Development of CAR T Cells Targeting U5 snRNP200 for the Treatment of Acute Myeloid and B-Lymphoid Leukemias

Currently there are no effective immunotherapies for AML outside of allogeneic transplant. This challenge is, in part, due to a lack of known antigens unique to AML and not present on normal hematopoietic stem or progenitor cells (HSPCs). To this end, several groups have recently made the surprising finding that a RNA helicase, termed U5 snRNP200, whose expression is normally restricted to the nucleus, is aberrantly localized to the cell surface of ~50% of AML patients and absent from normal HSPCs. This discovery was made following identification of naturally occurring donor-derived anti-U5 snRNP200 antibodies in AML patients in long-term remission following allogeneic transplant. While Fc-modified anti-U5 snRNP200 antibodies exhibit some therapeutic activity in AML preclinical models, here we reasoned that generation of chimeric antigen receptor (CAR) T cells targeting U5 snRNP200 would provide a more effective means to treat AML.

We converted 3 U5 snRNP200-directed antibodies into 6 single-chain variable fragments (scFvs) with heavy/light, or light/heavy chain orientation. We integrated these into a CD28-based CAR T cell platform which exhibited activation and proliferation of CAR T cells in response to antigen as well as clear cytotoxicity against human AML in vivo.

Prior work identified that U5 snRNP200 translocates to the cell surface in association with the Fc receptor CD32A. CD32A knockout in AML cell lines reduced the cytotoxicity of U5 snRNP200 CAR T cells by ~60%. At the same time, surface CD32A abundance is known to be augmented by IFNγ and we found that IFNγ exposure to AML cells upregulated both CD32A and U5 snRNP200 in a dose-dependent manner. The antigen gain due to IFNγ was leveraged to enhance U5 snRNP200 CAR T activity through addition of constitutive IL-18 (also known as IFNγ Inducing Factor) secretion. IL-18 armoring increased abundance of surface U5 snRNP200 and greatly enhanced activity of U5 snRNP200 CAR T cells compared to those without IL-18 expression. IL-18 secreting anti-U5 snRNP200 CAR T cells resulted in ~60% and ~40% long-term survival of mice engrafted with U937 human AML cells and an AML PDX from an adult (74y adverse risk) patient, respectively. Efficacy of IL-18 armored CAR T cells was dependent on IFNγ signaling as IFNγR1 knockout abrogated cytotoxicity of IL-18 armored U5 snRNP200 CAR T cells by ~80% in vitro.

The near complete sequence conservation of U5 snRNP200 and cross-reactivity of our scFvs across human and mouse enabled investigation of the safety of mouse IL-18 secreting anti-U5 snRNP200 CAR T cells in a fully syngeneic immunocompetent background. Mouse armored U5 snRNP200 CAR T cells greatly enhanced the survival of C57BL/6 mice engrafted with aggressive mouse MLL-AF9/NRas^G12D AML cells as 80% of mice were alive 90 days following CAR T cell infusion (compared with 100% lethality of control CAR mice at 30 days). Importantly, mouse armored CAR T cells had limited toxicity, both in the context of tumor and and in non-tumor-bearing wild-type C57BL/6 hosts infused with 300,000 and 1,000,000 armored CAR T cells, respectively.

Prior work has identified that U5 snRNP200, while absent on normal HSPCs, is present on normal mouse and human B cells. In fact, the only appreciable impact of U5 snRNP200 CAR T cells in syngeneic hosts was a reduction in B cells. We therefore next evaluated U5 snRNP200 CAR T cells in B acute lymphoblastic leukemia (B-ALL), where U5 snRNP200 is prominently expressed on blasts. Anti-U5 snRNP200 CAR T cells effectively eradicated a human B-ALL CDX (NALM6), as well as a PDX model from an adult (38y normal karyotype) B-ALL patient. Genome-wide CRISPR knockout screens identified a series of Golgi-localized enzymes required for N-linked glycosylation as regulators of surface U5 snRNP200 presentation on B-ALL cells.

Collectively, our studies identify a novel CAR T cell platform which targets an antigen that effectively eliminates AML while sparing normal myelopoiesis, and can also be utilized in B-ALL. The physical association between surface U5 snRNP200 and CD32A enabled us to augment antigen abundance on malignant cells through IL-18 secretion and thereby enhance the therapeutic activity of anti-U5 snRNP200 CAR T cells through “antigen-gain.” The tumor-selectivity and safety of this approach make this CAR T cell platform an exciting approach ripe to translate to the clinical setting for AML and B-ALL patients refractory to current therapies.