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Updated: Nov 7, 2025
Author Spotlight: Advancements in CAR-T Cell Manufacturing and Gene Therapy Production
Published on: August 18, 2023
Chimeric antigen receptor preparation from hybridoma to T-cell expression
Hakan Köksal1, Elizabeth Baken1, David John Warren2
1Department of Cellular Therapy, Department of Oncology, Oslo University Hospital-Radiumhospitalet, Oslo 0379, Norway.
Abstract:
The successful use of chimeric antigen receptor (CAR) for hematological cancer treatment has influenced the direction taken in translational research toward an increasing focus on personalized targeted immunotherapy. Thus, a growing number of labs worldwide are now interested in testing their old antibody collections in this format to broaden the spectrum of utility and improve safety and efficacy. We herein present a straightforward protocol for the identification of an antibody from a hybridoma and the design of the single chain fragment that will be placed on the extracellular part of the CAR construct. We further show how to test the expression and the activity of the construct in primary T cells. We illustrate our demonstration with two new CARs targeted against the B cell receptor, more precisely the light chains κ and λ, that represent potential alternatives to the CD19 CAR used in the treatment of B-cell malignancies.
Insights
Researchers developed a protocol for creating new chimeric antigen receptor (CAR) immunotherapies targeting B-cell malignancies. This method identifies antibodies and tests their efficacy in T cells, offering alternatives to CD19 CAR treatments.
Area of Science:
- Immunology
- Oncology
- Biotechnology
Background:
- Chimeric antigen receptor (CAR) therapy has shown success in treating hematological cancers.
- Translational research is increasingly focused on personalized targeted immunotherapy.
- There is growing interest in utilizing existing antibody collections for CAR development to enhance utility, safety, and efficacy.
Purpose of the Study:
- To present a straightforward protocol for identifying antibodies from hybridomas for CAR construct design.
- To demonstrate the process of designing single-chain fragments for the extracellular portion of CARs.
- To show how to test the expression and activity of novel CAR constructs in primary T cells.
Main Methods:
- Hybridoma antibody identification and single-chain fragment design for CARs.
- Construction of CARs incorporating identified antibody fragments.
- Assessment of CAR expression and functional activity in primary T cells.
Main Results:
- A protocol for antibody identification and CAR design was successfully established.
- Two novel CARs targeting B-cell receptor light chains (κ and λ) were generated.
- The expression and activity of these new CAR constructs in T cells were demonstrated.
Conclusions:
- The presented protocol offers a method for developing new CARs from existing antibody libraries.
- Novel CARs targeting B-cell receptor light chains (κ and λ) show potential as alternatives to CD19 CARs for B-cell malignancies.
- This approach facilitates the expansion of targeted immunotherapy options for hematological cancers.
