Induction of resistance to chimeric antigen receptor T cell therapy by transduction of a single leukemic B cell

Abstract

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.

Fig. 1|. Detection and characterization of B cell lymphoblastic leukemia expressing CAR19 in a patient treated with CTL019 therapy.

a, Dynamics of CTL019 T cells detected by flow cytometry and of CAR19 4–1BBζ transcripts in peripheral blood over time. b, Serial flow cytometry analysis of CAR19⁺ cells (either CD3⁺ or CD3^–) (top) compared to leukemic cells (gated on CD45^dim and showing CD10 and CD19) (bottom) in the bone marrow (BM). c, Flow cytometry phenotyping of the CAR19-expressing leukemic blasts (identified as the CD3^–CD10⁺CD22⁺CD45^dim population) at relapse. d, Results from IgH-seq of apheresis material and bone marrow at relapse. Allele 1 and allele 2 are depicted as in e and are boxed. e, Serial monitoring of IgH clonotypes over time in the bone marrow. f, Lentiviral integration site (LVIS) analysis of pre- and postinfusion samples from Patient #107; horizontal bars indicate abundance and location of LVIS, annotated by the nearest gene. g, Schematic of single-cell analysis of five genes in 71 relapsed leukemia cells. Nine cells showed the simultaneous presence of the integrations in both NRP1 and PCCA. The genomic locations and orientations of the two main integration sites observed in single leukemia cells at relapse are shown beneath the graph. For a–f, results are representative of two independent experiments.

Fig. 2|. Ectopic CAR19 expression on B-ALL cells masks CD19 and creates CTL019-resistant leukemia.

a, Immunohistochemistry analysis of CD19 (OTI3B10 antibody clone, which binds to the cytoplasmic domain of CD19) in bone marrow biopsies before CTL019 treatment and at relapse. The black arrows point to CD19⁺ B cells. Scale bars, 200 μm. b, Flow cytometry analysis of CAR19⁺ Patient #107 leukemia cells at baseline and relapse using two anti-CD19 antibodies (0TI3B10, intracellular; HIB19, extracellular). c, Confocal imaging of Patient #107 leukemic blasts at baseline and relapse using Dylight488-conjugated 0TI3B10 anti-CD19 antibody (green), Alexa647-conjugated anti-CAR19 idiotype antibody (red), and DAPI (blue). Scale bars,2 μm. In (a–c), representative data from two independent experiments are shown. d, Expression of CD19 and CAR19 on wild-type, CD19-knockout, and CAR19-transduced NALM-6 cells analyzed by flow cytometry (representative of 5 experiments). e, CD19 mRNA expression is maintained in CAR19⁺ NALM-6 as compared to wild-type NALM-6 cells, as determined by qPCR (ΔΔCt normalized to wild-type, shown as wild-type). K562 cells were used as controls (pooled data from three independent experiments). ***P< 0.0001 as determined using a two-sided unpaired Student’s t-test. f, Confocal imaging demonstrates the coexpression of the CAR19–mCherry and CD19–GFP fusion proteins on the surface of NALM-6 leukemic cells. CD19^– (CRISPR–Cas9 knocked-out) NALM-6 cells were cotransduced with CD19–GFP (green) and CAR19–mCherry (red) fusion constructs. DAPI was used to stain cell nuclei (representative of four independent experiments with similar results). g, Upon doxycycline treatment, CD19 surface expression (mCherry) was no longer detected in Tet-inducible CAR–transduced NALM-6 cells by anti-CD19 flow antibody (clone: HIB19; representative of four independent experiments). h, Wild-type NALM-6 cells but not CAR19⁺ NALM-6 cells are highly resistant to CAR19⁺ T cell killing in a murine xenograft model. Targeting the CD22 protein with CAR T cells prevents leukemia progression in both wild-type and CAR19⁺ NALM-6 cells (n = 5 mice per group; symbols depict the mean; error bars represent s.e.m.). i, CAR22⁺ T cells but not CAR19⁺ T cells are able to eradicate tumor cells in NOD–SCID gamma chain-deficient (NSG) mice engrafted with CAR19⁺ leukemia (derived from Patient #107 at relapse) (n = 5 mice per group; each symbol represents the mean; error bars depict s.e.m.). For h and i, results are representative of two independent experiments.