Genetic Ablation of HLA Class I, Class II, and the T-cell Receptor Enables Allogeneic T Cells to Be Used for Adoptive T-cell Therapy

Abstract

Adoptive immunotherapy can induce sustained therapeutic effects in some cancers. Antitumor T cell grafts are often individually prepared in vitro from autologous T cells, which requires an intensive workload and increased costs. The quality of the generated T cells can also be variable, which affects the therapy's antitumor efficacy and toxicity. Standardized production of antitumor T cell grafts from third-party donors will enable widespread use of this modality if allogeneic T cell responses are effectively controlled. Here, we generated HLA class I, class II, and T cell receptor triple knockout (tKO) T cells by simultaneous knockout of the B2M, CIITA and TRAC genes through Cas9/sgRNA ribonucleoprotein electroporation. Although HLA deficient T cells were targeted by natural killer cells, they persisted better than HLA sufficient T cells in the presence of allogeneic peripheral blood mononuclear cells (PBMC) in immunodeficient mice.When transduced with a CD19 chimeric antigen receptor (CAR) and stimulated by tumor cells, tKO CAR-T cells persisted better when cultured with allogeneic PBMC compared with TRAC and B2M double-knockout T cells. The CD19 tKO CAR-T cells did not induce graft-versus-host disease, but retained antitumor responses. These results demonstrated the benefit of HLA class I, class II, and TCR deletion in enabling allogeneic-sourced T cells to be used for off-the-shelf adoptive immunotherapy. SynopsisAntitumor T cell grafts concurrently ablated of HLA class I, class II, and TCR molecules evade allogeneic T cell responses and can be used as a universal T cell source for adoptive cancer immunotherapy.

Allogeneic CAR Cell Therapy—More Than a Pipe Dream

“…Kagoya et al. showed that B2M knockout to eliminate HLA class I as well as knockout of class II major histocompatibility complex transactivator (CIITA) to eliminate HLA class II improved CAR T cells persistence in vitro ( 79 ). Other strategies to mitigate immunogenicity rely on depleting resident immune cells.…”

Allogeneic CAR T Cells: An Alternative to Overcome Challenges of CAR T Cell Therapy in Glioblastoma

“…This issue probably could be avoided by genetic editing of the transcription factors regulatory factor X and CIITA (101,102). Allogeneic anti-CD19 CAR T cells triple-knockout for HLA class I (β2-microglobulin KO), class II (CIITA KO) and TCR (α-chain KO) showed better persistence than double-knockout (β2-microglobulin and TCR) cells in a mouse tumor model, with anti-tumor activity, but without GVHD (103). Other cells potentially mediating an allogeneic response are NK cells (104), even if NK were shown to be functionally impaired in some tumors, particularly from FIGURE 2 | Allogeneic CAR T cells must avoid host immune rejection and GVHD.…”

Current Perspectives on the Use of off the Shelf CAR-T/NK Cells for the Treatment of Cancer

“…To overcome this the HLA-class 1 molecules can be removed from the T cells [117]. To remove HLA-class 1 the Beta-2 microglobulin (B2M) gene locus can be disrupted as it is required for HLA-class 1 expression (Figure 2) [118][119][120]. Additionally, mechanisms utilised by viruses to evade immune recognition have been investigated.…”

“…Activated T cells also express HLA class II, which can be recognised by the host immune system and cause CAR T cell destruction [124]. Triple knockout of HLA class I, HLA class II, and T cell receptor by triple CRISPR/Cas9 targeting of the B2M, CIITA, and TRAC genes result in better persistence in in-vivo models compared to double knock out B2M and TRAC [119].…”