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. 2023 Apr;72(4):957-968.
doi: 10.1007/s00262-022-03303-4. Epub 2022 Oct 10.

Targeting FLT3-specific chimeric antigen receptor T cells for acute lymphoblastic leukemia with KMT2A rearrangement

Masaya Suematsu  1 , Shigeki Yagyu  2 , Hideki Yoshida  1 , Shinya Osone  1 , Yozo Nakazawa  3 , Kanji Sugita  4 , Toshihiko Imamura  1 , Tomoko Iehara  1
Affiliations

Targeting FLT3-specific chimeric antigen receptor T cells for acute lymphoblastic leukemia with KMT2A rearrangement

Masaya Suematsu et al. Cancer Immunol Immunother. 2023 Apr.

Abstract

CD19-specific chimeric antigen receptor T (CAR T) immunotherapy is used to treat B-cell malignancies. However, antigen-escape mediated relapse following CAR T therapy has emerged as a major concern. In some relapsed cases, especially KMT2A rearrangement-positive B-acute lymphoblastic leukemia (KMT2A-r B-ALL), most of the B-cell antigens are lost via lineage conversion to the myeloid phenotype, rendering multi-B-cell-antigen-targeted CAR T cell therapy ineffective. Fms-related tyrosine kinase-3 (FLT3) is highly expressed in KMT2A-r B-ALL; therefore, in this study, we aimed to evaluate the antitumor efficacy of CAR T cells targeting both CD19 and FLT3 in KMT2A-r B-ALL cells. We developed piggyBac transposon-mediated CAR T cells targeting CD19, FLT3, or both (dual) and generated CD19-negative KMT2A-r B-ALL models through CRISPR-induced CD19 gene-knockout (KO). FLT3 CAR T cells showed antitumor efficacy against CD19-KO KMT2A-r B-ALL cells both in vitro and in vivo; dual-targeted CAR T cells showed cytotoxicity against wild-type (WT) and CD19-KO KMT2A-r B-ALL cells, whereas CD19 CAR T cells demonstrated cytotoxicity only against WT KMT2A-r B-ALL cells in vitro. Therefore, targeting FLT3-specific CAR T cells would be a promising strategy for KMT2A-r B-ALL cells even with CD19-negative relapsed cases.

Keywords: CAR T cell therapy; FLT3; KMT2A gene rearrangement; Lineage switch; piggyBac transposon.

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Conflict of interest statement

The authors have no financial relationship to declare related to this study.

Figures

Fig. 1
Fig. 1
Generation of PB-FLT3 CAR T cells and their characteristics. a Schematic representation of a transposon plasmid expressing the FLT3-CAR construct. ITR, internal tandem repeat; TM, transmembrane domain; cyto, cytoplasmic domain. b Representative flow cytometry dot plots of PB-FLT3 CAR T cell characteristics regarding CAR expression, programmed cell death-1 (PD-1) expression, and differentiation profiles in CAR T cells on day 14 after transfection
Fig. 2
Fig. 2
Cytotoxic effects of PB-FLT3 CAR T cells on FLT3-positive ALL cells in vitro. a FLT3 expression in ALL cell lines analyzed using flow cytometry. b Cytotoxicity of PB-FLT3 CAR T cells co-cultured for 72 h with ALL cell lines at the indicated effector-to-target (E:T) ratio. The survival of ALL cells was assessed using flow cytometry (n = 3). c Cytokine levels in the co-culture supernatant containing CAR T cells and ALL cells after 24 h of co-culture (n = 3). All data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001
Fig. 3
Fig. 3
Cytotoxic effects of dual-targeted CAR T cells on CD19-knockout KMT2A-r ALL cells in vitro. a Flow cytometry analysis of CD19 expression in wild-type (WT) and CD19-KO KMT2A-r ALL cells. b Representative flow cytometry dot plots of CAR expression in dual CAR T cells. c Cytotoxicity of dual CAR T cells co-cultured for 72 h with leukemic cell lines at the indicated effector-to-target (E:T) ratio compared to CD19 CAR T cells. The survival of the leukemic cells was assessed using flow cytometry (n = 3). d Cytokine levels in the co-culture supernatant containing CAR T cells and CD19-KO KMT2A-r ALL cells after 24 h of co-culture (n = 3). All data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001
Fig. 4
Fig. 4
PB-FLT3 CAR T cells prolong the survival of CD19-knockout KMT2A-r ALL cells in vivo. We injected 5 × 105 KOCL44 CD19-KO-FFLuc cells into NSG mice via tail vein. Three and six days later, 7.5 × 106 FLT3 CAR T, dual CAR T, CD19 CAR T cells, or D-PBS was injected into the tail vein of each mouse. a Bioluminescence images of groups of five NSG mice after the first intravenous CAR T cell injection. b Tumor volumes of each mouse in each group measured as the total flux (p/s). The FLT3 CAR T cell group demonstrates a significant tumor reduction, measured as the mean total flux at day 27, compared with the CD19 CAR T cell group. P < 0.001. c The Kaplan–Meier plot of overall survival (n = 5 per group). The FLT3 CAR T cell group achieves prolonged tumor control compared to the CD19 CAR T cell group. Log-rank test: **P < 0.01. d Most long-lived mice (#12 and #15) were injected with FLT3 CAR T cells and leukemic cells. Representative dot plots of their bone marrow on days 24 and 48 showing decrease of leukemic cells and expansion of CAR T cells
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References

    1. Maude SL, Laetsch TW, Buechner J, Rives S, Boyer M, Bittencourt H, Bader P, Verneris MR, Stefanski HE, Myers GD, Qayed M, De Moerloose B, Hiramatsu H, Schlis K, Davis KL, Martin PL, Nemecek ER, Yanik GA, Peters C, Baruchel A, Boissel N, Mechinaud F, Balduzzi A, Krueger J, June CH, Levine BL, Wood P, Taran T, Leung M, Mueller KT, Zhang Y, Sen K, Lebwohl D, Pulsipher MA, Grupp SA. Tisagenlecleucel in children and young adults with B-Cell lymphoblastic leukemia. N Engl J Med. 2018;378:439–448. doi: 10.1056/NEJMoa1709866. - DOI - PMC - PubMed
    1. Park JH, Rivière I, Gonen M, Wang X, Sénéchal B, Curran KJ, Sauter C, Wang Y, Santomasso B, Mead E, Roshal M, Maslak P, Davila M, Brentjens RJ, Sadelain M. Long-term follow-up of CD19 CAR therapy in acute lymphoblastic leukemia. N Engl J Med. 2018;378:449–459. doi: 10.1056/NEJMoa1709919. - DOI - PMC - PubMed
    1. Shah NN, Lee DW, Yates B, Yuan CM, Shalabi H, Martin S, Wolters PL, Steinberg SM, Baker EH, Delbrook CP, Stetler-Stevenson M, Fry TJ, Stroncek DF, Mackall CL. Long-term follow-up of CD19-CAR T-cell therapy in children and young adults with B-ALL. J Clin Oncol. 2021;39:1650–1659. doi: 10.1200/jco.20.02262. - DOI - PMC - PubMed
    1. Zhang X, Lu XA, Yang J, Zhang G, Li J, Song L, Su Y, Shi Y, Zhang M, He J, Song D, Lv F, Li W, Wu Y, Wang H, Liu H, Zhou X, He T, Lu P. Efficacy and safety of anti-CD19 CAR T-cell therapy in 110 patients with B-cell acute lymphoblastic leukemia with high-risk features. Blood Adv. 2020;4:2325–2338. doi: 10.1182/bloodadvances.2020001466. - DOI - PMC - PubMed
    1. Ghorashian S, Kramer AM, Onuoha S, Wright G, Bartram J, Richardson R, Albon SJ, Casanovas-Company J, Castro F, Popova B, Villanueva K, Yeung J, Vetharoy W, Guvenel A, Wawrzyniecka PA, Mekkaoui L, Cheung GW, Pinner D, Chu J, Lucchini G, Silva J, Ciocarlie O, Lazareva A, Inglott S, Gilmour KC, Ahsan G, Ferrari M, Manzoor S, Champion K, Brooks T, Lopes A, Hackshaw A, Farzaneh F, Chiesa R, Rao K, Bonney D, Samarasinghe S, Goulden N, Vora A, Veys P, Hough R, Wynn R, Pule MA, Amrolia PJ. Enhanced CAR T cell expansion and prolonged persistence in pediatric patients with ALL treated with a low-affinity CD19 CAR. Nat Med. 2019;25:1408–1414. doi: 10.1038/s41591-019-0549-5. - DOI - PubMed

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