Strategies for overcoming bottlenecks in allogeneic CAR-T cell therapy

Abstract

Patient-derived autologous chimeric antigen receptor (CAR)-T cell therapy is a revolutionary breakthrough in immunotherapy and has made impressive progress in both preclinical and clinical studies. However, autologous CAR-T cells still have notable drawbacks in clinical manufacture, such as long production time, variable cell potency and possible manufacturing failures. Allogeneic CAR-T cell therapy is significantly superior to autologous CAR-T cell therapy in these aspects. The use of allogeneic CAR-T cell therapy may provide simplified manufacturing process and allow the creation of 'off-the-shelf' products, facilitating the treatments of various types of tumors at less delivery time. Nevertheless, severe graft-versus-host disease (GvHD) or host-mediated allorejection may occur in the allogeneic setting, implying that addressing these two critical issues is urgent for the clinical application of allogeneic CAR-T cell therapy. In this review, we summarize the current approaches to overcome GvHD and host rejection, which empower allogeneic CAR-T cell therapy with a broader future.

Keywords: T cell subsets; allogeneic CAR-T cell; gene-editing technology; non-gene editing technology; pluripotent stem cell.

Figure 1

Comparison of clinic manufacturing process between autologous and allogeneic CAR-T cells. The above (red) represents the process flows of patient-derived autologous CAR-T cells. T lymphocytes are firstly enriched and activated from peripheral blood mononuclear cells. Afterwards, CARs are introduced by viral transduction and the obtained CAR-T cells are expanded and frozen, which are available for the patient’s own use after reviving. The below (blue) represents the process flows of healthy donor-derived allogeneic CAR-T cells. T lymphocytes are same enriched and activated, followed by elimination of TCR at the time of CAR introduction. The TCR-negative CAR-T cells are then purified from expanded cell population and the final frozen cells are available for different patients.

Figure 2

Gene editing technologies for overcoming GvHD and host-mediated allorejection. The left (A) represents the strategy to overcome GvHD that inserting CAR gene into the TRAC/TRBC loci. The right (B–F) represents the strategies to overcome host rejection, showing a combination strategy by removing CD52 in CAR-T cells with lymphodepletion through Alemtuzumab (B), knockout of CD7 in CAR-T cells when treating CD7-positive T cell malignancies (C), HLA-E insertion in one allele of B2M while another allele is knocked out (D), construction of HLA-C-retained cells that disrupts both HLA-A and HLA-B alleles (E), and knockout of transcription factors like RFX5/CIITA to achieve elimination of HLA-II (F). TRAC, TCR α constant region; TRBC, TCR β constant region.

Figure 3

Non-gene editing technologies for overcoming GvHD and host-mediated allorejection. The above (A–C) represents the strategies to overcome GvHD, illustrating the knockdown of TCR complex through CD3ζ mRNA silencing (A), the downregulation of TCR complex through expressing a CD3ϵ-specific PEBL that retaining the complex in cytoplasm (B), and the inhibition of TCR signaling by expressing TIM molecules that can competitively replace CD3ζ when binding αβ TCR (C). The below (D) represents the strategies to overcome host rejection, in which approach 1-4 show the expression of ADR for evading the killing of host T and NK cells, the expression of NKi to inhibit the activation of host NK cells and thus to reduce NK cell-mediated cytotoxicity, the overexpression of CD47 to escape host NK cell-mediated rejection, the overexpression of CD64 to escape antibody-mediated rejection, respectively. The symbol “*” represents the general schematic of a molecule (e.g., labeled virus particles (blue) in A) and its specific structure or composition (e.g., labeled white box in A).

Figure 4

Two novel differentiation systems for generation T cells from pluripotent stem cells. The above (red) briefly summarizes the flow of the 3D organoid culture system. EMPs differentiated by PSCs are first co-cultured with DL4-expressing MS5 cells to form EMOs. After hematopoietic induction, ATOs are then formed. Finally, T-cell differentiation is fulfilled to generate single-positive T cells. The below (blue) represents the serum-free and stroma-free culture system. PSCs are first differentiated into EBs. CD34-positive HEs are then obtained by dissociating EBs, which later gradually differentiate into progenitor T cells, double-positive T cells and finally mature as single-positive T cells. EMPs, embryonic mesodermal progenitor cells; EMOs, embryonic mesodermal organoids; ATOs, artificial thymus organoids; EBs, embryoid bodies; HEs, hemogenic endothelial cells.