New cell sources for T cell engineering and adoptive immunotherapy

Abstract

The promising clinical results obtained with engineered T cells, including chimeric antigen receptor (CAR) therapy, call for further advancements to facilitate and broaden their applicability. One potentially beneficial innovation is to exploit new T cell sources that reduce the need for autologous cell manufacturing and enable cell transfer across histocompatibility barriers. Here we review emerging T cell engineering approaches that utilize alternative T cell sources, which include virus-specific or T cell receptor-less allogeneic T cells, expanded lymphoid progenitors, and induced pluripotent stem cell (iPSC)-derived T lymphocytes. The latter offer the prospect for true off-the-shelf, genetically enhanced, histocompatible cell therapy products.

Figure 1. Human T Lymphocyte Development

Hematopoietic stem cell-derived thymus-seeding progenitors (TSPs) migrate into the thymus and differentiate into an Early Thymic Progenitor (ETP) upon rearrangement of the diversity (D) and joining (J) regions of the TCR β locus. ETPs progress to a pre-T cell state expressing CD1a and CD5. At this stage, recombination of the variable (V) region of the TCR β locus to form a complete rearranged VDJ TCR β locus occurs almost simultaneously with the rearrangement of the gene segments encoding the γδ TCR. Depending on the outcome of the TCR segment rearrangements, the cells can then follow an αβ or a γδ differentiation path. A successful TCR β rearrangement leads to the process of β-selection and emergence of a CD4⁺ immature single positive (ISP) T cell. The CD4 ISP cell then develops into a double-positive (DP) cell that expresses both CD4 and CD8 and has begun to rearrange the V and J regions of the TCRα locus. The life span of DP thymocytes is limited as they quickly proceed to apoptosis if they do not receive a TCR-mediated survival signal provided by the self-HLA molecules of the thymic epithelium before maturing into CD4⁺CD8⁻ and CD4⁻ CD8⁺ single-positive (SP) T cells.

Figure 2. Antigen Receptors Used for T Cell Engineering

Left: Structure of the αβ heterodimeric T cell receptor (TCR) associated with the multi-chain CD3 complex (γ, δ, ε, and ζ) and flanked by CD28, a costimulatory receptor constitutively expressed in most αβ-T cells. Right: Structure of a prototypical second-generation chimeric antigen receptor (CAR), comprising an scFv for antigen recognition, the CD3ζ cytoplasmic domain for T cell activation, and the CD28 cytoplasmic domain to enhance T cell function and persistence (Maher et al., 2002).

Figure 3. General Schema of Autologous T Cell Manufacturing

The semi-closed system relies on the use of a cell washer to wash the apheresis product before freezing and after thawing, the capture of CD3+CD28+ T cells with magnetic beads or microbeads. For magnetic beads subsequent selection is performed on the ClinExVivo magnetic particle concentrator (MPC). Thereafter, T cells are transduced with a viral vector expressing a TCR or a CAR, expanded in a bioreactor (e.g., Wave Bioreactor), debeaded on the ClinExVivo MPC, and formulated. Adapted from Hollyman et al. (2009).

Figure 4. Perspectives for Synthetic TiPSC-Derived T Cell Generation

TiPSCs can be generated from one donor and utilized for the production of synthetic T lymphocytes engineered to possess optimized properties such as (1) antigen specificity through a CAR or TCR, (2) enhanced function (e.g., introducing costimulatory molecules), (3) elimination of alloreactivity and partial broadening of applicability (e.g., strategies to knock out the TCR expression without perturbing the T lymphoid differentiation process), and (4) broad histocompatibility by selection or genetic modification of the HLA loci.