Background
CAR-T cells use TCR signaling cassettes coupled to the recognition elements of antibodies. While CAR-T technology has achieved significant success in treatment of certain liquid cancers, many challenges hinder the development of this therapy. CAR-T cells must be produced from the patient’s own T cells, which are often of poor quality and in short supply after earlier first and second line treatments. The time taken to produce sufficient CAR-T cells for therapy is also problematic. ‘Off-the-shelf’ allo-CAR-T could solve these problems, since they could be produced in advance, in bulk, from healthy donors. However, these can potentially cause Graft versus Host Disease (GVHD), and can be rejected by the patient’s own immune system. Additionally, autologous CAR-T cells have not so far been very effective against solid tumors. Our understanding of CAR signaling, and how it may differ from TCR signaling, is still not very strong.
Methods
We directly compared a CAR and a TCR targeting the same pMHC antigen, analyzing various signaling parameters in vitro. CRISPR-Cas9 was used to knock-in CAR constructs into specific loci (LCK and TRAC) using lentivirus. In vivo studies used NSG mice adoptively transferred with modified primary human CD8+ T cells.
Results
Using a 2nd generation CD28-CAR and ab TCR recognizing the same HLA-A*02:01 LMP peptide complex, we found differences in strength and kinetics of signaling. Most interestingly the CD28-CAR signaling was triggered in the absence of the SRC family kinase (SFK) LCK, which is essential for TCR signaling. We found that LCK-disrupted CAR-T cells (disLCK-CAR-T) are strongly signaled through CAR and have significantly better in vivo efficacy in both liquid and solid tumor models. This is because of their enhanced persistence, induction of memory, and reduced exhaustion phenotype. The disLCK-CAR-T blocks activation through endogenous TCR equally as well as TCR KO CAR-T, and both methods block the development of GVHD. disLCK-CAR-T also shows superior in vivo persistence compared to TCR KO T cells, probably because of retained tonic signaling.
Conclusions
This non-canonical signaling in CAR-T cells provides new insight into the initiation of TCR and CAR signaling and has important clinical implications for improvement of both autologous and allogeneic CAR-T cells. disLCK-CAR-T is being further developed as an allogeneic CAR-T.
Acknowledgements
NMRC: MOH-000523; A*STAR Singapore Therapeutics Development Review (STDR) H23H9a0014 and H24H9a0005.
Ethics Approval
Blood collection protocol was approved by NUS IRB. Informed consent was obtained from all donors. NUS-IRB Reference Code: LH-19-026.