Targeting T-Cell Lymphoma Using CD70-Directed Cord Blood-Derived CAR-NK Cells

Abstract

T-cell lymphomas (TCL) are a heterogenous group of non-Hodgkin lymphoma characterized by resistance to conventional therapies posing an urgent need for safe and effective therapies. CAR-NK cells have emerged as a promising “off-the-shelf” cancer immunotherapy and are especially attractive for the treatment of T-cell malignancies. This is especially the case given the challenge of targeting T cell malignancies with CAR T cells due to shared antigens across the therapeutic, normal and malignant T cells posing the risk of CAR T cell fratricide, T-cell aplasia, and contamination of the CAR T products with malignant T cells. We have developed an FDA-approved system for ex vivo NK cell expansion, which reliably generates clinically relevant doses of GMP-grade NK cells from a cord blood (CB) unit. We have used this platform to engineer NK cells to express a CAR targeting CD19 using GMP-grade retroviral vectors and demonstrated the safety and efficacy of this platform in the clinic. We now propose to expand this approach to target TCL. CD70, a surface ligand of CD27, is overexpressed in a variety of cancers, including TCL. Signaling mediated by CD70-CD27 is thought to induce proliferation of TCL cells via activation of the NF-κB pathway. Thus, CD70 is an attractive target for CAR NK-cell therapy against TCL. We analyzed the publicly available primary TCL dataset (GSE19069) including anaplastic large cell lymphoma (30 samples), angioimmunoblastic TCL (37 samples), peripheral TCL (PTCL; 50 samples), and adult T cell leukemia/lymphoma (13 samples) for CD70 expression. The majority of the samples expressed CD70 transcript with a variable level of expression ( Figure 1). Using flow cytometry, we screened a panel of TCL cell lines with diverse histologic, cytogenetic and molecular properties for the expression of CD70. We demonstrated positive CD70 expression on most of the TCL cell lines profiled encompassing anaplastic large cell lymphoma (ALK+: KAR299; ALK-: MAC2A; fusion gene NPM-ALK+: SU-DHL1 and SUP-M2), hepatosplenic gamma-delta TCL (DERL-7), Sezary syndrome (SS; H9 and HuT78), and PTCL NOS (OCI-LY12). The CD70 intensity was highest on MAC2A, HuT78, KAR299 and SUP-M2, intermediate to low on DERL-7 and H9, and negative on SU-DHL1 and OCI-LY12. We designed a novel retroviral vector targeting CD70 that: 1) encodes the CD70-targeting CAR gene based on the CD27 extracellular domain; 2) ectopically produces interleukin (IL)-15 to support NK cell proliferation; and 3) expresses the suicide gene inducible caspase 9 ( iC9) that can be pharmacologically activated to eliminate transduced cells in the event of toxicity. The construct will be referred to as CAR.70/IL-15. Non-transduced (NT) NK cells and NK cells transduced with a construct that leads to IL-15 production without a CAR (IL-15 NK cells) were used as controls. We demonstrated that CAR.70/IL-15 NK cells had a significantly enhanced cytotoxicity compared to NT- and IL-15 NK cells against the CD70+ cell lines while there was no difference in cytotoxicity between the NK cell conditions in the negative cell lines pointing to a CD70-targeted fashion of anti-TCL NK cell cytotoxicity. Moreover, CAR.70/IL-15 NK cells exhibited greater cytotoxicity against CD70+ cell lines in long-term cytotoxicity assays ( Figure 2). In a tumor rechallenge assay against KAR299 where fresh tumor cells were added every 3-4 days, CAR.70/IL-15 NK cells showed superior potency in continuous tumor control. In addition, CAR.70/IL-15 NK cells exhibited higher levels of degranulation (CD107a) and cytokine production (TNFα and INF-γ) when cocultured with CD70+ TCL cell lines, compared to NT and IL-15 NK cells. Furthermore, these CAR NK cells targeting CD70 outperformed CAR NK cells engineered to target CD19, which is an irrelevant target for TCL, further confirming the specific CD70-targeted activity of CAR.70/IL-15 NK cells. CyTOF immunophenotyping revealed that CAR.70/IL-15 NK cells are characterized by the increased expression of markers of cytotoxicity such as granzyme-b, perforin, TRAIL; and activating coreceptors/proliferation markers such as DNAM, CD25 and Ki67. Taken together, our findings validate CD70 as a promising target for TCL treatment using CAR NK cells. Our approach provides a solid foundation for the clinical translation of CD70-targeting CAR NK-cell therapy for patients with TCL.