An Off-the-Shelf™ Fratricide-Resistant CAR-T for the Treatment of T Cell Hematologic Malignancies

Abstract

T cell malignancies represent a class of devastating hematologic cancers with high rates of relapse and mortality in both children and adults for which there are currently no effective or targeted therapies. Despite intensive multi-agent chemotherapy regimens, fewer than 50% of adults and 75% of children with T-ALL survive beyond five years. For those who relapse after initial therapy, salvage chemotherapy regimens induce remissions in 20-40% of cases. Allogeneic stem cell transplant, with its associated risks and toxicities, is the only curative therapy. T cells engineered to express a chimeric antigen receptor (CAR) are a promising cancer immunotherapy. Such targeted therapies have shown great potential for inducing both remissions and even long-term relapse-free survival in patients with B cell leukemia and lymphoma7-9. Thus, a targeted therapy against T cell malignancies represents a significant unmet medical need. However, several challenges have limited the clinical development of CAR-T cells against T cell malignancies. First, the shared expression of target antigens between T effector cells and T cell malignancies results in fratricide, or self-killing, of CAR-T cells. Second, harvesting adequate numbers of autologous T cells, without contamination by malignant cells is, at best, technically challenging and prohibitively expensive. Third, the use of genetically modified CAR-T cells from allogeneic donors may result in life-threatening graft-vs.-host disease (GvHD) when infused into immune-compromised HLA-matched or mismatched recipients. We hypothesized that deletion of CD7 and the T cell receptor alpha chain (TRAC) using CRISPR/Cas9 in CAR-T targeting CD7 (UCART7) would result in the efficient targeting and killing of malignant T cells without significant effector T cell fratricide or induction of GvHD. To generate the CD7 CAR, the anti-CD7 single chain variable fragment (scFv) was created using commercial gene synthesis and cloned into the backbone of a 3rd generation CAR with CD28 and 4-1BB internal signaling domains. The construct was modified to express CD34 via a P2A peptide to enable detection of CAR following viral transduction. Human primary T cells were activated using anti-CD3/CD28 beads for 48 hours prior to bead removal and electroporation with CD7 gRNA, TRAC gRNA, and Cas9 mRNA. On day three, T cells were transduced with lentivirus particles encoding either CD7 CAR or CAR CD19 control and allowed to expand for a further 6 days. Transduction efficiency and ablation of CD7 and TRAC were confirmed by flow cytometry. Multiplex CRISPR/Cas9 gene-editing resulted in the simultaneous bi-allelic deletion of both CD7 and TRAC in 72.8%±1.92 of cells, as determined by both non-homologous end joining (NHEJ) and FACS analyses. To prevent alloreactivity, CD3+ CAR-T were removed from the product by magnetic depletion. Of particular importance is that by using two distinct methods for assessing "off-target" nuclease activity across the entire human T cell genome (Guide-seq and probe capture), we could only detect one gene, an intronic modification of RMB33, that was inappropriately targeted using this approach. No obvious genomic rearrangements were detected by these analyses. UCART7 effectively expanded and killed T-ALL cell lines (CCRF-CEM, MOLT3, and HSB2) and human primary T-ALL blasts in vitro. Next, we tested the capacity of UCART7 to kill primary T-ALL in vivo without xenogeneic GvHD. Considerable expansion of alloreactive T cells, severe GvHD (mean clinical GvHD score = 5.66), and a robust graft vs. leukemia effect were observed in recipients of WT T cells. In contrast, GvHD was completely absent, T cells were undetectable, and considerable tumor burden was observed in mice receiving TRACΔ T cells. Mice receiving UCART7, however, had no GvHD and, unlike UCART19 controls, effectively cleared primary human T-ALL in NSG mice. Fratricide-resistant and allo-tolerant 'off-the-shelf' UCART7 signifies a novel strategy for treatment of relapsed and refractory T-ALL and non-Hodgkin's T cell lymphomas without a requirement for autologous T cells and represents the first clinically feasible adoptive T cell therapy for T cell malignancies. Disclosures No relevant conflicts of interest to declare.

Delivery technologies for T cell gene editing: Applications in cancer immunotherapy

“…Although autologous CAR T therapies have had successful clinical outcomes, their widespread application has been limited by the complexity and cost of manufacturing patient-derived CAR T cells [137]. These therapies heavily rely on the ability to harvest sufficient autologous T cells from cancer patients [138]. Although donor-derived CAR T cells could overcome many of the immune defects associated with cancer treatment and simplify the manufacturing process, current CAR T cell therapies use autologous T cells to prevent GVHD [6].…”

“…They also disrupted b-2 microglobulin (B2M) to suppress the HVG effect in allogeneic T cells [144]. CRISPR/Cas9 has also been used to prevent unintended CAR T cell fratricide caused by shared antigen expression between CAR T cells and malignant T cells [138]. Cooper et al used CRISPR/Cas9 to develop fratricide-resistant universal CD7-specific CAR T cells that targeted T cell ALL in vitro and in vivo without inducing GVHD [138].…”