Combining CD34+ stem cell selection with prophylactic pathogen and leukemia directed T-cell immunotherapy to simultaneously reduce graft versus host disease, infection, and leukemia recurrence after allogeneic stem cell transplant.

We designed a trial to simultaneously address the problems of graft versus host disease (GVHD), infection, and recurrence of malignancy after allogeneic stem cell transplantation. CD34+ stem cell isolation was used to minimize the development of acute and chronic GVHD. Two prophylactic infusions, one combining donor-derived cytomegalovirus, Epstein-Barr virus, and Aspergillus fumigatus specific T-cells and the other comprising donor-derived CD19 directed chimeric antigen receptor (CAR) bearing T-cells, were given 21-28 days after transplant. Two patients were transplanted for acute lymphoblastic leukemia from HLA identical siblings using standard doses of cyclophosphamide and total body irradiation without antilymphocyte globulin. Patients received no post-transplant immune suppression and were given no pre-CAR T-cell lymphodepletion. Neutrophil and platelet engraftment was prompt. Following adoptive T-cell infusions, there was rapid appearance of antigen-experienced CD8+ and to a lesser extent CD4+ T-cells. Tetramer-positive T-cells targeting CMV and EBV appeared rapidly after T-cell infusion and persisted for at least 1 year. CAR T-cell expansion occurred and persisted for up to 3 months. T-cell receptor tracking confirmed the presence of product-derived T-cell clones in blood targeting all three pathogens. Both patients are alive over 3 years post-transplant without evidence of GVHD or disease recurrence. Combining robust donor T-cell depletion with directed T-cell adoptive immunotherapy targeting infectious and malignant antigens permits independent modulation of GVHD, infection, and disease recurrence. The combination may separate GVHD from the graft versus tumor effect, accelerate immune reconstitution, and improve transplant tolerability.

Investigation of product-derived lymphoma following infusion of piggyBac-modified CD19 chimeric antigen receptor T cells.

We performed a phase 1 clinical trial to evaluate outcomes in patients receiving donor-derived CD19-specific chimeric antigen receptor (CAR) T cells for B-cell malignancy that relapsed or persisted after matched related allogeneic hemopoietic stem cell transplant. To overcome the cost and transgene-capacity limitations of traditional viral vectors, CAR T cells were produced using the piggyBac transposon system of genetic modification. Following CAR T-cell infusion, 1 patient developed a gradually enlarging retroperitoneal tumor due to a CAR-expressing CD4+ T-cell lymphoma. Screening of other patients led to the detection, in an asymptomatic patient, of a second CAR T-cell tumor in thoracic para-aortic lymph nodes. Analysis of the first lymphoma showed a high transgene copy number, but no insertion into typical oncogenes. There were also structural changes such as altered genomic copy number and point mutations unrelated to the insertion sites. Transcriptome analysis showed transgene promoter-driven upregulation of transcription of surrounding regions despite insulator sequences surrounding the transgene. However, marked global changes in transcription predominantly correlated with gene copy number rather than insertion sites. In both patients, the CAR T-cell-derived lymphoma progressed and 1 patient died. We describe the first 2 cases of malignant lymphoma derived from CAR gene-modified T cells. Although CAR T cells have an enviable record of safety to date, our results emphasize the need for caution and regular follow-up of CAR T recipients, especially when novel methods of gene transfer are used to create genetically modified immune therapies. This trial was registered at www.anzctr.org.au as ACTRN12617001579381.

CAR T Cell Generation by piggyBac Transposition from Linear Doggybone DNA Vectors Requires Transposon DNA-Flanking Regions.

CD19-specific chimeric antigen receptor (CAR19) T cells, generated using viral vectors, are an efficacious but costly treatment for B cell malignancies. The nonviral piggyBac transposon system provides a simple and inexpensive alternative for CAR19 T cell production. Until now, piggyBac has been plasmid based, facilitating economical vector amplification in bacteria. However, amplified plasmids have several undesirable qualities for clinical translation, including bacterial genetic elements, antibiotic-resistance genes, and the requirement for purification to remove endotoxin. Doggybones (dbDNA) are linear, covalently closed, minimal DNA vectors that can be inexpensively produced enzymatically in vitro at large scale. Importantly, they lack the undesirable features of plasmids. We used dbDNA incorporating piggyBac to generate CAR19 T cells. Initially, expression of functional transposase was evident, but stable CAR expression did not occur. After excluding other causes, additional random DNA flanking the transposon within the dbDNA was introduced, promoting stable CAR expression comparable to that of using plasmid components. Our findings demonstrate that dbDNA incorporating piggyBac can be used to generate CAR T cells and indicate that there is a requirement for DNA flanking the piggyBac transposon to enable effective transposition. dbDNA may further reduce the cost and improve the safety of CAR T cell production with transposon systems.

PiggyBac-Engineered T Cells Expressing CD19-Specific CARs that Lack IgG1 Fc Spacers Have Potent Activity against B-ALL Xenografts.

Clinical trials of CD19-specific chimeric antigen receptor (CAR19) T cells have demonstrated remarkable efficacy against relapsed and refractory B cell malignancies. The piggyBac transposon system offers a less complex and more economical means for generating CAR19 T cells compared to viral vectors. We have previously optimized a protocol for the generation of CAR19 T cells using the piggyBac system, but we found that CAR19 T cells had poor in vivo efficacy and persistence, probably due to deleterious FcγR interactions with the CAR's IgG1 Fc-containing spacer domain. We therefore designed three CD19-specifc CARs that lacked the IgG1 Fc region, and we incorporated combinations of CD28 or 4-1BB transmembrane and co-stimulatory domains. PiggyBac-generated CAR19 T cells expressing these re-designed constructs all demonstrated reactivity in vitro specifically against CD19+ cell lines. However, those combining CD28 transmembrane and co-stimulatory domains showed CD4 predominance and inferior cytotoxicity. At high doses, CAR19 T cells were effective against B-ALL in a xenograft mouse model, regardless of co-stimulatory domain. At diminishing doses, 4-1BB co-stimulation led to greater potency and persistence of CAR19 T cells, and it provided protection against B-ALL re-challenge. Production of potent CAR T cells using piggyBac is simple and cost-effective, and it may enable wider access to CAR T cell therapy.