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

Abstract

We performed a Phase I clinical trial of donor derived CD19-specific chimeric antigen receptor T-cells (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, one patient developed a gradually enlarging retroperitoneal tumor due to a CAR expressing CD4+ T-cell lymphoma. Screening of other patients led to the detection of a second CAR T-cell tumor in thoracic para-aortic lymph nodes in an asymptomatic patient. 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 one patient died. We describe the first two 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. The trial was registered at www.anzctr.org.au as ACTRN12617001579381.

Gene editing to enhance the efficacy of cancer cell therapies

“…78 , 79 Furthermore, two cases of T cell lymphoma have been observed as a result of insertional mutagenesis using the Piggybac transposon platform. [80][81][82] These results verify that integration into host genes can lead to transformation and leukemogenesis and thereby confirm the risk of random vector integration. These safety issues, in addition to the expense and laborious manufacturing process involved in generating viral vectors, have led investigators to develop viral-free CRISPR-Cas9 platforms for gene delivery.…”

The evolving need for patient reported outcome data in determining choice of immune therapy treatment

“…Risk of secondary malignancies with CAR-T cellular therapy has been a concern with only rare cases of clonal malignancies outside of skin cancers, perhaps related to the manufacture of CAR-T cells. 9 Two recently described cases of malignant lymphoma derived from CAR gene modified T cells produced using the piggyBac transposon system have been reported, highlighting the need for caution and regular followup with CAR-T recipients, especially when novel methods of gene transfer are used.…”

Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular Consensus on genetically modified cells. VII. Present and future of technologies for production of CAR cell therapies

“…Importantly, two cases of malignant lymphoma derived from CAR gene modified T-cells were described in patients that received CAR-T cells anti-CD19 prepared with PB vector, 39 emphasizing the need for regular monitoring of new gene transfer methods used in clinical immunotherapy.…”

“… Sleeping Beauty ( SB) Good results in T-cells, antitumor activity both in vitro and in vivo, SB100X was initiate to treat multiple myeloma patients Low integration rate in a large scale Piggy bac Properties more similar to viral-based vectors, also with CAR anti-CD73, MSLN, EGFRvIII, and PSMA to treat solid tumors. Two cases of malignant lymphoma derived from CAR gene modified T-cells prepared with PB vector were reported 39 In vitro transcribed (IVT) mRNA Low risk of insertional mutagenesis transient expression of CAR Nanoplasmids Low risk of insertional mutagenesis, long-term transgene expression suitable for large scale production Difficult to produce 4) Expansion T-Flasks Most affordable consumables Time consuming, handling from trained operators, contamination risk, no agitation Culture bag Low risk of contamination Handling from trained operators, no agitation G-Rex Less timing consuming from operators, superior final volume Handling from trained operators, no agitation Rocking motion bioreactor Closed system, low probability of contamination, cells kept in constant agitation, final volume up to 25 liters Not indicated for cells sensible to shear stress …”