A New Specific Promoter Allow Hematopoietic Stem Cell Immunotherapy Approach Against Acute Lymphoblastic Leukemia Using Chimeric Antigen Receptor

Abstract

acting form of recombinant human interleukin-7 fused with hybrid Fc (rhIL-7-hyFc, NT-I7) in vivo using a CD19 + lymphoma xenograft model. Methods: To create anti-CD19 universal CART (UCART19), we activated human T cells on CD3/CD28 beads, electroporated the T cells with Cas9 mRNA and a TRAC gRNA, and virally transduced an anti-CD19 scFv 3 rd generation CAR containing a peptidase 2A-cleaved human CD34 construct for both purification and tracking in vivo. Residual TRAC+ cells were depleted using magnetic selection. For xenograft tumor modeling in vivo, we injected NSG mice with 5 £ 10 5 Ramos CBR-GFP cells four days prior to UCART19 (2 £ 10 6 cells). Mice were treated with NT-I7 (10mg/kg SC) on days +1, +15 and +29 post UCART19 infusion. Results: Ramos CBR-GFP mice receiving NT-I7 without UCART19 (NT-I7 only group) survived marginally longer (24 day med survival) than mice receiving Ramos CBR-GFP cells alone (No tx group) (21 day medium survival, p=0.018, NT-I7 only vs. No Tx). While Ramos CBR-GFP mice treated with UCART19 alone (UCART19 group) survived 33 days, 100% of Ramos CBR-GFP mice treated with UCART19 and NT-I7 (UCART19+NT-I7 group) were alive at 80 days (Fig 1a), with no mouse showing signs of xenogeneic GVHD (p<0.0001, UCART19+NT-I7 vs. UCART19). At three weeks post UCART19 infusion, bioluminescent imaging (BLI) revealed minimal tumor signal in UCART19+NT-I7 treated mice (10 8 vs. 10 10 photon flux/s, p<0.05, UCART19+NT-I7 vs. UCART19) and near-undetectable photon flux/s at four weeks (10 7 vs 10 11 photon flux/s, p<0.0001, UCART19+NT-I7 vs. UCART19). Quantitative 17-parameter flow cytometric analyses of the blood, bone marrow, and spleens revealed an up to »8000-fold increase in UCART19 cells in NT-I7-treated mice four weeks post UCART19 infusion (Fig 1a). These UCART19 cells demonstrated a predominantly effector and effector memory phenotype. Discussion: Here, we demonstrate that a pharmacological grade long-acting interleukin-7 agonist can potentiate adoptive cellular therapies. Specifically, NT-I7 can dramatically enhance gene modified T cell proliferation, persistence and tumor killing in vivo, resulting in enhanced survival. Unlike genetic strategies to potentiate IL-7 signaling in CART, NT-I7 provides a tunable clinic-ready adjuvant for reversing suboptimal CART activity in vivo.