Solving the mystery of the FMC63-CD19 affinity

  • Sci Rep. 2023 Dec 27;13(1):23024. doi: 10.1038/s41598-023-48528-0.
Jacqueline Seigner  1  2 ,  Charlotte U Zajc  1  3 ,  Sarah Dötsch  4 ,  Caroline Eigner  1 ,  Elisabeth Laurent  5 ,  Dirk H Busch  4 ,  Manfred Lehner  3  6  7 ,  Michael W Traxlmayr  8  9
Affiliations
  • 1. Department of Chemistry, Institute of Biochemistry, University of Natural Resources and Life Sciences, Vienna, Austria.
  • 2. Department of Biotechnology, Institute of Animal Cell Technology and Systems Biology, University of Natural Resources and Life Sciences, Vienna, Austria.
  • 3. CD Laboratory for Next Generation CAR T Cells, Vienna, Austria.
  • 4. Institute for Medical Microbiology, Immunology and Hygiene, Technical University of Munich, Munich, Germany.
  • 5. BOKU Core Facility Biomolecular and Cellular Analysis, University of Natural Resources and Life Sciences, Vienna, Austria.
  • 6. St. Anna Children's Cancer Research Institute, CCRI, Vienna, Austria.
  • 7. Department of Pediatrics, St. Anna Kinderspital, Medical University of Vienna, Vienna, Austria.
  • 8. Department of Chemistry, Institute of Biochemistry, University of Natural Resources and Life Sciences, Vienna, Austria. [email protected].
  • 9. CD Laboratory for Next Generation CAR T Cells, Vienna, Austria. [email protected].
PMID: 38155191 DOI: 10.1038/s41598-023-48528-0
Abstract

The majority of approved CAR T cell products are based on the FMC63-scFv directed against CD19. Surprisingly, although antigen binding affinity is a major determinant for CAR function, the affinity of the benchmark FMC63-scFv has not been unambiguously determined. That is, a wide range of affinities have been reported in literature, differing by more than 100-fold. Using a range of techniques, we demonstrate that suboptimal experimental designs can cause artefacts that lead to over- or underestimation of the affinity. To minimize these artefacts, we performed SPR with strictly monomeric and correctly folded soluble CD19, yielding an FMC63-scFv affinity of 2-6 nM. Together, apart from analyzing the FMC63-scFv affinity under optimized conditions, we also provide potential explanations for the wide range of published affinities. We expect that this study will be highly valuable for interpretations of CAR affinity-function relationships, as well as for the design of future CAR T cell generations.

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