1159Multiplexed Engineering of CD19CAR T Cells for Post-Transplant

Consolidative Immunotherapy
Clinical Allogeneic and Autologous Transplantation: Late Complications and Approaches to Disease Recurrence
Program: Oral and Poster Abstracts
Type: Oral
Session: 723. Clinical Allogeneic and Autologous Transplantation: Late Complications and Approaches to Disease
Recurrence: Immunotherapy to Prevent or Treat Relapse After Transplantation

Monday, December 5, 2016: 4:30 PM

Grand Hall C (Manchester Grand Hyatt San Diego)

Corinne Summers, MD1 , Alexandra Grier 1*, Rebecca Gardner, MD2*, Colleen Delaney, MD, MSc3 and Michael C Jensen,
MD4*
1 SeattleChildren's Research Institute, Seattle, WA
2 SeattleChildren's Hospital, Seattle, WA
3 Fred Hutchinson Cancer Research Center, Seattle, WA
4 Ben Towne Center for Childhood Cancer Research, Seattle Children's Research Institute, Seattle, WA

Introduction: Relapse of CD19 + acute lymphoblastic leukemia (ALL) post-hematopoietic cell transplant (HCT) portends a
dismal prognosis and opportunities to intensify the anti-tumor potency of HCT are limited by regimen related toxicities.
While pre-transplant CD19 chimeric antigen receptor (CAR) T cell therapy can increase the numbers of patients who
achieve minimal residual disease negative remissions, there remains an unmet need to further reduce the incidence of
early post-HCT relapses through the use of adoptive immunotherapy. However, the use of CAR T cell therapies in this
setting is severely limited as patients are on immunosuppressive therapy (IST) to prevent graft versus host disease
(GVHD). Here, we report on a multiplexed engineering strategy to generate donor-derived CD19CAR T cells that are
resistant to combinations of cyclosporine (CSA)/Tacrolimus (FK506) and/or mycophenolate mofetil (MMF), and devoid of
GVHD reactivity for this patient population.

Methods and Results: We developed CNA22 or CNA12-P2A-IMPDH2-T2A-Her2tG vectors using the CNA22, CNA12
[Brewin M, et al, Blood 2009] and IMPDH2 [Jonnalagadda J, et al, PLoS One 2013] mutants conferring CSA, FK506 and
MMF resistance, respectively. Her2tG is a truncated HER2 extracellular protein developed in our lab and used for
selection and transduction efficiency determination using Herceptin. The drug resistant lentivirus packaged vector was
used to co-transduce human T cells with our CD19CAR vector which includes a 4-1BB co-stimulatory domain and
truncated EGFR protein (EGFRt) for cell selection, transduction efficiency and in vivo suicide using Erbitux [Wang, X, et al,
Blood 2011]. We coupled dual transduction with mRNA TALEN transfection technology using a TALEN pair targeting the
first exon of the TcR constant region to transfect T cells for efficient knockout of the endogenous T cell receptor (TcR)
thus eliminating the risk of causing GVHD. Our multiplexed technologies yield CD3/TcR knockouts rates greater than
80% with successful dual transduction of both vectors demonstrated by EGFRt and Her2tG expression (Figure A). Cells
demonstrated MMF resistance in vitro as evidenced by increased Her2tG expression for culture purification following
mycophenolic acid (MPA, active metabolite of MMF) exposure. In addition, prolonged MPA drug exposure at multiple
drug concentrations (0-10uM) demonstrated improved culture expansion and viability for cultures containing cells
transduced to express IMPDH2 over a 21 day culture period (Figure B). CD19CAR expressing cells demonstrated CD19
antigen targeted cell lysis as evaluated by chromium release assay. Furthermore, cytokine evaluations of cells following
co-culturing with target cells at multiple CSA concentrations (0-1000ng/ml) yielded sustained IL-2, IFNg and TNFa
secretion by CNA22 expressing cells following CD19CAR activation (Figure C). In vivo experiments are in progress to
assess the functional attributes of these multiplexed engineered CAR T cells in relevant murine model systems.
Conclusions: We successfully used multiplexed engineering strategies to generate CD3/TcR- CD19CAR +EGFRt +CNA22-
IMPDH2-HER2tG + T cells. Cells expressing CNA22 and IMPDH2 mutants exhibited activation and expansion in vitro in
the presence of immunosuppressive agents CSA and MPA with targeted CD19CAR activity. These studies suggest the
ability to generate modified T cells that remain functional in vivo even in the presence of IST and without induction of
GVHD, allowing for this approach in the post-transplant setting to prevent/treat disease relapse.

Figure A) Cells underwent successful dual transduction followed by mRNA TALEN transfection for development of
CD3/TcR- CD19CAR +EGFRt + CNA22-IMPDH2-HER2tG + T cells. B) Transduced T cells underwent selection for their
respective markers, were stimulated and cultured for 21 days at varying MPA concentrations. Cells transduced to express
IMPDH2 exhibited improved expansion as compared to CD19CAR and mock control cells. Data was normalized to the
cell group's no drug control expansion. C) Following co-culture with K562-CD19 antigen cells with CSA concentrations
of 0-1000ng/ml CD19CAR cells transduced with the drug resistant vector secreted significantly increased IFNg and IL-2
levels compared to CD19CAR only cells. NS = not significant; Horizontal bars indicate statistical significance defined as
p-value <0.05.

Disclosures: Jensen: Juno Therapeutics, Inc: Consultancy, Equity Ownership, Membership on an entity's Board of
Directors or advisory committees, Research Funding.

See more of: 723. Clinical Allogeneic and Autologous Transplantation: Late Complications and Approaches to Disease
Recurrence: Immunotherapy to Prevent or Treat Relapse After Transplantation
See more of: Clinical Allogeneic and Autologous Transplantation: Late Complications and Approaches to Disease
Recurrence
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*signifies non-member of ASH