Early transduction produces highly functional chimeric antigen receptor-modified virus-specific T-cells with central memory markers: a Production Assistant for Cell Therapy (PACT) translational application.
BACKGROUND
Virus-specific T-cells (VSTs) proliferate exponentially after adoptive transfer into hematopoietic stem cell transplant (HSCT) recipients, eliminate virus infections, then persist and provide long-term protection from viral disease. If VSTs behaved similarly when modified with tumor-specific chimeric antigen receptors (CARs), they should have potent anti-tumor activity. This theory was evaluated by Cruz et al. in a previous clinical trial with CD19.CAR-modified VSTs, but there was little apparent expansion of these cells in patients. In that study, VSTs were gene-modified on day 19 of culture and we hypothesized that by this time, sufficient T-cell differentiation may have occurred to limit the subsequent proliferative capacity of the transduced T-cells. To facilitate the clinical testing of this hypothesis in a project supported by the NHLBI-PACT mechanism, we developed and optimized a good manufacturing practices (GMP) compliant method for the early transduction of VSTs directed to Epstein-Barr virus (EBV), Adenovirus (AdV) and cytomegalovirus (CMV) using a CAR directed to the tumor-associated antigen disialoganglioside (GD2).


RESULTS
Ad-CMVpp65-transduced EBV-LCLs effectively stimulated VSTs directed to all three viruses (triVSTs). Transduction efficiency on day three was increased in the presence of cytokines and high-speed centrifugation of retroviral supernatant onto retronectin-coated plates, so that under optimal conditions up to 88% of tetramer-positive VSTs expressed the GD2.CAR. The average transduction efficiency of early-and late transduced VSTs was 55 ± 4% and 22 ± 5% respectively, and early-transduced VSTs maintained higher frequencies of T cells with central memory or intermediate memory phenotypes. Early-transduced VSTs also had higher proliferative capacity and produced higher levels of TH1 cytokines IL-2, TNF-α, IFN-γ, MIP-1α, MIP-1β and other cytokines in vitro.


CONCLUSIONS
We developed a rapid and GMP compliant method for the early transduction of multivirus-specific T-cells that allowed stable expression of high levels of a tumor directed CAR. Since a proportion of early-transduced CAR-VSTs had a central memory phenotype, they should expand and persist in vivo, simultaneously protecting against infection and targeting residual malignancy. This manufacturing strategy is currently under clinical investigation in patients receiving allogeneic HSCT for relapsed neuroblastoma and B-cell malignancies (NCT01460901 using a GD2.CAR and NCT00840853 using a CD19.CAR).
Center for Cell and Gene Therapy Baylor College of Medicine Texas Children's Hospital Houston Methodist Hospital, Houston, TX 77030 USA.|Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX 77030 USA.
Jiali
J
Sun
Center for Cell and Gene Therapy Baylor College of Medicine Texas Children's Hospital Houston Methodist Hospital, Houston, TX 77030 USA.
Leslie E
LE
Huye
Center for Cell and Gene Therapy Baylor College of Medicine Texas Children's Hospital Houston Methodist Hospital, Houston, TX 77030 USA.|Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX 77030 USA.
Natalia
N
Lapteva
Center for Cell and Gene Therapy Baylor College of Medicine Texas Children's Hospital Houston Methodist Hospital, Houston, TX 77030 USA.
Maksim
M
Mamonkin
Center for Cell and Gene Therapy Baylor College of Medicine Texas Children's Hospital Houston Methodist Hospital, Houston, TX 77030 USA.
Manasa
M
Hiregange
Center for Cell and Gene Therapy Baylor College of Medicine Texas Children's Hospital Houston Methodist Hospital, Houston, TX 77030 USA.
Brandon
B
Ballard
Center for Cell and Gene Therapy Baylor College of Medicine Texas Children's Hospital Houston Methodist Hospital, Houston, TX 77030 USA.
Olga
O
Dakhova
Center for Cell and Gene Therapy Baylor College of Medicine Texas Children's Hospital Houston Methodist Hospital, Houston, TX 77030 USA.
Darshana
D
Raghavan
Center for Cell and Gene Therapy Baylor College of Medicine Texas Children's Hospital Houston Methodist Hospital, Houston, TX 77030 USA.
April G
AG
Durett
Center for Cell and Gene Therapy Baylor College of Medicine Texas Children's Hospital Houston Methodist Hospital, Houston, TX 77030 USA.
Serena K
SK
Perna
Center for Cell and Gene Therapy Baylor College of Medicine Texas Children's Hospital Houston Methodist Hospital, Houston, TX 77030 USA.
Bilal
B
Omer
Center for Cell and Gene Therapy Baylor College of Medicine Texas Children's Hospital Houston Methodist Hospital, Houston, TX 77030 USA.
Lisa A
LA
Rollins
Center for Cell and Gene Therapy Baylor College of Medicine Texas Children's Hospital Houston Methodist Hospital, Houston, TX 77030 USA.|Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX 77030 USA.|Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030 USA.
Ann M
AM
Leen
Center for Cell and Gene Therapy Baylor College of Medicine Texas Children's Hospital Houston Methodist Hospital, Houston, TX 77030 USA.|Department of Medicine, Baylor College of Medicine, Houston, TX 77030 USA.
Juan F
JF
Vera
Center for Cell and Gene Therapy Baylor College of Medicine Texas Children's Hospital Houston Methodist Hospital, Houston, TX 77030 USA.|Department of Medicine, Baylor College of Medicine, Houston, TX 77030 USA.
Gianpietro
G
Dotti
Center for Cell and Gene Therapy Baylor College of Medicine Texas Children's Hospital Houston Methodist Hospital, Houston, TX 77030 USA.|Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030 USA.
Adrian P
AP
Gee
Center for Cell and Gene Therapy Baylor College of Medicine Texas Children's Hospital Houston Methodist Hospital, Houston, TX 77030 USA.|Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030 USA.|Department of Medicine, Baylor College of Medicine, Houston, TX 77030 USA.
Malcolm K
MK
Brenner
Children's Mercy Hospitals and Clinics, Kansas City, MO 64108 USA.
Douglas G
DG
Myers
Center for Cell and Gene Therapy Baylor College of Medicine Texas Children's Hospital Houston Methodist Hospital, Houston, TX 77030 USA.|Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX 77030 USA.|Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030 USA.|Department of Molecular Virology and Immunology, Baylor College of Medicine, Houston, TX 77030 USA.
Cliona M
CM
Rooney
Chimeric antigen receptor; Clinical grade T-cell manufacture; Virus-specific T cells