Genome editing of donor-derived T-cells to generate allogenic chimeric antigen receptor-modified T cells: Optimizing αβ T cell-depleted haploidentical hematopoietic stem cell transplantation

Abstract

Allogeneic hematopoietic stem cell transplantation is an effective therapy for high-risk leukemias. In children, graft manipulation based on the selective removal of aβ T cells and B cells has been shown to reduce the risk of acute and chronic graft-versus-host disease, thus allowing the use of haploidentical donors which expands the population of recipients in whom allogeneic hematopoietic stem cell transplantation can be used. Leukemic relapse, however, remains a challenge. T cells expressing chimeric antigen receptors can potently eliminate leukemia, including those in the central nervous system. We hypothesized that by engineering the donor aβ T cells that are removed from the graft by genome editing to express a CD19-specific chimeric antigen receptor, while simultaneously inactivating the T-cell receptor, we could create a therapy that enhances the anti-leukemic efficacy of the stem cell transplant without increasing the risk of graft-versus-host disease. Using genome editing with Cas9 ribonucleoprotein and adeno-associated virus serotype 6, we integrated a CD19-specific chimeric antigen receptor inframe into the TRAC locus. More than 90% of cells lost T-cell receptor expression, while >75% expressed the chimeric antigen receptor. The initial product was further purified with less than 0.05% T-cell receptorpositive cells remaining. In vitro, the chimeric antigen receptor T cells efficiently eliminated target cells and produced high cytokine levels when challenged with CD19+ leukemia cells. In vivo, the gene-modified T cells eliminated leukemia without causing graft-versus-host disease in a xenograft model. Gene editing was highly specific with no evidence of off-target effects. These data support the concept that the addition of aβ T-cell-derived, genome-edited T cells expressing CD19-specific chimeric antigen receptors could enhance the anti-leukemic efficacy of aβ T-celldepleted haploidentical hematopoietic stem cell transplantation without increasing the risk of graft-versus-host disease.

Figure 1.

T-cell therapy approaches in combination with T-cell receptor αβ⁺/CD19⁺-depleted haploidentical stem cell transplantation aiming to decrease relapse rates. (A) The protocol for haploidentical hematopoietic stem cell transplantation (HSCT) with TCRαβ⁺/CD19⁺-depletion, which establishes a backbone for additional cellular immunotherapies. (B) In order to improve immune reconstitution and enhance antileukemic activity, a specified number of T cells is transfused to the patient separate from the graft. (C) In order to retain control over the T cells and be able to intervene in the case of severe graft-versus-host disease (GvHD), the T cells can be transduced with a safeguard system such as herpes simplex virus-derived thymidine kinase or inducible caspase 9. (D) The αβT cells are removed from the graft before transplantation and can be used as starting material to create genome-edited chimeric antigen receptor (CAR) T cells by targeted integration of a CD19-CAR into the TRAC locus, in order to target residual leukemia after HSCT without causing GvHD. sgRNA: single guide RNA; CAR: chimeric antigen receptor; GvHD: graftversus- host disease; HSPC: hematopoietic stem and progenitor cells; TCR; T-cell receptor; NK cells: natural killer cells: TRAC: T-cell receptor alpha chain; HLA: human leukocyte antigen.

Figure 2.

Targeted integration of a CD19-specific chimeric antigen receptor into the TRAC locus. (A) Targeting strategy using Cas9 ribonucleoprotein and recombinant adeno-associated virus serotype 6. (B) Representative fluorescence activated cell sorting (FACS) plots for cells treated as indicated 4 days after targeting to evaluate the efficiency of T-cell receptor (TCR)αβknockout and nerve growth factor receptor (NGFR) expression. (C) Quantification of the populations after targeting of T cells from 11 different donors. (D) Representative FACS plot of the cells stained for NGFR and a CD19-CAR idiotype-specific antibody. (E) FACS plot showing NGFR and TCRαβexpression after depletion of cells expressing the αβT-cell receptor. (F) Quantification of αβTCR depletion efficiency for four different replicates, plotted as mean ± standard deviation. (G) Expansion of T cells during the 7 days after gene editing compared to numbers before electroporation using optimized conditions for adeno-associated virus transduction. (H) Expansion of T cells (compared to numbers before gene editing) cultured at different densities after electroporation. RNP: ribonucleoprotein; rAAV6: recombinant adeno-associated virus serotype 6; NGFR: nerve growth factor receptor; pA: poly-adenylation signal; 2A: 2A peptide.

Figure 3.

In vitro functionality of chimeric antigen receptor T cells engineered from αβ⁺ T cells. (A) Interleukin-2 and interferon-γ concentrations in cell culture supernatant after culture of control cells or chimeric antigen receptor (CAR) T cells alone, or co-cultures of CAR T cells with Nalm6 or Raji cells. Control cells were treated with ribonucleoprotein only (TRAC knockout without CAR expression). Bars and error bars represent mean ± standard deviation (SD) from three biological replicates. Asterisks depict levels of significance compared to control cells as analyzed by t tests. (B) In vitro cytotoxicity assay of CAR T cells co-cultured for 20 h with Nalm6 cells or Raji cells (both CD19⁺ and GFP⁺) at different effector-to-target ratios. Counts of viable cells were assessed for target cells co-cultured with control cells or CAR T cells and the fraction of target cells killed was calculated using samples without effector cells as reference. Bars and error bars represent means ± SD from three biological replicates and asterisks depict levels of significance (t tests). (C) Quantification of B cells for differentially treated cell populations on day 1 and day 4 after gene editing, for cell populations that had undergone gene targeting (RNP + AAV) or control treatments. Groups were compared by t tests and levels of significance are indicated by asterisks. (D-G) Phenotyping of the CAR T-cell product, gated on NGFR⁺ cells. (D) Distribution of CD4⁺ and CD8⁺ cells. (E) Expression of memory and effector T-cell markers among CD4⁺ and CD8⁺ cells. (F) Quantification of CD4/CD8 distribution from four biological replicates. Bars and error bars represent mean ± SD. (G) Quantification of the memory/effector populations on cells from four different donors. Bars represent mean ± SD. CAR: chimeric antigen receptor; IFN: interferon; IL: interleukin; RNP: ribonucleoprotein; AAV: adeno-associated virus, NGFR: nerve growth factor receptor.

Figure 4.

Antileukemic activity of genome-edited chimeric antigen receptor T cells in vivo. (A) Bioluminescence imaging of Nalm6 xenografts in NSG mice treated with genome-edited CD19-specific chimeric antigen receptor (CAR) T cells (1x10⁶) that were manufactured from αβ-TCR⁺ T cells. The experiment was repeated at the dose level of 5x10⁶ cells per mouse with comparable outcome. (B) Kaplan-Meier survival plot of mice treated with control T cells or CAR T cells. Asterisks indicate levels of significance of the CAR T-cell group compared to the respective control group (mock) of the same cell dose using log-rank tests. CAR: chimeric antigen receptor.

Figure 5.

Evaluation of endonuclease specificity. (A) Putative off-target (OT) sites in the human genome (hg38) determined by COSMID and sorted by predicted relevance in descending order. Mismatches to the target site are marked red. OT 1-37 have three relevant mismatches in the protospacer region without insertions/deoetopms and OT sites 38-40 have two relevant mismatches and a protospacer-adjacent motif (PAM) mismatch. The nucleotide furthest from the PAM was ignored for sorting due to mismatch tolerance at this location by Cas9. (B) Human T cells from six different donors were electroporated with the high-fidelity Cas9 protein complexed with the sgRNA targeting the TRAC locus (or mock electroporated to determine background). Next-generation sequencing was performed on all predicted OT sites. The dotted line depicts the sensitivity limit attributed to this method of 0.1%. sgRNA: single guide RNA, PAM: protospacer-adjacent motif; OT: off-target.