Development of a compact bidirectional promoter-driven dual chimeric antigen receptor (CAR) construct targeting CD19 and CD20 in the Sleeping Beauty (SB) transposon system

Abstract

Background: A bidirectional promoter-driven chimeric antigen receptor (CAR) cassette provides the simultaneous expression of two CARs, which significantly enhances dual antigen-targeted CAR T-cell therapy.

Methods: We developed a second-generation CAR directing CD19 and CD20 antigens, incorporating them in a head-to-head orientation from a bidirectional promoter using a single Sleeping Beauty transposon system. The efficacy of bidirectional promoter-driven dual CD19 and CD20 CAR T cells was determined in vitro against cell lines expressing either, or both, CD19 and CD20 antigens. In vivo antitumor activity was tested in Raji lymphoma-bearing immunodeficient NOD-scid IL2Rgamma^null (NSG) mice.

Results: Of all tested promoters, the bidirectional EF-1α promoter optimally expressed transcripts from both sense (CD19-CAR) and antisense (GFP.CD20-CAR) directions. Superior cytotoxicity, cytokine production and antigen-specific activation were observed in vitro in the bidirectional EF-1α promoter-driven CD19/CD20 CAR T cells. In contrast, a unidirectional construct driven by the EF-1α promoter, but using self-cleaving peptide-linked CD19 and CD20 CARs, showed inferior expression and in vitro function. Treatment of mice bearing advanced Raji lymphomas with bidirectional EF-1α promoter-driven CD19/CD20 CAR T cells effectively controlled tumor growth and extended the survival of mice compared with group treated with single antigen targeted CAR T cells.

Conclusion: The use of bidirectional promoters in a single vector offers advantages of size and robust CAR expression with the potential to expand use in other forms of gene therapies like CAR T cells.

Keywords: Cell Engineering; Hematologic Neoplasms; Immunotherapy, Adoptive; Receptors, Chimeric Antigen.

Figure 1

Different bidirectional promoter-driven dual CD19/20 CAR T-cell generation. (A) Schematic illustration of the Sleeping Beauty backbone bearing four different eukaryotic promoters (EF-1α, hPGK, RPBSA and CMV). (B) Schematic design of CAR T-cell generation. (C) Flow cytometry plot representing forward CAR19 expression on day 14 after transfection. (D) Flow cytometry plot representing GFP expression on day 14 after transfection. (E) Flow cytometry plot representing double CAR19⁺ and GFP⁺ cell per cent expression on day 14 after transfection. (F) Transposition efficiency of CAR19, GFP and CAR19⁺GFP⁺ on day 14 after transfection. (G) Mean fluorescent intensity of CAR19 and GFP on day 14 after transfection. All results are summarized as the mean±SEM from at least three independent healthy blood donors. Significant differences were determined by two-way analysis of variance, and asterisks indicate significant p values as follows: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. CAR, chimeric antigen receptor; CD, cluster of differentiation; FBS, fetal bovine serum; GFP, green fluorescent protein; IL, interleukin; P2A, 2A self-cleaving peptide; PBMCs, peripheral blood mononuclear cells; TM, transmembrane.

Figure 2

Bidirectional CD19/20 CAR T-cell expansion and phenotype. (A) Expansion of total cell numbers. Cell numbers were determined by trypan blue exclusion assay on day 0, day 7 and day 14 after cell transfection. (B) Mean percentages of helper CD3⁺/CD4⁺ T cells and cytotoxic CD3⁺/CD8⁺ T cells on day 14 after transfection. (C) Flow cytometric representation of CD3⁺ T-cell memory phenotypes. (D) Memory phenotype of CD3+T cells on day 14 after transfection showing the percentage of naïve (T_N), central memory (T_CM), effector memory (T_EM), and terminal effector (T_E) T cells. All results are summarized as the mean±SEM from at least three independent healthy blood donors. Significant differences were determined by Student’s t-tests and two-way analysis of variance. Asterisks indicate significant p values as follows: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 3

Bidirectional CD19/20 CAR T-cell cytotoxic function in vitro. Flow cytometry-based cytotoxicity assay were performed using (A) Nalm-6, (B) Raji, (C) CD19-K562 and (D) CD20-K562. CAR T cells and target cells were co-cultured overnight at different E:T ratios, X-axis. All results are summarized as the mean±SEM from at least three independent healthy blood donors. Significant differences were determined by two-way analysis of variance. Asterisks indicate significant p values as follows: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. CAR, chimeric antigen receptor; E:T, effector to target.

Figure 4

Comparisons of CAR20.GFP.EF1.CAR19, EF1.GFP.CAR19.CAR20 and EF1.CAR19 CAR T-cell efficiencies. (A) Schematic illustration of CAR20.GFP.EF1.CAR19 (B) Schematic illustration of EF1.GFP.CAR19.CAR20 (C) Schematic illustration of EF1.CAR19. (D) Flow cytometry plot representing CAR19 expression from CAR20.GFP.EF1.CAR19, EF1.GFP.CAR19.CAR20 and EF1.CAR19-transfected CD3⁺ T cells. (E) Flow cytometry plot representing GFP expression from CAR20.GFP.EF1.CAR19, and EF1.GFP.CAR19.CAR20-transfected CD3⁺ T cells. (F) Flow cytometry plot representing double CAR19⁺ and GFP⁺ cells. (G) Transfection efficiency of CAR19−, GFP− and CAR19⁺GFP⁺ cells on day 14 after transfection. (H) MFI of CAR19-positive and GFP-positive cells on day 14 after transfection. (I) Transfection efficiency of CAR19−, GFP− and CAR19⁺GFP⁺ cells on day 30 after transfection. (J) MFI of CAR19-positive and GFP-positive cells on day 30 after transfection. All results are summarized as the mean±SEM from at least three independent healthy blood donors. Significant differences were determined by Student’s t-test and two-way analysis of variance. Asterisks indicate significant p values as follows: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. CAR, chimeric antigen receptor; GFP, green fluorescent protein; MFI, mean fluorescent intensity; P2A, 2A self-cleaving peptide.

Figure 5

Comparisons of the cytolytic activity of CAR20.GFP.EF1.CAR19, EF1.GFP.CAR19.CAR20 and EF1.CAR19 CAR T cells. (A) Fluorescence microscopy of K562 cells expressing dTomato. (B) Flow cytometric representation of CAR20.GFP.EF1.CAR19, EF1.GFP.CAR19.CAR20 and EF1.CAR19 CAR T cells co-cultured with Raji cells. (C) Per cent killing of CAR20.GFP.EF1.CAR19, EF1.GFP.CAR19.CAR20 and EF1.CAR19 CAR T cells to Raji cell line after 24 and 48 hours of co-culture. (D) Flow cytometric representation of CAR20.GFP.EF1.CAR19, EF1.GFP.CAR19.CAR20 and EF1.CAR19 CAR T cells co-cultured with CD19-K562 cells. (E) Flow cytometric representation of CAR20.GFP.EF1.CAR19, EF1.GFP.CAR19.CAR20 and EF1α. CAR19 CAR T cells co-cultured with CD20-K562 cells. (F) Per cent killing of CAR20.GFP.EF1.CAR19, EF1.GFP.CAR19.CAR20 and EF1.CAR19 CAR T cells to CD19-K562, CD20-K562 and only K562 after 48 hours of co-culture. (G) Flow cytometric representation of CAR20.GFP.EF1.CAR19, EF1.GFP.CAR19.CAR20 and EF1.CAR19 CAR T cells co-cultured with mixed cultures of CD19-K562 and CD20-K562, CD19-K562 and CD20-K562 cells possessed dTomato; however, CD19-K562 cells were stained with anti-CD19 antibody, cells positive for anti-CD19 antibody and dTomato were CD19-K562, and only dTomato-positive cells were CD20-K562 (as represented in the template). All results are summarized as the mean±SE from at least three independent healthy blood donors. Significant differences were determined by one-way analysis of variance, two-way analysis of variance and Student’s t-test, and asterisks indicate significant p values as follows: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. CAR, chimeric antigen receptor; GFP, green fluorescent protein.

Figure 6

Cytokine production, activation and exhaustion markers of CAR20.GFP.EF1.CAR19, EF1.GFP.CAR19.CAR20 and EF1.CAR19 CAR T cells in response to Raji cells. (A) Secretion levels of the cytokines IL-2, IFN-γ and TNF-α from CAR20.GFP.EF1.CAR19, EF1.GFP.CAR19.CAR20 and EF1.CAR19 CAR T cells. (B) Expression of the early activation marker CD69 in CD3⁺ T cells before and after co-culture. (C) Expression of the late activation marker CD25 in CD3⁺ T cells before and after co-culture. (D) Expression of TIM-3 in CD3⁺ T cells before 24 hours of co-culture. (E) LAG-3 expression in CD3+T cells before 24 hours of co-culture. (F) PD-1 expression in CD3⁺ T cells before 24 hours of co-culture. (G) TIGIT expression in CD3⁺ T cells before 24 hours of co-culture. All results are summarized as the mean±SEM from at least three independent healthy blood donors. Significant differences were determined by Student’s t-test or two-way analysis of variance, and asterisks indicate significant p values as follows: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. CAR, chimeric antigen receptor; GFP, green fluorescent protein; IFN, interferon; IL, interleukin; PD-1, Programmed cell death protein 1; LAG-3, Lymphocyte activation gene-3; TIGIT, T cell immunoreceptor with Ig and ITIM domains; TIM-3, T cell immunoglobulin and mucin domain-containing protein 3; TNF, tumor necrosis factor.

Figure 7

In vivo activity of CAR T constructs. (A) Schematic diagram of the in vivo experiment. (B) Bioluminescent images of the tumor burden in mice treated with CAR20.GFP.EF1.CAR19 and EF1.CAR19 CAR T constructs on days 5, 13, 20, 27, and 34 post tumor injection. (C) Bioluminescent intensity of total flux photons/second, post tumor injection. Six mice per group were studied. The results are summarized as the mean±SD. Statistical differences were determined by two-way analysis of variance. (D) Fold change in total flux post-CAR T-cell injection. Statistical differences were analyzed by multiple t-tests. (E) Kaplan-Meier survival curve. Statistical differences were analyzed by the Gehan-Breslow-Wilcoxon test. Asterisks indicate significant p values as follows: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. CAR, chimeric antigen receptor; GFP, green fluorescent protein; IV, intravenous.; SC, subcutaneous.