Background CD28-derived co-stimulation modules are frequently used as part of the signaling domain of chimeric antigen receptors (CAR). These modules contain well characterized tyrosine-containing moieties (YMNM and PYAP) that become phosphorylated upon CAR activation, resulting in IL-2 secretion and T cell expansion.^1,2 Previous work by our group has shown that an additional tyrosine residue, located in the C-terminus of CD28 (Y218), was also phosphorylated upon CAR activation.³ In this work we sought to evaluate the functional relevance and molecular mechanism of CD28-Y218 phosphorylation in CAR-T cells.

Methods We first analyzed the kinetics of Y218 phosphorylation. To that end, prostate stem cell antigen (PSCA) specific CAR-T cells were stimulated through co-culture with HPAC pancreatic cancer cells for 0, 1, 10, 30 and 60 minutes. Phosphorylation of Y218 was analyzed by Western blotting using a phospho-specific antibody. To determine the functional relevance of this phosphorylation event, we generated mutant CARs where the tyrosine residue was replaced with a non-phosphorylatable amino acid (Y218F) and evaluated its effects in vivo and in vitro. CAR-T cell cytotoxicity was monitored using the xCELLigence Real Time Cytotoxicity Assay (RTCA). A CD19-specific CAR was used as an alternative model, where NALM6 cells expressing firefly luciferase served as target cells. Luciferase activity was used as surrogate for target cell viability. In both models, we monitored cytokine production following overnight co-culture with target cells using the ELLA system (Biotechne, CA, USA). To evaluate in vivo antitumor effect of PSCA-specific CAR-T cells, NSG mice were injected (s.c.) with 1 HPAC cells and infused 14 days later with 5 CAR-T cells (i.v.). Tumor growth was monitored for 35 days.

Results We observed an antigen-dependent phosphorylation of Y218 which reached maximum levels approximately 10 min post-stimulation. Mutation of the CD28-Y218 residue did not affect CAR expression, cytotoxicity in vitro, or INFγ production. However, this mutation was associated with reduced IL-2 and TNFα secretion. In vivo, this mutation completely abrogated the therapeutic effect of these cells in the pancreatic cancer model.

Conclusions These results show that CD28-Y218 plays a role in CAR-T cell function, with direct impact on IL-2 secretion and antitumor efficacy. Future experiments will determine the specific signaling events that are triggered by Y218 phosphorylation.

Acknowledgements This work has been supported in part by the Flow Cytometry Core Facility at Moffitt Cancer Center, an NCI designated Comprehensive Cancer Center (P30-CA076292).

References

1. Esensten JH, Helou YA, Chopra G, Weiss A, Bluestone JA. CD28 costimulation: From mechanism to therapy. Immunity. 2016; 44:973-988.

2. Dong R, Libby KA, Blaeschke F, Fuchs W, Marson A, Vale RD, Su X. Rewired signaling network in T cells expressing the chimeric antigen receptor (CAR). EMBO J. 2020; 39:e104730.

3. Ramello MC, Benzaïd I, Kuenzi BM, Lienlaf-Moreno M, Kandell WM, Santiago DN, Pabón-Saldaña M, Darville L, Fang B, Rix U, Yoder S, Berglund A, Koomen JM, Haura EB, Abate-Daga D. An immunoproteomic approach to characterize the CAR interactome and signalosome. Sci Signal. 2019; 12:eaap9777.

Ethics Approval This study was approved by the Institutional Animal Care and Use Committee (IACUC); approval number R ISO00010727.