Accelerating clinical scale production of BCMA CAR T cells with defined maturation stages

Chimeric antigen receptor (CAR)-T cells already showed impressive clinical regressions in leukemia and lymphoma. However, the development of CAR-T cells against solid tumors lags behind. Here we present the clinical-scale production of CAR-T cells for the treatment of melanoma under full GMP compliance. In this approach a CAR, specific for chondroitin sulfate proteoglycan 4 (CSPG4) is intentionally transiently expressed by mRNA electroporation for safety reasons. The clinical-scale protocol was optimized for: (i) expansion of T cells, (ii) electroporation efficiency, (iii) viability, (iv) cryopreservation, and (v) potency. Four consistency runs resulted in CAR-T cells in clinically sufficient numbers, i.e., 2.4 × 109 CAR-expressing T cells, starting from 1.77x108 PBMCs, with an average expansion of 13.6x, an electroporation efficiency of 88.0% CAR-positive cells, a survival of 74.1% after electroporation, and a viability of 84% after cryopreservation. Purity was 98.7% CD3+ cells, with 78.1% CD3+/CD8+ T cells and with minor contaminations of 1.2% NK cells and 0.6% B cells. The resulting CAR-T cells were tested for cytolytic activity after cryopreservation and showed antigen-specific and very efficient lysis of tumor cells. Although our work is descriptive rather than investigative in nature, we expect that providing this clinically applicable protocol to generate sufficient numbers of mRNA-transfected CAR-T cells will help in moving the field of adoptive cell therapy of cancer forward.

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Allogenic T-Cells Targeting CD123 for Adoptive Immunotherapy of Acute Myeloid Leukemia (AML)

Blood ◽

10.1182/blood.v124.21.1116.1116 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1116-1116 ◽

Cited By ~ 1

Author(s):

Roman Galetto ◽

Celine Lebuhotel ◽

Agnes Gouble ◽

Cecile Schiffer-Mannioui ◽

Julianne Smith

Keyword(s):

T Cells ◽

Acute Myeloid Leukemia ◽

Mouse Model ◽

Adoptive Immunotherapy ◽

Myeloid Leukemia ◽

Large Scale ◽

Scale Production ◽

Car T Cells ◽

Car T ◽

Acute Myeloid

Abstract Adoptive immunotherapy using autologous T-cells endowed with chimeric antigen receptors (CARs) has emerged as a promising new approach to treating cancer. However, a limitation of this approach is that CAR T-cells must be generated on a bespoke basis. To overcome this limitation, we have developed an allogeneic based platform for large scale production of “off-the-shelf” CAR T-cells from unrelated 3rd party donors. This platform utilizes Transcription Activator-Like Effector Nuclease (TALENTM) gene editing technology to inactivate the TCRα constant (TRAC) gene, eliminating the potential for T-cells bearing alloreactive TCR’s to mediate Graft versus Host Disease (GvHD). We have previously demonstrated that editing of the TRAC gene can be achieved at high frequencies, obtaining up to 80% of TCRαβ negative cells. This allows us to efficiently produce TCR-deficient T-cells that have been shown to no longer mediate alloreactivity in a xeno-GvHD mouse model. Furthermore, the capacity to perform efficient multiplex genome editing using TALENTM offers the possibility of simultaneously rendering cells resistant to standard chemotherapy or to tumor evasion mechanisms. In this work we present the adaptation of this allogeneic platform to the production of T cells targeting CD123, the transmembrane alpha chain of the interleukin-3 receptor, which is expressed in tumor cells from the majority of patients with Acute Myeloid Leukemia (AML). In a first step, we have screened human primary T-cells harboring CARs with different antigen recognition domains in the context of multiple CAR architectures in order to identify candidates displaying specific activity against cell lines expressing variable levels of the CD123 antigen. To provide proof of concept for the general applicability of the allogeneic approach we have manufactured a TCR-deficient CD123 CAR T-cell (UCART123) and demonstrated that this product maintains a potent anti-tumoral activity in vitro. Experiments in an orthotopic AML mouse model using UCART123 cells are currently ongoing, in order to establish the absence of alloreactivity and the anti-tumoral activity in vivo. The ability to carry out large scale manufacturing of allogeneic, non alloreactive CD123 specific T Cells from a single healthy donor could thus offer the possibility of an off-the-shelf treatment that would be immediately available for administration to a large number of AML patients. Disclosures Galetto: Cellectis SA: Employment. Lebuhotel:Cellectis SA: Employment. Gouble:Cellectis SA: Employment. Schiffer-Mannioui:Cellectis SA: Employment. Smith:Cellectis SA: Employment.

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