compare patterns to known superagonistic antibodies

• Assess time-course of cytokine release (6, 24, 48, 72 hours)

In Vitro Superagonistic Activity Assessment:

• Culture human PBMCs with plate-bound CD28 antibodies (3 μg/ml)

• Measure activation without TCR/CD3 stimulation

• Include multiple donors to account for variability

• Compare results with known superagonistic antibodies (e.g., TGN1412)

In Vivo Model Selection:

• Use humanized mouse models (e.g., NSG mice with human PBMC engraftment)

• Ensure antibody cross-reactivity with the model species

• Remember that conventional animal models failed to predict TGN1412 toxicity

Clinical Signs and Histopathology:

• Monitor for signs of systemic inflammation

• Examine tissues for lymphocyte infiltration

• Correlate cytokine levels with pathological findings

How does epitope mapping inform the development of safer CD28 antibodies?

Epitope mapping has proven crucial for developing safer CD28 antibodies, as demonstrated with E1P2 development:

Epitope-Function Relationship:

Strategic Selection Approaches:

• For E1P2 development, phage display selection was designed to favor binders toward the apex of CD28 by attaching biotin-streptavidin-magnetic bead complexes near the superagonistic epitope region

• This effectively masked the C''D loop during selection, directing antibody development toward safer epitopes

Mapping Methodologies:

• Competition binding assays with known ligands (CD80/CD86)

• Hydrogen-deuterium exchange mass spectrometry

• X-ray crystallography or cryo-EM

• Targeted mutagenesis of potential epitope residues

By understanding epitope-function relationships, researchers can direct antibody development toward safer epitopes that require TCR/CD3 co-engagement for T-cell activation, rather than epitopes that confer superagonistic properties .

What strategies optimize the combination of CD28 antibodies with CD3 bispecific T-cell engagers?

Combining CD28 antibodies with CD3 bispecific T-cell engagers requires optimized protocols to enhance efficacy while maintaining safety:

Experimental Protocol for Combination Testing:

• Prepare target cells expressing tumor antigens (e.g., WI-38 cells expressing fibronectin EDB)

• Add human PBMCs at defined effector-to-target ratios (5:1)

• Include anti-CD3/anti-tumor antigen BiTE at various concentrations (10, 1, 0.1 nM)

• Add CD28 antibody at fixed concentration (50 nM)

• Incubate for 4 days

• Measure:

  • Target cell killing (live/dead staining)

  • T-cell proliferation (absolute CD3+ counts)

  • Activation status (CD25 expression)

  • Cytokine production (IFN-γ ELISA)

Optimization Parameters:

• Dose ratio between CD28 and CD3 components

• Timing of administration (sequential vs. simultaneous)

• Format options (separate molecules vs. bispecific constructs)

Results from E1P2 Studies:
When E1P2 was combined with CD3 bispecific antibodies targeting EDB, researchers observed enhanced tumor cell killing and T-cell proliferation compared to CD3 bispecific antibodies alone, demonstrating the value of CD28 co-stimulation in cancer immunotherapy applications .

How can researchers distinguish CD28 antibody-induced cytokine release syndrome from other immune-related adverse events?

Differentiating CD28 antibody-induced cytokine release syndrome (CRS) from other immune-related adverse events requires comprehensive analysis:

Temporal and Cytokine Profile Analysis:

Cellular Analysis Protocol:

• Collect peripheral blood at multiple timepoints

• Perform flow cytometry for:

  • T-cell activation markers (CD25, CD69)

  • T-cell subset distribution

  • Monocyte/macrophage activation status

• Compare patterns with established CRS profiles

In Vivo Differentiation:
E1P2 demonstrated no signs of CRS in humanized NSG mice, while TGN1412 induced significant cytokine release, highlighting the importance of proper epitope selection in developing safer CD28 antibodies .

Researchers should perform predictive in vitro screening for superagonistic properties through stimulation of PBMCs without TCR/CD3 co-engagement as a critical step to identify antibodies with potential to cause CRS before advancing to in vivo studies .

How can phage display technology be optimized for isolating safer CD28 antibodies?

The successful development of E1P2 provides valuable insights into optimizing phage display for safer CD28 antibodies:

Phage Display Protocol Optimization:

• Clone and express the extracellular domain of human CD28 with an Fc tag for homo-dimerization

• Add an AviTag™ for site-specific biotinylation near the C-terminal region

• Attach biotin to the membrane-proximal part of CD28, near the C''D loop (superagonistic epitope)

• Use streptavidin-coated magnetic beads to immobilize the antigen

• Pre-incubate phage library with non-specific human IgG1 to eliminate Fc binders

• Perform multiple rounds of selection with appropriate washing steps

Strategic Considerations:

• Epitope masking: Position biotin-streptavidin complexes to shield unwanted epitopes

• Alternative approach: Include competing antibodies during selection to block unwanted epitopes

• Screen clones by ELISA against both CD28-Fc and human IgG1 to exclude Fc binders

• Sequence positive clones to identify consensus in CDR regions

The E1P2 development demonstrated that phage display can successfully isolate non-superagonistic CD28 antibodies when selection strategies are designed to favor binding to specific regions of the target protein .

What are the essential controls and validation steps for CD28 antibody characterization in immunotherapy research?

Comprehensive characterization of CD28 antibodies for immunotherapy requires rigorous controls and validation:

Basic Characterization Controls:

• Isotype-matched control antibodies

• CD28-negative cell lines for specificity testing

• Multiple T-cell donors to account for response variability

Functional Validation Protocol:

• In vitro superagonistic testing:

  • Plate-bound antibody without TCR/CD3 stimulation

  • Compare to known superagonistic antibodies (TGN1412)

  • Test with multiple PBMC donors

• Co-stimulation activity assessment:

  • Combine with anti-CD3 or BiTE molecules

  • Include conditions with CD3 stimulation alone

  • Measure multiple parameters (activation, proliferation, cytokines)

• In vivo safety validation:

  • Test in humanized mouse models

  • Include positive control (TGN1412)

  • Monitor cytokine release and clinical signs

Epitope Characterization:

• Competition binding with natural ligands

• Epitope mapping to confirm binding region

• Cross-reactivity testing with mouse CD28 to enable preclinical studies

Through this comprehensive validation approach, E1P2 was confirmed to be non-superagonistic while maintaining effective costimulatory properties when combined with CD3 stimulation, demonstrating the importance of thorough characterization before advancing to clinical applications .