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. 2022 Oct 25;6(20):5634-5640.
doi: 10.1182/bloodadvances.2022007705.

Discovery of a novel genomic alteration that renders leukemic cells resistant to CD19-targeted immunotherapies

Armin Ghobadi  1 , Jack H Landmann  1 , Alun Carter  1 , Matthew L Cooper  1 , Mehmet Emrah Selli  1 , Jufang Chang  1 , Matthew Baker  2   3 , Christopher A Miller  1 , Francesca Ferraro  1 , David Y Chen  1 , Amanda M Smith  1 , Taylor A LaValle  1 , Eric J Duncavage  1 , Justin Chou  4 , Victor Tam  4 , Joseph M Benoun  4 , Jenny Nater  4 , Nathalie Scholler  4 , Francesca Milletti  4 , Remus Vezan  4 , Adrian Bot  4 , John M Rossi  4 , Nathan Singh  1
Affiliations

Discovery of a novel genomic alteration that renders leukemic cells resistant to CD19-targeted immunotherapies

Armin Ghobadi et al. Blood Adv. .
No abstract available

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Conflict of interest statement

Conflict-of-interest disclosure: A.G. declares a consulting or advisory role for Kite, a Gilead Company, Amgen, Atara, Wugen, and Celgene; research funding from Kite and Amgen; and honoraria from Kite. M.L.C. is employed by and has stock or other ownership in Wugen; reports a consultancy or advisory role for Rivervest Ventures; and reports patents, royalties, or other intellectual property from Wugen and NeoImmuneTech. F.F. has patents and receives royalties from Magenta (unrelated to this work). A.C. is employed by and has stock or other ownership in Wugen. J. Chou, F.M., and A.B. are employed by Kite. V.T., J.M.B., R.V., and J.M.R. were formerly employed by Kite. J.M.B. has stock or other ownership in Gilead Sciences, Allogene, Aurinia Pharma, and TG Therapeutics. J.N. has stock or other ownership in Kite. N. Scholler is employed by Gilead; was formerly employed by with Kite; and reports stock or other ownership in Gilead Sciences, Bristol-Myers Squibb, and Seattle Genetics. N. Singh has several patents related to CAR T cells and other cellular immunotherapies. The remaining authors declare no competing financial interests.

Figures

Figure 1.
Figure 1.
Emergence of a leukemic clone with a deletion of Tyr260 in CD19. (A) Diagnostic flow cytometry demonstrates loss of surface CD19 during treatment with CD19-targeted immunotherapies. Scatter plots of pretreatment (bone marrow) and postblinatumomab (peripheral blood) lymphoblasts stained with the anti-CD19 antibody FMC63 and costained with antibodies against CD10 (left). Histograms of CD19 and CD10 expression by lymphoblasts and other hematopoietic cells pre- and postblinatumomab treatment (right). (B) Predictive protein structural modeling of wild-type (WT) and ΔY260 CD19. Residues essential for FMC63 binding are shown in blue, and residues surrounding Tyr260 are shown in orange. (C) Schematic representation of inferred evolutionary trajectory of CD19ΔY260. Dark blue ellipse represents a clone that was detectable at diagnosis but disappeared after initial chemotherapy. Table below reflects allele frequency of each clone over time normalized to percentage of cells collected that were malignant (∼90% for clone 3 across time points). (D) Quantification of B-cell lineage transcripts in diagnosis, postblinatumomab, and post–CAR T-cell treatment ALL cells.
Figure 2.
Figure 2.
Loss of Tyr260 results in resistance to CAR T cells by preventing surface expression of CD19. (A) Percentage of Jurkat cells engineered to express either WT or CD19ΔY260 that were killed by CAR T cells. Cocultures were established at several effector-to-target ratios, and measurements were taken after 48 hours. (B) Jurkat cells engineered to express either WT or ΔY260 with a GFP marker were combined with nonengineered CD19− GFP− Jurkats at a ratio of 1:1, and these mixed cell pools were combined with CD19 CAR T cells. Data represent composition of cocultures after 48 hours. (C-D) Jurkat cells were engineered to express either WT or ΔY260 CD19 with a GFP marker, and binding of anti-CD19 antibodies SJ25C1 (C) and HIB19 (D) was evaluated by flow cytometry. (E-F) Jurkat cells were engineered to express either WT or ΔY260 CD19 that was linked to an N-terminal FLAG tag, and expression of CD19 and FLAG was determined by extracellular staining (E) or intracellular staining (F) and analysis by flow cytometry. (G) Jurkat cells engineered with nothing (CD19−), WT, or ΔY260 CD19 underwent fractionated cellular lysis. Lysates from the whole cell (WCL) or cytoplasmic or membrane compartments were separated by electrophoresis, and membranes were stained for CD19 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH; loading control). (H) Peptide:N-glycosidase (PNGase) F–treated whole-cell lysates from Jurkats expressing either WT or ΔY260 CD19 were probed for CD19 or GAPDH (loading control).
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