Artificial Thymic Organoids Permit Allelic Exclusion and Efficient Generation of Naïve TCR-Engineered T-Cells from Human Hematopoietic Stem Cells In Vitro

Engineered adoptive immunotherapies have shown unprecedented activity in the treatment of cancer and chronic viral infections. Current approaches rely on individualized ex vivo genetic modification of autologous T cells due to the risk of graft-versus-host disease from allogeneic T cells. These processes furthermore require activation and prolonged expansion of T cells, which may reduce in vivo efficacy and persistence. Direct in vitro differentiation of engineered T cells from hematopoietic stem and progenitor cells (HSPCs) may overcome these problems by permitting the suppression of endogenous TCR expression through allelic exclusion, and the de novo generation of naïve antigen-specific T cells. Existing methods of in vitro human T cell differentiation are subject to wide experimental variability and do not adequately support the positive selection of immature T cell precursors to mature T cells, and thus have not been suitable for clinical-scale production of engineered T cells.

We report here the preclinical development of an artificial thymic organoid (ATO) system using off-the-shelf, serum-free components and a standardized stromal cell line that supports highly efficient in vitro differentiation and positive selection of native and TCR-engineered human T cells from cord blood (CB), bone marrow, and mobilized peripheral blood CD34+ HSPCs, and purified CD34+CD38- hematopoietic stem cells. ATOs closely recapitulated thymic T cell commitment and differentiation, resulting in greater than 80% CD7+CD5+ T-lineage cells and 50% CD4+CD8+ double positive (DP) T cell precursors by 4 weeks. By 6 weeks, 30-40% of ATO cells were CD3+TCRαβ+ T cells, of which 20-30% were mature CD8 single positive (SP) T cells. CD4SP cells were generated at a lower frequency and later in culture (2-14% of CD3+TCRαβ+ cells). ATO-derived T cells exhibited a naïve CD45RA+CD27+CCR7+CD62L+ phenotype, a diverse, thymic-like TCR repertoire, and robust TCR-dependent cytokine release and proliferation.

Transduction of CB CD34+ HSPCs with an HLA-A*02:01-restricted αβ TCR specific for NY-ESO-1 resulted in a markedly increased cell output per ATO (>400-fold, relative to input HSPCs) and enhanced generation of naïve CD3+TCRαβ+CD8αβ+ conventional T cells, the majority of which were antigen-specific by tetramer staining. Positive selection of TCR-engineered naïve T cells could be further enhanced by expression of cognate HLA-A*02:01 in ATO stromal cells. ATO-derived TCR-engineered T cells exhibited a near complete lack of endogenous TCR Vβ expression, consistent with induction of allelic exclusion by the exogenous TCR during T cell development. ATO-derived engineered T cells underwent antigen-specific cytotoxic priming, polyfunctional cytokine release, and proliferation in response to artificial APCs; and exhibited antigen-specific killing of NY-ESO-1+ tumor cells in vitro and in vivo.

ATOs thus present a highly efficient off-the-shelf platform for the generation of clinically relevant numbers of naïve and potentially non-alloreactive engineered T cells for adoptive immunotherapy. Clinical translation of the ATO system will be aided by its simplicity, scalability, use of serum-free components, and compatibility with irradiated stromal cells. In addition, genetic manipulation of stem or stromal cell components can be easily incorporated into the system to further enhance downstream T cell engraftment or function.