Abstract
Background
T-cell Acute Lymphoblastic Leukemia (T-ALL) / Lymphoblastic Lymphoma (LBL) represent a class of devastating hematologic cancers with high rates of relapse and mortality in both children and adults. Development of CAR-T cell therapies for T-cell cancers has been complicated by induction of fratricide and the high risk of malignant cell contamination of the drug product in the autologous setting. Previously, Cooper et. al., demonstrated that CRISPR/Cas9 gene-editing to delete CD7 prevented self-killing, and deletion of the T-cell receptor alpha constant (TRAC) enabled the use of healthy donor-derived T-cells to manufacture CD7-targeted CAR-T cells without risk of malignancy and mitigating the risk of GvHD. Here we present preclinical data supporting the safety and efficacy of WU-CART-007, an IND ready, off-the-shelf, and fratricide resistant CD7-targeted CAR-T cell for the treatment of CD7+ T-cell malignancies.
Methods
WU-CART-007 was manufactured using T cells isolated from healthy donors by deletion of CD7 and TRAC, followed by CAR transduction, cell expansion, depletion of residual TCRa/b+ cells and cryopreservation. Donors were confirmed negative for a panel of adventitious viruses. CD7/TRAC deletion and CAR transduction were confirmed by flow cytometry. Off-target editing profile was assessed by GUIDE-Seq. The binding kinetics to human CD7 were conducted by bio-layer interferometry and CD7 selectivity was confirmed by cell microarray with a library of HEK-293 cells expressing approximately 6000 human proteins. The in vitro activity of WU-CART-007 was interrogated by co-culture with human CD7+ CCRF-CEM T-ALL cells and the potential on-target, off-tumor activity was assessed by co-culture with a panel of immune and non-immune primary human cells. In vivo anti-tumor functionality was confirmed in immunocompromised NSG mice after the establishment of low or high tumor burden CCRF-CEM xenografts engineered to express green fluorescent protein (GFP) and click beetle red (CBR) luciferase. The impact of WU-CART-007 on normal hematopoiesis was assessed using CD34+ humanized NCG mice.
Results
Several successful full-scale manufacturing runs were completed with consistently high dual CD7/TRAC deletion, transduction efficiency, and cell viability. Drug product was primarily composed of a T cell memory phenotype. Off- target nuclease analysis by GUIDE-seq and targeted NGS confirmed no evidence of off-target editing events. The WU-CART-007 scFv exhibited high affinity and exquisite specificity for human CD7. In vitro co-incubation experiments confirmed strong cytotoxicity against CD7-expressing cells including CCRF-CEM T-ALL cells, primary T and NK cells, but not CD7- cells such as myeloid cells, B cells, hepatocytes, astrocytes, cardiomyocytes, epithelial cells, and endothelial cells. Importantly, no cytotoxicity was observed against hematopoietic progenitor cells in human bone marrow or cord blood following co-incubation with WU-CART-007. Similarly, WU-CART-007 treatment of a non-tumor bearing humanized mouse model resulted in transient reductions in CD7+ cells (T-cells and NK cells) but not CD7- cells (myeloid and B cells), and the impacted cells recovered after circulating WU-CART-007 cells were no longer detectable. Assessment of in vivo anti-tumor efficacy revealed that WU-CART-007 effectively inhibited tumor progression (>99% TGI) in both low and high burden CCRF-CEM tumor models and improved survival in a dose-dependent manner, while CAR- cells were inactive, confirming CD7-dependent activity.
Conclusions
These preclinical studies support the use of WU-CART-007 in clinical trials and highlight the potential of WU-CART-007 to be a well-tolerated and active therapy for patients with CD7+ T-cell malignancies. A first in human Phase 1/2 trial in patients with R/R T-ALL/LBL is currently open for enrollment (NCT# 04984356).
No relevant conflicts of interest to declare.
