Chimeric Antigen Receptor Signaling Domains Differentially Regulate Proliferation and Native T Cell Receptor Function in Virus-Specific T Cells

Abstract

The efficacy of T cells expressing chimeric antigen receptors (CARs) for solid tumors has been limited by insufficient CAR T cell expansion and persistence. The use of virus-specific T cells (VSTs) as carriers for CARs may overcome this limitation since CAR-VSTs can be boosted by viral vaccines or oncolytic viruses. However, there is limited understanding of the optimal combination of endodomains and their influence on the native T cell receptor (TCR) in VSTs. We therefore compared the function of GD2.CARs expressing the TCR zeta chain (ζ) alone or combined with endodomains from CD28 and 4-1BB in varicella zoster virus-specific (VZV) T cells. VZVSTs expressing GD2-CARs recognized VZV-derived peptides and killed GD2-expressing tumor cells. However, after repeated stimulation through their native TCR, the expansion of GD2-CAR.CD28ζ-VZVSTs was 3.3-fold greater (p < 0.001) than non-transduced VZVSTs, whereas GD2-CARζ- and GD2-CAR.41BBζ inhibited VZVST expansion (p < 0.01). Compared to control VZVSTs, GD2-CAR.ζ VZVSTs showed a greater frequency of apoptotic (p < 0.01) T cells, whereas prolonged downregulation of the native αβ TCR was observed in GD2-CAR.41BBζ VZVSTs (p < 0.001). We confirmed that CD28ζ can best maintain TCR function by expressing GD2.CARs in Epstein-Barr virus-specific T cells and CD19-CARs in VZVSTs. In response to CAR stimulation VSTs with CD28ζ endodomains also showed the greatest expansion (6 fold > GD2-CAR.41BBζ VZVSTs (p < 0.001), however anti-tumor efficacy was superior in GD2-CAR.41BBζ-VZVSTs. These findings demonstrate that CAR signaling domains can enhance or diminish the function of the native TCR and indicate that only CD28ζ may preserve the function of the native TCR in tonically signaling CAR-VSTs.

Chimeric antigen receptor T cell persistence and memory cell formation

“…Virus‐specific T cells represent a convenient source of memory cells in peripheral blood that can be expanded with antigen‐presenting cells pulsed with peptide pools, for example, to Epstein–Barr virus or varicella zoster. Repeated stimulation for expansion via the native (virus‐specific) TCR was best achieved using CD28 endodomains, demonstrating that the choice of costimulator domains is critical for maintaining CAR T cell proliferation via the endogenous TCR . However, 41BB‐based CAR gave superior in vivo anti‐tumor responses, as compared to CD28‐based CAR …”

“…Repeated stimulation for expansion via the native (virus-specific) TCR was best achieved using CD28 endodomains, demonstrating that the choice of costimulator domains is critical for maintaining CAR T cell proliferation via the endogenous TCR. 9,37 However, 41BB-based CAR gave superior in vivo anti-tumor responses, as compared to CD28-based CAR. 37 Virtual memory T (T VM ) cells are memory T cells differentiated from naive T cells via interactions with MHC: self-peptide.…”

“…9,37 However, 41BB-based CAR gave superior in vivo anti-tumor responses, as compared to CD28-based CAR. 37 Virtual memory T (T VM ) cells are memory T cells differentiated from naive T cells via interactions with MHC: self-peptide. This self-recognition upregulates the IL-15-receptor, which in turn provides signals from endogenous IL-15 for memory cell development.…”

Engineering for Success: Approaches to Improve Chimeric Antigen Receptor T Cell Therapy for Solid Tumors

“…CARs have also been engrafted onto virus-specific T cells with the rationale that in vivo antigen stimulation and co-stimulation received after engagement of their native T cell receptor (TCR) will promote persistence of CAR T cells. However, there is an intricate interplay between the expressed CAR and native virus-specific TCR requiring detailed analysis [ 43 ]. These bi-specific T cells have accordingly shown increased expansion and persistence in neuroblastoma patients compared with T cells expressing the same CAR but lacking viral specificity [ 44 ].…”

Rational Tuning of CAR Tonic Signaling Yields Superior T-Cell Therapy for Cancer

“…CAR engineering efforts thus far have revealed several design parameters that influence CAR-T cell function (Chang and Chen, 2017;Hong et al, 2020). These include co-stimulatory domains that booster T-cell activation upon antigen stimulation (Omer et al, 2018;Wijewarnasuriya et al, 2020;Zhao et al, 2015), extracellular domains that provide structural support for optimal T-cell/target-cell conjugation (Hudecek et al, 2013;Srivastava and Riddell, 2015), binding affinity between the CAR and the targeted antigen (Drent et al, 2019;Liu et al, 2015), as well as CAR-independent parameters such as antigen expression level on target cells (Majzner et al, 2020;Watanabe et al, 2015). However, these parameters cannot fully explain the difference between the CD19 CAR and other constructs such as CD20 CARs, which have similarly high binding affinities, contain the same co-stimulatory domains, and bind an antigen that is also highly expressed on the same type of tumor cells as targeted by the CD19 CAR.…”