Discovery and preclinical development of a SdAb-based CAR-T technology for targeting CD33 in AML
Franco Bernasconi-Bisio1, Eva Molina1, Vianca Ibarra1
1Therapeutic Innovation Program, Cima Universidad de Navarra, 31008 Pamplona, Spain.
Abstract:
Chimeric antigen receptor T cell (CAR-T) therapies have revolutionized cancer immunotherapy. Traditional single-chain variable fragments (ScFvs) used as CAR recognition moieties face challenges such as high tonic signaling, compromised binding epitopes, and suboptimal affinity. Single-domain antibodies (SdAbs) offer an attractive alternative due to their smaller size, stability, and reduced immunogenicity. In this work, we developed an SdAb-CAR-T cell discovery platform integrating generation, characterization, and selection of SdAbs based on various properties. This approach was demonstrated by developing CAR-T cells with SdAbs against CD33, a target for acute myeloid leukemia (AML). We identified diverse SdAbs against CD33, with affinities ranging from 3.9-115 nM, and characterized their binding kinetics and epitope recognition. Using SdAb-based second-generation CARs, we assessed tonic signaling, T cell phenotypes, cytotoxicity and cytokine release in vitro, resulting in reduced tonic signaling and increased cytokine production. In vivo, SdAb-based CAR-T cells exhibited enhanced efficacy at lower doses, in a xenograft AML mouse model, demonstrating advantages over ScFv-based CD33 CAR-T cells.
Insights
Single-domain antibodies (SdAbs) offer improved chimeric antigen receptor T-cell (CAR-T) therapies for acute myeloid leukemia (AML). SdAb-CAR-T cells show reduced signaling and enhanced efficacy in AML models compared to traditional ScFv-CAR-T cells.
Area of Science:
- Immunology
- Biotechnology
- Oncology
Background:
- Chimeric antigen receptor T-cell (CAR-T) therapies are transformative in cancer immunotherapy.
- Traditional single-chain variable fragments (ScFvs) in CARs present limitations including tonic signaling and suboptimal affinity.
- Single-domain antibodies (SdAbs) offer potential advantages due to smaller size, stability, and lower immunogenicity.
Purpose of the Study:
- To develop a platform for generating and selecting single-domain antibodies (SdAbs) for CAR-T cell applications.
- To engineer and evaluate SdAb-based CAR-T cells targeting CD33 for acute myeloid leukemia (AML).
- To compare the efficacy and safety profile of SdAb-CAR-T cells against ScFv-CAR-T cells.
Main Methods:
- Established an SdAb discovery platform integrating generation, characterization, and selection.
- Developed and characterized diverse anti-CD33 SdAbs with varying affinities (3.9-115 nM).
- Engineered second-generation CAR-T cells using selected SdAbs and evaluated their in vitro and in vivo performance.
Main Results:
- Identified multiple anti-CD33 SdAbs with detailed kinetic and epitope characterization.
- SdAb-CAR-T cells demonstrated reduced tonic signaling and increased cytokine production in vitro.
- In vivo studies in a xenograft AML model showed enhanced efficacy of SdAb-CAR-T cells at lower doses compared to ScFv-CAR-T cells.
Conclusions:
- SdAb-based CAR-T cells represent a promising advancement over ScFv-based CAR-T cells for AML treatment.
- The developed SdAb discovery platform facilitates the creation of novel and effective CAR-T cell therapies.
- SdAb-CAR-T cells offer improved therapeutic potential with enhanced efficacy and potentially better safety profiles.
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