The Development of Allogeneic Tips-Derived TCR^- CAR⁺ CD8αβ T Cells

Adoptive CAR T cell therapy has revolutionized the treatment of relapsed/refractory hematopoietic malignancies. However, the autologous nature of the treatment imposes manufacturing constraints that delay chimeric antigen receptor (CAR) T cell therapy availability, may result in production failure, and are costly. The development of allogeneic, "off-the-shelf” CAR T cells aims to resolve some of these challenges. However, due to the risk of graft-versus-host disease (GvHD) with allogeneic immune cells, careful donor-matching and T cell receptor (TCR)-specificity selection is required. Alternatively, genome editing may be used to eliminate TCR expression.

Induced pluripotent stem cells (iPSCs) are an attractive source for allogeneic, "off-the-shelf” cellular immunotherapy. We have previously shown that T cell-derived iPSCs (TiPS) can provide a self-renewing source for in vitro CAR T cell production (Themeli, Nat Biotechnol, 2013), resulting in a homogenous CAR⁺TCR⁺ TiPS-derived T (iT) cell population. However, in vitro differentiation yields iT cells with suboptimal features, generating cells that eschew the CD4⁺CD8αβ⁺ double-positive (DP) stage and divert towards a CD8αα innate-like phenotype. We have shown that premature expression of the T cell receptor (TCR) causes this diversion towards an innate-like phenotype. This can be partially averted by disabling the TCR but does not allow for maturation of the cells to a CD8αβ single-positive stage due to the inability to effect positive selection through TCR engagement (van der Stegen, Nat Biomed Eng, 2022).

We have demonstrated that in the absence of a TCR, careful control of CAR expression timing and calibrated signaling strength can uniquely drive adaptive iT cell differentiation to the CD4⁺CD8αβ⁺ DP stage. CAR engagement at the DP stage subsequently facilitates maturation to CD8αβ single positive T cells, substituting for the TCR. The resulting TCR^-CAR⁺ CD8αβ iT cells are similar to healthy-donor peripheral blood-derived CD8αβ T cells in displaying CD19 antigen specificity and dose-dependent cytotoxicity, as well as achieving effective tumor control in a systemic mouse model of leukemia, but do not cause GvHD due to complete TCR ablation (van der Stegen, Nat Biomed Eng 2022).

Although the resulting TCR^-CAR⁺ iT cells are similar to their CD8⁺ peripheral blood counterparts, the cells require higher treatment doses, and exogenous cytokine support to achieve that result. To further increase the potency of TCR^-CAR⁺ iT cells and remove the need for exogenous cytokine support, we enhanced iT cell function through engineering in enhanced costimulatory support. When overexpressing 4-1BBL in TCR^-CAR⁺ iT cells, in vitro proliferation upon weekly antigen restimulation increased compared to the parental iT cells relying on solely CD28-based costimulation through the CAR. In a xenograft tumor model of disseminated leukemia, 41BBL⁺ TCR^-CAR⁺ iT cells persist longer in the bone marrow (Figure 1A, measured 12 days after 4x10⁶ iT cell injection), resulting in improved in vivo survival rates (Figure 1B).

Collectively, we show that CAR expression can drive adaptive T cell maturation in absence of a TCR, facilitating large-scale development of antigen-specific allogeneic CD8αβ iT cells devoid of allo-reactive potential and GvHD, and that TCR^-CAR⁺ iT cell persistence and function can be further increased through developmentally regulated complementary costimulation.

Figure 1. Increased persistence and function of 41BBL-iT cells. a) Bone marrow counts of human CD45⁺ CAR⁺ iT cells 12 days post iT cell injection. b) overall survival of mice treated with 4x10⁶ iT cells 3 days post 1x10⁵ Nalm6 injection.

van der Stegen:Fate Therapeutics Inc.: Research Funding. Petrovic:Fate Therapeutics Inc.: Research Funding. Lindenbergh:Fate Therapeutics Inc.: Research Funding. Diop:Fate Therapeutics Inc.: Research Funding. Alexeeva:Fate Therapeutics Inc.: Research Funding. Xie:Fate Therapeutics Inc.: Research Funding. Clarke:Fate Therapeutics: Current Employment. Valamehr:Fate Therapeutics: Current Employment. Rivière:Akron Biotechnology, LLC: Other: Provision of Services; Centre for Commercialization of Cancer Immunotherapy: Other: Provision of Services; Fate Therapeutics: Current equity holder in publicly-traded company, Other: Provision of Services, Patents & Royalties; FloDesign Sonics: Current equity holder in private company, Other: Provision of Services; Juno Therapeutics: Patents & Royalties; Mnemo Therapeutics: Current equity holder in private company, Other: Provision of Services (uncompensated), Patents & Royalties; The Georgia Tech Research Corporation (GTRC): Other: Provision of Services (uncompensated). Sadelain:Atara Biotherapeutics: Consultancy, Patents & Royalties, Research Funding; Fate Therapeutics: Research Funding; Mnemo Therapeutics: Research Funding; Takeda Pharmaceuticals: Research Funding.