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A cell-based immunogenicity assay to detect antibodies against chimeric antigen receptor expressed by
Bernd Potthoff1, Fraser McBlane1, Sebastian Spindeldreher2
1Novartis Pharma AG, Basel, Switzerland.
Abstract:
The use of T-cells expressing Chimeric Antigen Receptors (CARs) offers new opportunities for cancer treatment, as well as new challenges for the bioanalysis of this new class of drugs. The analysis of humoral immunogenicity (anti-drug antibodies) against CARs could be performed with a bridging ELISA, using labeled CAR fragments. However, outside of its native cell membrane environment and without potential interaction partners on the cell surface, a labeled or coated recombinant CAR fragment may structurally differ from the membrane-bound CAR expressed on CAR-T cells. Consequently, immunogenicity to CARs may be missed due to the artificial nature of a ligand binding assay setup. T-cell lines expressing the CAR offer the opportunity to measure anti-drug antibodies to the CAR in its natural cell environment, as an alternative to ligand-binding assays. Here we describe a novel, flow cytometry-based humoral immunogenicity assay for tisagenlecleucel (CTL019, Kymriah®) using a human T-cell line that expresses murine CAR19. The assay described here was fully validated according to health authority guidelines for the development and validation of immunogenicity assays and has a sensitivity of 100 ng/mL. A good correlation of screening assay signal strengths to titer assay results was observed while exploring options to increase titration assay throughput. Pre-existing antibodies against the cell line used in the assay as well as against the CAR itself complicate the assay and data interpretation.
Insights
A novel flow cytometry assay accurately measures anti-drug antibodies against CAR-T therapies. This method uses T-cells expressing chimeric antigen receptors (CARs) in their natural environment, overcoming limitations of traditional assays.
Area of Science:
- Immunology
- Biotechnology
- Pharmacology
Background:
- Chimeric antigen receptor (CAR)-T cell therapy represents a significant advancement in cancer treatment.
- Bioanalysis of CAR-based therapeutics presents unique challenges, particularly in assessing humoral immunogenicity (anti-drug antibodies).
- Traditional ligand-binding assays using recombinant CAR fragments may not accurately reflect the immunogenicity of membrane-bound CARs due to altered structure outside the native cell environment.
Purpose of the Study:
- To develop and validate a novel, flow cytometry-based assay for measuring humoral immunogenicity against tisagenlecleucel (Kymriah®).
- To establish an alternative to ligand-binding assays by assessing anti-drug antibodies in the natural cellular context of CAR-T cells.
- To ensure the assay meets health authority guidelines for immunogenicity assay development and validation.
Main Methods:
- A human T-cell line engineered to express murine CAR19 was utilized.
- A flow cytometry-based assay was developed to detect anti-drug antibodies against the CAR in its native cellular environment.
- The assay was fully validated according to established health authority guidelines.
Main Results:
- The developed assay demonstrated high sensitivity, with a limit of detection of 100 ng/mL.
- A strong correlation was observed between screening assay signal strengths and titration assay results.
- Challenges in assay interpretation were noted due to pre-existing antibodies against the CAR and the cell line.
Conclusions:
- A validated flow cytometry assay provides a robust method for assessing humoral immunogenicity of CAR-T therapies like tisagenlecleucel.
- This cell-based approach offers a more biologically relevant assessment of anti-drug antibodies compared to assays using isolated CAR fragments.
- Further optimization is needed to address potential interferences from pre-existing antibodies for improved assay interpretation and throughput.
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