Optimization of universal allogeneic CAR-T cells combining CRISPR and transposon-based technologies for treatment of acute myeloid leukemia

Abstract

Despite the potential of CAR-T therapies for hematological malignancies, their efficacy in patients with relapse and refractory Acute Myeloid Leukemia has been limited. The aim of our study has been to develop and manufacture a CAR-T cell product that addresses some of the current limitations. We initially compared the phenotype of T cells from AML patients and healthy young and elderly controls. This analysis showed that T cells from AML patients displayed a predominantly effector phenotype, with increased expression of activation (CD69 and HLA-DR) and exhaustion markers (PD1 and LAG3), in contrast to the enriched memory phenotype observed in healthy donors. This differentiated and more exhausted phenotype was also observed, and corroborated by transcriptomic analyses, in CAR-T cells from AML patients engineered with an optimized CAR construct targeting CD33, resulting in a decreased in vivo antitumoral efficacy evaluated in xenograft AML models. To overcome some of these limitations we have combined CRISPR-based genome editing technologies with virus-free gene-transfer strategies using Sleeping Beauty transposons, to generate CAR-T cells depleted of HLA-I and TCR complexes (HLA-I^KO/TCR^KO CAR-T cells) for allogeneic approaches. Our optimized protocol allows one-step generation of edited CAR-T cells that show a similar phenotypic profile to non-edited CAR-T cells, with equivalent in vitro and in vivo antitumoral efficacy. Moreover, genomic analysis of edited CAR-T cells revealed a safe integration profile of the vector, with no preferences for specific genomic regions, with highly specific editing of the HLA-I and TCR, without significant off-target sites. Finally, the production of edited CAR-T cells at a larger scale allowed the generation and selection of enough HLA-I^KO/TCR^KO CAR-T cells that would be compatible with clinical applications. In summary, our results demonstrate that CAR-T cells from AML patients, although functional, present phenotypic and functional features that could compromise their antitumoral efficacy, compared to CAR-T cells from healthy donors. The combination of CRISPR technologies with transposon-based delivery strategies allows the generation of HLA-I^KO/TCR^KO CAR-T cells, compatible with allogeneic approaches, that would represent a promising option for AML treatment.

Keywords: AML; CRISPR; allogeneic CAR-T; transcriptomics (RNA sequencing); transposon.

Figure 1

Phenotypic characterization of T cells from AML patients. (A) FACS analysis of T cell phenotype in AML patients (n=21), adult (below 30 years; n=5) and aged matched (senior; n=5) healthy donors. T cell subpopulations within CD4⁺ and CD8⁺ cells are depicted. T_N: naïve; T_SCM: stem central memory; T_CM: central memory; T_EM: effector memory; T_E: effector. (B) Percentage of Naïve (left) and effector (right) T cell subpopulations in CD8⁺ T cells from AML patients (n=21), adult (n=5) and senior (n=5) healthy donors. (C) Analysis of the expression of CD69, HLA-DR, PD1 and LAG3 in CD8⁺ T cells from AML patients (n=21), adult (n=5) and senior (n=5) healthy donors. Mean ± SEM for each group is depicted. Kruskal-Wallis test with Dunn’s multiple comparisons test. ns, not significant; *p<0.05; **p<0.01; ***p<0.001.

Figure 2

Phenotypic characterization of CD33-CAR-T cells from AML patients. (A) Population doublings of CAR-T cells generated from AML patients (n=7), adult (n=5) and senior (n=5) healthy donors during CAR-T cell production. (B) Percentage of transduced cells (CAR⁺) at the end of each CAR-T cell production. (C) Analysis of the phenotype of CAR-T cells at resting state for each group. CAR-T cell subpopulations within CD4⁺ and CD8⁺ cells are depicted. T_N, naïve; T_SCM, stem central memory; T_CM, central memory; T_EM, effector memory; T_E, effector. (D) Analysis of the expression of CD69, HLA-DR, PD1 and LAG3 in CD8⁺ T cells from AML patients (n=7), adult (n=5) and senior (n=5) healthy donors. Mean ± SEM for each group is depicted. 2-way ANOVA with Tukey’s multiple comparisons test (A), Kruskal-Wallis test with Dunn’s multiple comparisons test (B, D). ns, not significant; *p<0.05; **p<0.01.

Figure 3

Functional characterization of CD33-CAR-T cells from AML patients. (A) Quantification of the cytotoxic activity of CAR-T and UTD cells generated from AML patients (n=7), adult (n=5) and senior (n=5) healthy donors, against CD33⁺ (left) and CD33 knock-out (right) MOLM-13 AML cell line at different E:T ratio. The percentage of specific lysis for each CAR-T cell production is depicted. (B) Quantification of IFN-γ levels in supernatants from cytotoxic assays (ratio 1:3) measured by ELISA. The cytokine concentration (ng/ml) for each CAR-T cell production is depicted. Analysis of the expression of PD1 and LAG3 in CD4⁺ (C) and CD8⁺ (D) CAR-T cells from AML patients, adult, and senior healthy donors, before (basal) and after continuous repeated in vitro stimulation (reest) for 21 days with MOLM-13 tumoral cells. (E) Cytotoxic activity of CAR-T cells from AML patients (n=7), adult (n=5) and senior (n=5) healthy donors after continuous repeated in vitro stimulation for 21 days with MOLM-13 tumoral cells. (F) Survival of mice treated with CAR-T cells from AML patients, adult, and senior healthy donors. Untreated animals or treated with UTD cell form same groups were use as control. All groups included 12 animals (6 male and 6 female). Mean ± SEM of the average of three technical replicates for each group is depicted. Kruskal-Wallis test with Dunn’s multiple comparisons test (C, D), 2-way ANOVA with Tukey’s multiple comparisons test (E), Logrank test (F). ns, not significant; *p<0.05; **p<0.01; ***p<0.001.

Figure 4

Transcriptomic characterization of CD33-CAR-T cells from AML patients. The transcriptomic landscape of CAR-T cells generated from AML patients (n=4), adult (n=3) and senior (n=3) healthy donors was profiled using high-throughput RNA sequencing (RNA-seq) (A) RNA-seq principal components (PC) analysis, corrected by patient heterogeneity, of sorted CD4⁺ and CD8⁺ CAR-T cell subsets. Percentage of variance explained by PC1 and PC2 are depicted. (B) Left: Heatmap of differentially expressed genes associated to stem cell memory and T cell activation shared between CD8⁺ CAR-T cells from AML patients and senior healthy donors (age-related) compared to adult CAR-T cells. Right: Quantification of CD28 and CIITA gene expression. (C) Left: Heatmap of differentially expressed genes specific for CD8⁺ CAR-T cells from AML patients (AML-specific) compared to adult and senior CAR-T cells. Right: Quantification of LAG3, NR4A1, CCR7 and OAS1 gene expression. (D) Quantification of CD81, CCL5, IRF1 and KLF2 gene expression as example of genes with disrupted expression pattern in AML CAR-T cells after stimulation with tumoral cells. Mean ± SEM for each group is depicted. Kruskal-Wallis test with Dunn’s multiple comparisons test (B, C), 2-way ANOVA with Tukey’s multiple comparisons test (D). ns, not significant; *p<0.05; **p<0.01; ***p<0.001.

Figure 5

Characterization of HLA-I^KO/TCR^KO CD33-CAR-T cells. Selected CRISPR RNPs were combined with the Sleeping Beauty transposon system to generate HLA-I^KO/TCR^KO CD33-CAR-T (CAR-T^KO) cells from healthy donors (n=8 independent samples) (A) Distribution of the different edited populations observed after simultaneous HLA-I and TCR targeting of CAR-T and UTD cells with CRISPR systems. (B) Percentage of transduced cells (CAR⁺) at the end of CAR-T and CAR-T^KO cell production. (C) Percentage of HLA-I^KO/TCR^KO double negative cells in CAR-T and UTD cells before and after selection. (D) Percentage of transduced cells (CAR⁺) at the CAR-T^KO cell production before and after selection of HLA-I^KO/TCR^KO double negative cells. (E) Population doublings of CAR-T and CAR-T^KO cells during CAR-T cell production. UTD and UTD^KO cells were use as control. (F) Analysis of CD4/CD8 ratio in CAR-T and CAR-T^KO cells. (G) Analysis of the phenotype of CAR-T and CAR-T^KO cells at resting state for each group. CAR-T cell subpopulations within CD4⁺ and CD8⁺ cells are depicted. T_N, naïve; T_SCM, stem central memory; T_CM, central memory; T_EM, effector memory; T_E, effector. (H) Quantification of the cytotoxic activity of CAR-T and CAR-T^KO cells against CD33⁺ MOLM-13 AML cell line at different E:T ratio. The percentage of specific lysis (average of three technical replicates) for each CAR-T cell production is depicted. UTD and UTD^KO cells were used as control. (I) Survival of mice treated with CAR-T and CAR-T^KO. Untreated animals or treated with UTD and UTD^KO cells were used as control. Mean ± SEM for each group is depicted. Kruskal-Wallis test with Dunn’s multiple comparisons test (B, D), 2-way ANOVA with Tukey’s multiple comparisons test (E, H), Logrank test (I). ns, not significant; *p<0.05; ***p<0.001.

Figure 6

Safety analysis of HLA-I^KO/TCR^KO CD33-CAR-T cells. (A) Analysis of the SB copy number integrations in CAR-T and CAR-T^KO cells (n=8 independent productions). (B) Histogram plot showing the genomic annotation of SB integration sites in CAR-T and CAR-T^KO cells (n=3 independent productions. (C) Sequences of cleavage sites identified by iGUIDE for B2M (left) and TRAC (right) sgRNAs annotated by on target or off target, with the total number of unique alignments associated with the site. Wilcoxon matched-pairs signed rank test (A). ns, not significant.

Figure 7

Preclinical production of HLA-I^KO/TCR^KO CD33-CAR-T cells. (A) Percentage of HLA-I^KO/TCR^KO double negative cells before and after selection (n=3 independent productions). (B) Percentage of transduced cells (CAR⁺) at the CAR-T^KO cell production after selection of HLA-I^KO/TCR^KO double negative cells (n=3 independent productions). (C) Population doublings of CAR-T^KO cells during CAR-T cell production (n=3 independent productions). (D) Quantification of total number of CAR-T^KO cells obtained at the end of the production after HLA-I^KO/TCR^KO double negative selection (n=3 independent productions). (E) Quantification of the cytotoxic activity of CAR-T^KO cells against CD33⁺ MOLM-13 AML cell line at different E:T ratio. The percentage of specific lysis (average of three technical replicates) for each CAR-T cell productions (n=3) is depicted. (F) Quantification of IFN-γ levels in supernatants from cytotoxic assays (ratio 1:3) measured by ELISA. The cytokine concentration (ng/ml; average of three technical replicates) for each CAR-T cell production (n=3) is depicted. Mann Whitney test (B, C, F), 2-way ANOVA with Tukey’s multiple comparisons test (E). ns, not significant.