title: Manufacture of clinical-grade CD19-specific T cells stably expressing chimeric antigen receptor using Sleeping Beauty system and artificial antigen presenting cells.
abstract: Adoptive transfer of T cells expressing a CD19-specific chimeric antigen receptor (CAR) is being evaluated in multiple clinical trials. Our current approach to adoptive immunotherapy is based on a second generation CAR (designated CD19RCD28) that signals through a CD28 and CD3-ζ endodomain. T cells are electroporated with DNA plasmids from the Sleeping Beauty (SB) transposon/transposase system to express this CAR. Stable integrants of genetically modified T cells can then be retrieved when co-cultured with designer artificial antigen presenting cells (aAPC) in the presence of interleukin (IL)-2 and 21. Here, we reveal how the platform technologies of SB-mediated transposition and CAR-dependent propagation on aAPC were adapted for human application. Indeed, we have initiated clinical trials in patients with high-risk B-lineage malignancies undergoing autologous and allogeneic hematopoietic stem-cell transplantation (HSCT). We describe the process to manufacture clinical grade CD19-specific T cells derived from healthy donors. Three validation runs were completed in compliance with current good manufacturing practice for Phase I/II trials demonstrating that by 28 days of co-culture on γ-irradiated aAPC ∼10(10) T cells were produced of which >95% expressed CAR. These genetically modified and propagated T cells met all quality control testing and release criteria in support of infusion.
authors.affiliation: Division of Pediatrics, Children's Cancer Hospital, The University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America.
authors.forename: Harjeet
authors.initials: H
authors.lastname: Singh
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Figliola
authors.forename: Margaret J
authors.initials: MJ
authors.lastname: Dawson
authors.forename: Simon
authors.initials: S
authors.lastname: Olivares
authors.forename: Ling
authors.initials: L
authors.lastname: Zhang
authors.forename: Ge
authors.initials: G
authors.lastname: Yang
authors.forename: Sourindra
authors.initials: S
authors.lastname: Maiti
authors.forename: Pallavi
authors.initials: P
authors.lastname: Manuri
authors.forename: Vladimir
authors.initials: V
authors.lastname: Senyukov
authors.forename: Bipulendu
authors.initials: B
authors.lastname: Jena
authors.forename: Partow
authors.initials: P
authors.lastname: Kebriaei
authors.forename: Richard E
authors.initials: RE
authors.lastname: Champlin
authors.forename: Helen
authors.initials: H
authors.lastname: Huls
authors.forename: Laurence J N
authors.initials: LJ
authors.lastname: Cooper
mesh_terms: D000938:Antigen-Presenting Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D018941:Antigens, CD19 / Q000235:genetics* / Q000276:immunology; D018106:CD28 Antigens / Q000235:genetics / Q000276:immunology; D017252:CD3 Complex / Q000235:genetics / Q000276:immunology; D049109:Cell Proliferation; D002986:Clinical Trials as Topic; D018920:Coculture Techniques; D004251:DNA Transposable Elements / Q000235:genetics* / Q000276:immunology; D018274:Electroporation; D015870:Gene Expression; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D018380:Hematopoietic Stem Cell Transplantation; D006801:Humans; D016219:Immunotherapy, Adoptive / Q000379:methods; D007376:Interleukin-2 / Q000235:genetics / Q000276:immunology; D020014:K562 Cells; D015448:Leukemia, B-Cell / Q000276:immunology / Q000473:pathology / Q000628:therapy*; D008213:Lymphocyte Activation; D050506:Mutant Chimeric Proteins / Q000235:genetics / Q000276:immunology; D013601:T-Lymphocytes / Q000166:cytology* / Q000276:immunology / Q000378:metabolism* / Q000637:transplantation; D019895:Transposases / Q000235:genetics / Q000276:immunology
chemical_list: D018941:Antigens, CD19; D018106:CD28 Antigens; D017252:CD3 Complex; C121704:CD3 antigen, zeta chain; D004251:DNA Transposable Elements; D007376:Interleukin-2; D050506:Mutant Chimeric Proteins; D019895:Transposases