Date of Graduation

12-2015

Document Type

Dissertation (PhD)

Program Affiliation

Immunology

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Dean Anthony Lee MD, PhD

Committee Member

Laurence JN Cooper M.D, PhD

Committee Member

Richard Eric Davis, M.D

Committee Member

Dat Tran M.D

Committee Member

Kenneth Tsai M.D, PhD

Committee Member

Elizabeth Shpall, M.D

Abstract

ABSTRAC

REDIRECTING T CELLS WITH CHIMERIC ANTIGEN RECEPTORS TO TARGET CD123+ LEUKEMIA

Radhika Thokala, Ph.D*

Advisory Professor: Dean Anthony Lee, M.D, Ph.D

CD123 or interleukin receptor alpha (IL-3Rα) is expressed on hematological malignancies such as acute myeloid leukemia (AML) and some acute lymphoblastic leukemia (ALL). Significantly, CD123 is over-expressed on leukemic stem cells (LSCs) compared to normal hematopoietic stem cells and thus targeting this tumor- associated antigen (TAA) provides the potential to prevent relapse. The prototyical chimeric antigen receptor (CAR) is fashioned by combining the variable light (V L) and heavy (VH) as a scFv derived from a single monoclonal antibody (mAb) specific for the TAA. We describe a new approach for generating CD123-specific CARs generating a chimeric scFv that is made up of the VL and VH harvested from two mAbs that are each specific for CD123. The hypothesis is VL and VH from different antibodies to the same TAA can be recombined to form unique binding domains that retain antigen specificity but may have altered binding characteristics. This non-homologous recombination of antibody binding domain may be used to select CAR for optimal anti-tumor characteristics, such as increasing the therapeutic index. The chimeric scFvs were derived by fusing the VL and VHchains derived from mAbs 26292, 32701, 32703, 32716 specific to CD123. Sleeping Beauty (SB) was employed as a non-viral gene transfer s ystem to stably express 2nd generation CARs in T cells derived from peripheral blood mononuclear cells (PBMC). The CARs were co-expressed with inducible Caspase 9 (iCaspase9) for conditional ablation of T cells in case of off-target toxicities. The SB plasmids coding for two CARs (transposons) activated T cells via chimeric CD28 with CD3-zeta and CD137 with CD3-zeta were electroporated into PBMC. Following electrotransfer of the SB system the genetically modified T cells were preferentially propagated on activating and propagating cells (AaPC) designated as Clone 1-CD123. The AaPC were derived from K562 cells genetically modified to co-express co-stimulatory molecules (CD86 and CD137L), a membrane bound cytokine (IL-15 fused to IL-15Rα), and the TAAs CD123 and CD19. CAR+ T cells specifically produced IFN-γ and lysed CD123+ leukemic cell lines and primary AML patient samples, but did not lyse D123neg tumor cells. The addition of a chemical dimerizer to activate iCaspase9 resulted in destruction of genetically modified T cells. Both populations of CAR+ T cells produced and eliminated leukemic tumors in vivo. We observed no difference in the anti-tumor effects whether the CARs triggered T cells via CD28 or CD137. These studies suggest that CD123 can be targeted by CAR+ T cells and that the hybrid arrangement of VL and VH maintained specificity for CD123.

Keywords

CD123, Chimeric Antigen Receptors, Sleeping Beauty System

Available for download on Friday, December 16, 2016

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