Broadly reactive anti-VHH antibodies for characterizing, blocking, or activating nanobody-based CAR-T cells
Scott McComb1,2,3,4, Bianca Dupont2, Alex Shepherd2
1Human Health Therapeutics Research Centre, National Research Council Canada, Montreal, QC, H4P 2R2, and Ottawa, ON, K1A 0R6, Canada.
Background:
Production of chimeric antigen receptor T cell (CAR-T) therapies depends on antibody reagents to label, isolate, and expand T cell products. We sought to create antibody tools specific for the variable domain of heavy-chain only antibodies (VHHs), also known as nanobodies, used in some CARs.
Methods:
We immunized a mouse with VHH and selected two murine monoclonal antibodies (mAbs) that bind to distinct epitopes in conserved framework regions of llama-derived VHHs, and not to human VH domains. Anti-VHH mAbs were characterized by enzyme-linked immunosorbent assay, surface plasmon resonance, and hydrogen-deuterium exchange mass spectrometry; were then tested for cell/tissue labeling and for modulating cellular activity in VHH-CAR-T cells.
Results:
We produced a high-quality dual-clonal anti-VHH antibody product and confirmed reactivity to over 98% of VHH proteins regardless of their antigenic specificity, with no reactivity to human or mouse IgG and reduced reactivity to conventional llama or alpaca IgG. Anti-VHH binding did not disrupt VHH/antigen interaction, and thus was appropriate for secondary labeling to assess cellular or tissue reactivity of VHH molecules. Despite not interfering with antigen binding, anti-VHH antibodies (Abs) potently blocked VHH-CAR-T activation and cytolytic killing of target cells. When immobilized, anti-VHH Abs induced strong activation and expansion of VHH CAR-T cells; with 730-fold mean expansion, >94% CAR purity, and retained CD8/CD4 heterogeneity. Functionally, anti-VHH antibody-expanded CAR-T cells maintained strong antigen-specific activity without functional exhaustion.
Conclusions:
Overall, these data identify useful anti-VHH mAbs that can be applied to better understand and manipulate VHH-based CAR-T cells or other VHH-based immunotherapies.
Insights
Researchers developed novel anti-VHH antibodies for chimeric antigen receptor T cell (CAR-T) therapies. These antibodies effectively label, activate, and expand VHH-CAR-T cells without hindering antigen binding, improving CAR-T cell therapy development.
Area of Science:
- Immunology
- Biotechnology
- Antibody Engineering
Background:
- Chimeric antigen receptor T cell (CAR-T) therapies rely on antibodies for T cell manipulation.
- Variable domain of heavy-chain only antibodies (VHHs), or nanobodies, are utilized in some CAR designs.
- Development of specific antibody tools for VHHs is crucial for advancing CAR-T therapies.
Purpose of the Study:
- To create specific antibody tools targeting the variable domain of heavy-chain only antibodies (VHHs).
- To assess the utility of these anti-VHH antibodies in labeling, modulating, and expanding VHH-CAR-T cells.
- To enhance the understanding and manipulation of VHH-based immunotherapies.
Main Methods:
- Immunization of mice with VHH and selection of murine monoclonal antibodies (mAbs).
- Characterization of anti-VHH mAbs using techniques like ELISA, SPR, and HDX-MS.
- Testing of anti-VHH mAbs for cell/tissue labeling and modulation of VHH-CAR-T cell activity.
Main Results:
- A dual-clonal anti-VHH antibody product demonstrated high reactivity (>98%) to VHH proteins with no cross-reactivity to human IgG.
- Anti-VHH antibodies did not interfere with VHH/antigen binding but could block VHH-CAR-T cell activation.
- Immobilized anti-VHH antibodies induced significant VHH-CAR-T cell expansion (730-fold) and purity (>94%) while maintaining functionality.
Conclusions:
- Novel anti-VHH monoclonal antibodies have been identified as valuable tools for CAR-T cell research.
- These antibodies can be used for secondary labeling and functional modulation of VHH-CAR-T cells.
- The developed anti-VHH antibodies facilitate better understanding and manipulation of VHH-based CAR-T cells and immunotherapies.
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