Characterization and Functional Analysis of CD44v6.CAR T Cells Endowed with a New Low-Affinity Nerve Growth Factor Receptor-Based Spacer

Abstract

Effectiveness of adoptively transferred chimeric antigen receptor (CAR) T cells strongly depends on the quality of CAR-mediated interaction of the effector cells with the target antigen on tumor cells. A major role in this interaction is played by the affinity of the scFv for the antigen, and by the CAR design. In particular, the spacer domain may impact on the CAR T cell function by affecting the length and flexibility of the resulting CAR. This study addresses the need to improve the manufacturing process and the antitumor activity of CD44v6-specific CAR T cells by defining the optimal structure of a spacer region derived from the extracellular domain of the human low affinity nerve growth factor receptor (LNGFR). We tailored the LNGFR spacer to modulate CAR length in order to efficiently recognize distal or proximal epitopes and to allow selection of transduced CAR T cells by the use of clinical grade validated manufacturing systems. The different LNGFR spacers investigated in this study are responsible for the generation of CAR T cells with a different memory phenotype, which is mainly related to the level of CAR expression and the extent of the associated tonic signaling. In particular, the CD44v6-NWN2.CAR T cells are enriched in central memory cells and show improved in vitro functions in term of killing capability, and in vivo antitumor activity against hematological and solid tumors.

Optimized NGFR-derived hinges for rapid and efficient enrichment and detection of CAR T cells in vitro and in vivo

“…In a more recent publication, the same group reported three new variants of the non-mutated full-length NWL hinge by shortening the S/R-rich stalk. 17 The shortest of these constructs, NWN2, with a length of 173 aa, was functionally almost undistinguishable from the NWL isoform in all assays. Strikingly, however, the CD44v6.NWN2 CAR T cells could also not be efficiently selected with ME20.4-based MACS microbeads; instead, the authors relied once again on a two-step procedure with ME20.4-PE staining followed by sheep-anti-mouse IgG1-coated microbeads, leading to purities of 90%, but yields for NWL and NWN2 CAR T cells of only 40% and 33%, respectively.…”

“… 2 , 3 Although the hinge can theoretically influence the function of CAR constructs, only few candidates for this domain in CARs have been thoroughly investigated. 3 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 One key aspect to consider for the length and flexibility of a hinge is the location of the epitope recognized by the CAR scFvs within the three-dimensional structure of the target antigen. 5 , 13 In addition, incorporating a specific hinge can improve the protein expression and stability of the CAR and can modulate the expansion, proliferation, and stimulation of the CAR T cells.…”

“… 12 In a follow-up study, the same group further improved the hinge regarding cytotoxicity by including additional amino acids from the stalk, but still failed to efficiently enrich their NGFR-hinged CAR T cells with GMP-compatible microbeads. 17 …”

“…Here, we describe the successful establishment of two novel NGFR-derived hinges termed N3 and N4, which mediate efficient selection of CAR T cells against hematological cancers and are functionally indistinguishable from a clinically well-established human CD8-derived hinge 12 , 17 in vitro and in vivo in immunodeficient mice.…”

Patient selection for CAR T or BiTE therapy in multiple myeloma: Which treatment for each patient?

“…Thus, by modulating signaling threshold, the hinge region is important to modulate on-target off-tumor toxicities too. A spacer is not always required: Its necessity depends on the distance of target epitopes from the cell membrane [ 21 – 24 ]. The majority of CARs targeted against MM cells contain a hinge region derived from short amino acid sequences of CD8 or CD28.…”