Redirecting T Cells With Chimeric Antigen Receptors to Target Cd123+ Leukemia

<h1>ABSTRAC</h1> <h1><strong> R</strong><strong>E</strong><strong>D</strong><strong>I</strong><strong>R</strong><strong>E</strong><strong>C</strong><strong>T</strong><strong>I</strong><strong>N</strong><strong>G T CELLS WITH CHIMERIC ANTIGEN RECEPTORS TO TARGET CD123</strong><strong>+ </strong><strong>LE</strong><strong>U</strong><strong>K</strong><strong>E</strong><strong>M</strong><strong>I</strong><strong>A</strong></h1> <p>Radhika Thokala, Ph.D*</p> <p>Advisory Professor: Dean Anthony Lee, M.D, Ph.D</p> <p>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 (V_H) 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 V_L and V_H harvested from two mAbs that are each specific for CD123. The hypothesis is V_L and V_Hfrom 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 V_L and V_Hchains derived from mAbs 26292, 32701, 32703, 32716 specific to CD123. <em>S</em><em>l</em><em>ee</em><em>p</em><em>i</em><em>n</em><em>g Beauty</em> (SB) was employed as a non-viral gene transfer s ystem to stably express 2^nd 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 <em>i</em><em>n vivo. </em>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 V_L and V_H maintained specificity for CD123.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation (PhD)

Year dc:date.available

2015

Author dc:creator

• Thokala, Radhika

Contributors dc:contributor

• Dean Anthony Lee MD, PhD
• Laurence JN Cooper M.D, PhD
• Richard Eric Davis, M.D

• CD123
• Chimeric Antigen Receptors
• Sleeping Beauty System
• Medicine and Health Sciences
• Translational Medical Research

Repository record dc:identifier

https://digitalcommons.library.tmc.edu/utgsbs_dissertations/644

OAI identifier oai:identifier

oai:digitalcommons.library.tmc.edu:utgsbs_dissertations-1679