Generation and Signaling Function of CD19 Chimeric Antigen Receptor Modified CD8+ T Cells Derived From Virus-Specific Central Memory Cells for Adoptive Therapy After Allogeneic Hematopoietic Stem Cell Transplant
Authors: Terakura Seitaro , Yamamoto Tori N. , Gardner Rebecca A. , Turtle Cameron J. , Jensen Michael C. , Riddell Stanley R. 
Published: October 2019

Abstract
Abstract 2978
Allogeneic hematopoietic stem cell transplantation (allo-HCT) is the most effective post-consolidation therapy for high-risk B cell acute lymphocytic leukemia (ALL) in pediatric and adult patients. However, relapse after allo-HCT remains a common cause of failure, and treatment of ALL that has recurred after transplant, either with additional chemotherapy or with donor lymphocyte infusions to enhance a graft-versus-leukemia effect is mostly unsuccessful and can cause graft-versus-host disease (GVHD). The adoptive transfer of donor T cells that have been genetically modified to recognize leukemia could prevent or treat leukemia relapse after allo-HCT. However, adoptive therapy after allo-HCT should be performed with T cells that have a defined endogenous T cell receptor (TCR) specificity to avoid GVHD, and with T cells that have the capacity to persist in vivo to ensure leukemia eradication. Our lab has previously shown that donor virus-specific T cells can be adoptively transferred without causing GVHD, and identified a role for cell intrinsic properties of central memory T (TCM) cells in determining cell persistence after adoptive transfer. Thus, we developed a strategy for deriving virus-specific T cells from CD45RA−CD62L+CD8+ TCM cells purified from donor blood with clinical grade reagents, and redirecting their specificity to the B-cell lineage marker CD19 through lentiviral transfer of a gene encoding an anti-CD19 ectodomain, a CD3 ζ and CD28 endodomain, and a truncated epidermal growth factor receptor as a transduction marker downstream of a T2A sequence. CD8+ TCM were enriched by first depleting CD4+, CD14+, and CD45RA+ cells from PBMC using clinical grade monoclonal antibodies (mAbs) conjugated to paramagnetic beads, and then positively selecting CD62L+ cells from the depleted subset with a biotinylated anti-CD62L mAb and clinical grade anti-biotin beads. After this two-step selection, the enriched virus-specific TCM were selectively transduced by exposure to the CD19-CAR lentivirus after stimulation with viral peptides. This procedure resulted in a selective expansion of bi-specific (CD19-CAR+/virus-specific) T cells that could be readily expanded by stimulation with CD19+ EBV-transformed lymphoblastoid cells (LCL). The bi-specific subset was further enriched to high purity using reversible class I MHC Streptamers (Stage Cell Therapeutics) folded with viral peptides. The frequency of tetramer positive and CD19-CAR+ cells in the final cell product was ∼90% (range, 80.7–96.3%) and ∼85% (range, 77.9–95.3%) respectively, and this high frequency of bi-specific T cells was maintained through additional in vitro expansion. With this procedure, large numbers of highly pure bi-specific TE cells that are derived from CD8+ TCM precursors and engineered to express a CD19-CAR could be readily generated for adoptive immunotherapy of allo-HCT recipients in ∼30–35 days from 400 ml of donor blood.
CAR binding to its ligand is structurally distinct from that of a TCR, and a direct comparison of effector functions and signaling through an introduced CAR and the endogenous TCR on the same cell could reveal differences that might affect cellular functions and therapeutic efficacy. To compare functions after CAR- and TCR-stimulation, we transduced K562 tumor cells with the costimulatory molecules CD80 and CD86, and with CD19 or HLA molecules to serve as antigen presenting cells. Activation of bi-specific T cells through the CAR or the virus-specific TCR elicited comparable lytic function and induced equivalent phosphorylation of downstream signaling molecules (CD3 ζ, Zap70, p38, ERK, JNK) with similar kinetics. Signaling through either CAR or TCR induced comparable production of IL-2, IFN- γ, and TNF- α as assessed by intracellular cytokine staining and Luminex assay, and T cell proliferation as assessed by CFSE dye dilution and absolute cell count. These studies identify a strategy for tumor-specific immunotherapy with CAR-modified T cells after allo-HCT, and for comparative studies of signaling through the TCR and CARs derived from antibodies of different affinities and containing distinct signaling domains.

Disclosures:
No relevant conflicts of interest to declare.