Phase I trials using Sleeping Beauty to generate CD19-specific CAR T cells

Abstract

Background: T cells expressing antigen-specific chimeric antigen receptors (CARs) improve outcomes for CD19-expressing B cell malignancies. We evaluated a human application of T cells that were genetically modified using the Sleeping Beauty (SB) transposon/transposase system to express a CD19-specific CAR.

Methods: T cells were genetically modified using DNA plasmids from the SB platform to stably express a second-generation CD19-specific CAR and selectively propagated ex vivo with activating and propagating cells (AaPCs) and cytokines. Twenty-six patients with advanced non-Hodgkin lymphoma and acute lymphoblastic leukemia safely underwent hematopoietic stem cell transplantation (HSCT) and infusion of CAR T cells as adjuvant therapy in the autologous (n = 7) or allogeneic settings (n = 19).

Results: SB-mediated genetic transposition and stimulation resulted in 2,200- to 2,500-fold ex vivo expansion of genetically modified T cells, with 84% CAR expression, and without integration hotspots. Following autologous HSCT, the 30-month progression-free and overall survivals were 83% and 100%, respectively. After allogeneic HSCT, the respective 12-month rates were 53% and 63%. No acute or late toxicities and no exacerbation of graft-versus-host disease were observed. Despite a low antigen burden and unsupportive recipient cytokine environment, CAR T cells persisted for an average of 201 days for autologous recipients and 51 days for allogeneic recipients.

Conclusions: CD19-specific CAR T cells generated with SB and AaPC platforms were safe, and may provide additional cancer control as planned infusions after HSCT. These results support further clinical development of this nonviral gene therapy approach.

Trial registration: Autologous, NCT00968760; allogeneic, NCT01497184; long-term follow-up, NCT01492036.

Funding: National Cancer Institute, private foundations, and institutional funds. Please see Acknowledgments for details.

Figure 1. High-throughput sequencing reveals distribution and genomic location of CAR integrants after SB-mediated transposition in primary T cells.

(A) The integration site for CAR insertions was determined in genomic DNA libraries (n = 33) isolated from independent T cell populations following genetic modification with the SB system and propagation on AaPCs with cytokines to stably express CD19RCD28 CAR. From greater than 7 million raw sequences, 696,059 nonredundant sequences were obtained. The inverted repeats (IRs) and direct repeats (DRs) were digitally removed, leaving 571,533 unique integrations, of which 100,000 were mapped. The percentages in AT-rich regions and in intragenic regions are displayed in the right-hand graphics. The green bar and associated text display the percentage of intragenic integrations located in introns (light green) vs. exons (dark green). (B) The location of each SB integration (CAR) is mapped onto the 23 human chromosome scaled (as shown in black and white inset) representations. Boxes denote constitutive heterochromatic regions that could not be analyzed. Each integration is noted with a bar (chromosomes 1, 2, and 23) or diamond. Integrations are widely dispersed throughout the genome, without hotspots.

Figure 2. Characterization of genetically modified T cells after electrotransfer of SB plasmids to introduce CAR and coculture on AaPCs with cytokines.

(A) Lines represent the numerical expansion of SB-transformed CAR T cells cultured on AaPCs in the presence of cytokines. Blue bars represent exposure to IL-21 in culture, and green bars IL-2. Red arrows indicate the addition of γ-irradiated AaPCs to the coculture. Allogeneic and autologous samples are presented in the left and right panels, respectively, as labeled. Each line represents a specific study patient sample. (B) Graph represents CAR copy number per cell of genetically modified allogeneic (n = 19) and autologous (n = 7) CAR T cells for patients by qPCR. Bar, average ± SD. (C) The plot on the left shows the percentage of modified and expanded lymphocytes expressing CD3 for allogeneic (left side) and autologous (right side) lymphocytes prior to infusion. The heavy line for all similar panels reflects the mean value and the whiskers represent the SD. Numbers beside each marker identify the subject whose data are represented. The percentage of CD3⁺ lymphocytes expressing CD8 (middle panel) and CD4 (right panel) is shown. (D) PCA 3D plot of the first 3 principal components of the 41 differentially expressed genes in 6 samples; the first 3 principal components account for approximately 98.1% of the total variance (90.5%, 4.8%, and 2.8%) (red, day 0; green, day 28). (E) Cluster analysis of differentially expressed genes by gene pattern is shown, with up- and downregulated genes in culture-day-28 cells compared to unmanipulated culture-day-0 CD3⁺ T cells. Red, highest level of expression; white/pink, moderate level; blue, lowest level of expression.

Figure 3. Survival of patients after HSCT after planned infusion of CAR T cells.

(A) Flow chart describing numbers of autologous and allogeneic patients enrolled in the 2 trials (NCT00968760 and NCT01497184, respectively). Enrollment occurred to obtain peripheral blood for manufacture of the genetically modified T cells and then the prospective patients were required to meet eligibility to administer the T cells. Some potential recipients were deemed ineligible to receive a successfully manufactured product, which highlights the medical fragility and advanced malignant disease of the enrollees. Some potential recipients were unable to receive T cells due to difficulties associated with their manufacture. All patients who received genetically modified T cells were included in survival analyses. Overall (blue line) and progression-free (red line) survival for (B) autologous as well as (C) allogeneic recipients and (D) the subset of allogeneic recipients from haploidentical donors.

Figure 4. Sustained persistence of infused genetically modified T cells determined by qPCR and ddPCR in serially collected PBMCs after single infusion of genetically modified T cells.

DNA from PBMCs from (A) autologous and (B) allogeneic recipients was tested by qPCR for the presence of CAR sequences. Values at each time point are the average transgene copy number with standard error from all available patient samples at that time point.

Figure 5. Cytokine milieu at the time of planned T cell infusions.

(A) Expression of cytokine receptors on administered T cells. The percentage of expanded CAR T cells expressing IL-15R (left panel) and IL-21R (right panel), determined by flow cytometry, is shown. Data are displayed as per similar panels of Figure 2. (B) Abundance of soluble cytokines that signal through the common cytokine receptor γ chain. The box and whiskers plots display the mean levels of each cytokine (pg/ml) in patient sera, while dots reflect the levels from individual patients, 12 hours after single CAR T cell infusions. Data from autologous and allogeneic recipients are shown on the left and right, respectively.