The CD19 antigen, expressed on most B-cell acute lymphoblastic leukemias (B-ALL), can be targeted with chimeric antigen receptor–armed T cells (CART-19), but relapses with epitope loss occur in 10% to 20% of pediatric responders. We detected hemizygous deletions spanning the CD19 locus and de novo frameshift and missense mutations in exon 2 of CD19 in some relapse samples. However, we also discovered alternatively spliced CD19 mRNA species, including one lacking exon 2. Pull-down/siRNA experiments identified SRSF3 as a splicing factor involved in exon 2 retention, and its levels were lower in relapsed B-ALL. Using genome editing, we demonstrated that exon 2 skipping bypasses exon 2 mutations in B-ALL cells and allows expression of the N-terminally truncated CD19 variant, which fails to trigger killing by CART-19 but partly rescues defects associated with CD19 loss. Thus, this mechanism of resistance is based on a combination of deleterious mutations and ensuing selection for alternatively spliced RNA isoforms. Significance CART-19 yield 70% response rates in patients with B-ALL, but also produce escape variants. We discovered that the underlying mechanism is the selection for preexisting alternatively spliced CD19 isoforms with the compromised CART-19 epitope. This mechanism suggests a possibility of targeting alternative CD19 ectodomains, which could improve survival of patients with B-cell neoplasms.

No abstract

Potent CD19-directed immunotherapies, such as chimeric antigen receptor T cells (CART) and blinatumomab, have drastically changed the outcome of patients with relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL). However, CD19-negative relapses have emerged as a major problem that is observed in approximately 30% of treated patients. Developing approaches to preventing and treating antigen-loss escapes would therefore represent a vertical advance in the field. Here, we found that in primary patient samples, the IL-3 receptor α chain CD123 was highly expressed on leukemia-initiating cells and CD19-negative blasts in bulk B-ALL at baseline and at relapse after CART19 administration. Using intravital imaging in an antigen-loss CD19-negative relapse xenograft model, we determined that CART123, but not CART19, recognized leukemic blasts, established protracted synapses, and eradicated CD19-negative leukemia, leading to prolonged survival. Furthermore, combining CART19 and CART123 prevented antigen-loss relapses in xenograft models. Finally, we devised a dual CAR-expressing construct that combined CD19- and CD123-mediated T cell activation and demonstrated that it provides superior in vivo activity against B-ALL compared with single-expressing CART or pooled combination CART. In conclusion, these findings indicate that targeting CD19 and CD123 on leukemic blasts represents an effective strategy for treating and preventing antigen-loss relapses occurring after CD19-directed therapies.

Key Points• Targeting of CD123 via CAR-engineered T cells results in rejection of human AML and myeloablation in mouse models.Many patients with acute myeloid leukemia (AML) are incurable with chemotherapy and may benefit from novel approaches. One such approach involves the transfer of T cells engineered to express chimeric antigen receptors (CARs) for a specific cell-surface antigen. This strategy depends upon preferential expression of the target on tumor cells. To date, the lack of AML-specific surface markers has impeded development of such CARbased approaches. CD123, the transmembrane a chain of the interleukin-3 receptor, is expressed in the majority of AML cells but is also expressed in many normal hematopoietic cells. Here, we show that CD123 is a good target for AML-directed CAR therapy, because its expression increases over time in vivo even in initially CD123 dim populations, and that human CD123-redirected T cells (CART123) eradicate primary AML in immunodeficient mice. CART123 also eradicated normal human myelopoiesis, a surprising finding because anti-CD123 antibody-based strategies have been reportedly well tolerated. Because AML is likely preceded by clonal evolution in "preleukemic" hematopoietic stem cells, our observations support CART123 as a viable AML therapy, suggest that CART123-based myeloablation may be used as a novel conditioning regimen for hematopoietic cell transplantation, and raise concerns for the use of CART123 without such a rescue strategy. (Blood. 2014;123(15):2343-2354

We report a patient relapsing 9 months after CD19-targeted CAR T cell (CTL019) infusion with CD19- leukemia that aberrantly expressed the anti-CD19 CAR. The CAR gene was unintentionally introduced into a single leukemic B cell during T cell manufacturing, and its product bound in cis to the CD19 epitope on the surface of leukemic cells, masking it from recognition by and conferring resistance to CTL019.