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

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

Masaya Suematsu 1, Shigeki Yagyu 1,✉, Hideki Yoshida 1, Shinya Osone 1, Yozo Nakazawa 2, Kanji Sugita 3, Toshihiko Imamura 1, Tomoko Iehara 1
PMCID: PMC10991605  PMID: 36214866

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.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00262-022-03303-4.

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

Introduction

CD19-targeted chimeric antigen receptor (CAR) T cell therapies have radically improved the outcomes for children and young adults with relapsed/refractory B-acute lymphoblastic leukemia (B-ALL); however, despite impressive initial responses, long-term remission occurs only in half of the patients with B-ALL [1–5]. CD19-negative relapse with CD19 surface expression loss is the predominant cause of treatment failure in patients treated with CD19-targeted CAR T cells; therefore, multi-B-cell antigen-targeted CAR T cells have been developed to overcome this issue [6–11], including CD19/CD22 [7], CD19/CD20 [9], and CD19/BAFF-R [11] dual-specific CAR T cells.

Despite improvements in the clinical management and survival of B-ALL, B-ALL patients with 11q23 translocations/KMT2A rearrangement (KMT2A-r), especially infants, continue to demonstrate the poorest outcomes [12–15]. Furthermore, relapsed cases with loss of B-cell antigens due to a lineage switch from B-ALL to the myeloid phenotype in KMT2A-r positive ALL (KMT2A-r ALL) following CD19-targeted immunotherapy have been reported [16–19]. In such relapsed cases, because of the loss of B lymphoid lineage, B-cell antigen-targeted immunotherapies including CAR T treatment are ineffective.

Fms-related tyrosine kinase-3 (FLT3) is highly expressed in KMT2A-r B-ALL [20, 21]. Using CRISPR-interference functional genomic screening, Nix et al. [22] revealed that FLT3 is the only protein that shows specific upregulation and significant genetic dependence in KMT2A-r B-ALL; therefore, FLT3 is an optimal immunotherapeutic target for KMT2A-r B-ALL. This study aimed to generate CAR T cells with dual targeting of CD19 and FLT3 and evaluate their antitumor efficacy in KMT2A-r B-ALL both in vitro and in vivo.

Materials and methods

Ethics approval statement

This study was approved by the Institutional Review Board of Kyoto Prefectural University of Medicine (Approval Numbers: ERB-C-669 and ERB-C-1406), and recombinant DNA experiments were approved by the safety committee of the Kyoto Prefectural University of Medicine (Approval Numbers 2019-111 and 2019-112). All experiments involving human participants were performed in accordance with the Declaration of Helsinki, and all participants provided written informed consent. All animal experiments and procedures were approved by the Kyoto Prefectural University of Medicine Institutional Review Board (Approval Number: M2020-13).

Blood sample collection and cell lines

Blood samples from healthy donors were obtained with written informed consent (3 donors). Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood samples using density gradient centrifugation and lymphocyte separation medium 1077 (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), followed by multiple washes with Dulbecco’s phosphate-buffered saline (D-PBS; Nakarai Tesque Inc., Kyoto, Japan). The number of living cells was determined using standard trypan blue staining and an automated cell counter (model R1; Olympus, Tokyo, Japan). The human B-cell precursor leukemia (REH), human acute myeloid leukemia (AML) with KMT2A-r (THP-1 and MV4-11), and human T cell leukemia (JURKAT) cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). Infant B-acute lymphoblastoid leukemia with KMT2A-r cell lines (KOCL44 and KOCL58) were established in the department of pediatrics, school of medicine, university of Yamanashi [23]. MV4-11 had FLT3-internal tandem duplication (ITD) mutations, whereas other cell lines had no FLT3 mutations. All cell lines expressing firefly luciferase (FFLuc) and green fluorescent protein (GFP) were obtained by introducing piggyBac transposon (PB)-based pIRII-FFLuc-puroR-GFP [24] into the cells and subsequent fluorescent-activated cell sorting. All cells were cultured in RPMI-1640 medium (Nacalai Tesque Inc.) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and maintained in a humidified incubator at 37 °C in a 5% CO2 atmosphere.

Generation of CD19-knockout cell lines

CD19-knockout cell lines (KOCL44 CD19-KO and KOCL58 CD19-KO) were generated using the CRISPR-Cas9 system and CD19 sgRNA CRISPR All-in-One Lentivirus product (Applied Biological Materials Inc., Richmond, Canada) according to the manufacturer’s instructions. Briefly, a lentiviral plasmid (pLenti-U6-sgRNA-SFFV-Cas9-2A-Puro) was transfected into cells via lentiviral infection. The infected cells were selected for stable expression using puromycin selection and expanded. CD19-deficient clones were sorted, and stable CD19-KO cells were verified using flow cytometry. These CD19-KO cell lines were also transfected with the PB-based pIRII-FFLuc-puroR-GFP plasmid to express FFLuc and GFP.

FLT3 CAR design

We generated PB transposon-mediated transgenes pIRII-FLT3 scFv-CAR-28z and pIRII-FLT3LG-CAR-28z, which enable transduced cells to express FLT3-CAR. For the specific recognition of FLT3, we utilized either FLT3 single-chain fragment variable (scFv) or the natural ligand of FLT3, FLT3LG. We synthesized cDNA containing the VH and VL chains from the single-chain variable regions (scFv) of the FLT3 monoclonal antibody (EB10) [25] fused with the IgG1-derived CH2CH3 sequence and the extracellular domain of human FLT3LG (Accession Number: NP_001450) encoded by FLT3LG (Accession Number: NM_001459), respectively. These were cloned into the PB transposon vector by replacing the CD19 scFv portion of pIRII-CD19-CAR (Supplementary Fig. 1) [26]. To reduce the non-specific recognition of the spacer region by FcγR-expressing macrophages, we replaced the IgG1 CH2CH3 spacer region of the FLT3-CAR vectors with either the IgG1 CH3 region (CH3) [27] or three repeated sequences of Gly-Gly-Gly-Gly-Ser (G4S)3 [28, 29]. The truncated CD34 sequence (Accession Number: NM_001025109) was cloned into pIRII-FLT3-G4S with T2A self-cleaving sites for the detection of CAR expression (Supplementary Fig. 2a). To produce antigen-presenting feeder cells, we generated an antigen-presenting feeder plasmid (pIRII-tFLT3-CD80-41BBL) that enabled transduced cells to express truncated FLT3 protein, CD80, and 4-1BBL. The truncated FLT3 sequence encoded the extracellular, transmembrane, and 20 amino-acid long intracellular portions of the FLT3 protein (Accession Number: NM_004119) and was cloned into the PB transposon vector by replacing the CD19 portion of pIRII-tCD19-CD80-41BBL (Supplementary Fig. 1) [26].

Generating PB-mediated CAR T cells

PB-FLT3 CAR T cells were generated from healthy donor PBMCs using PB transposon-mediated gene transfer, as described previously [24, 26]. Briefly, approximately 20 × 106 PBMCs were electroporated with 7.5 µg of the PB transposase and pIRII-FLT3 CAR transposon plasmids (Supplementary Fig. 2a) using the P3 Primary Cell 4D-Nucleofector™ X kit (Lonza, Basel, Switzerland, Program; FI-115). Concurrently, the antigen-presenting feeder plasmid (pIRII-tFLT3-CD80-41BBL; 15 μg) (Supplementary Fig. 1) was introduced into approximately 10 × 106 PBMCs via electroporation. For FLT3-CD19 dual CAR T cells, approximately 20 × 106 PBMCs were electroporated with 5 µg of the PB transposase plasmid, 7 µg of the pIRII-FLT3 CAR transposon plasmid, and 3 µg of the pIRII-CD19-28z CAR transposon plasmid. Approximately 10 × 106 PBMCs were electroporated with 10 µg of the pIRII-tFLT3-CD80-41BBL plasmid and 5 µg of the pIRII-tCD19-CD80-41BBL plasmid for feeder cells (Supplementary Fig. 1). Following electroporation, CAR T and feeder cells were cultured in a complete culture medium consisting of ALyS™705 Medium (Cell Science & Technology Institute Inc., Miyagi, Japan) supplemented with 5% artificial serum (Animal-free; Cell Science & Technology Institute Inc., Miyagi, Japan), IL-7 (10 ng/mL; Miltenyi Biotec, Bergisch Gladbach, Germany), and IL-15 (5 ng/mL; Miltenyi Biotec, Bergisch Gladbach, Germany). Feeder cells were irradiated with ultraviolet light to inactivate them 24 h after electroporation and co-cultured with CAR T cells for 14 days as described previously [24, 26, 30]. CAR T cells redirected to the EPHB4 receptor were generated using the PB transposon system as described previously [30]; these cells were used as control CAR T cells in the in vitro experiments (Supplementary Fig. 1).

Flow cytometry

The expression of FLT3-CH3-CAR on the T cell surface was measured using flow cytometry with a goat anti-human IgG Fc fragment specific antibody (Merck Millipore, Burlington, MA, USA) and an anti-goat IgG specific antibody conjugated to phycoerythrin (PE) to detect the CH3 region. The expression of FLT3-G4S-CAR was measured using fluorescein an isothiocyanate (FITC)-conjugated anti-CD34 antibody (BioLegend, San Diego, CA, USA). CD19-CAR expression was measured using an FITC-labeled monoclonal anti-FMC63 scFv antibody (ACROBiosystems Inc., Newark, DE, USA). Allophycocyanin (APC)-conjugated anti-PD-1, FITC-conjugated anti-CD45RA, and APC-conjugated anti-CCR7 antibodies (all from BioLegend) were used to characterize CAR T-cell phenotypes. PE-conjugated anti-FLT3 and FITC-conjugated anti-CD19 antibodies were used to determine expression on cell line surfaces, and APC-conjugated anti-CD3 antibody was used to identify CAR T cells in vivo and in vitro (all from BioLegend). All flow cytometry data were acquired using BD Accuri™ C6 Plus (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed using FlowJo™ software (BD Biosciences).

In vitro cytotoxicity

FLT3 CAR T or EPHB4 CAR T (control CAR T) cells and GFP-expressing tumor cells were co-cultured with effector-to-target (E:T) ratios of 0:1 (tumor cells only), 1:10, 1:5, 1:2, and 1:1 in 24-well cell culture plates. Three days later, the cytotoxicity of CAR T cells was assessed using flow cytometry, and the survival of tumor cells was determined by measuring GFP expression and defined by the exclusion of 7-amino-actinomycin D and the absence of CD3 expression.

Cytokine production assay

The levels of interferon (IFN)-γ, tumor necrosis factor (TNF), and IL-2 were measured using a cytometric bead array (CBA) kit (BD Biosciences). Briefly, CAR T cells were co-cultured with tumor cells at a ratio of 1:1. After 24 h of co-culture, the cell culture supernatant was collected, and cytokine levels were determined and analyzed. Data were acquired with a BD Accuri C6 Plus system (BD Biosciences) and analyzed using FCAP Array v.3.0 (BD Biosciences).

In vivo experiments

Six-week-old female NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and housed at the Kyoto Prefectural University of Medicine for over a week before the start of the experiments. Food and water were provided ad libitum. Then, 5 × 105 KOCL44 CD19-KO-FFLuc-GFP cells suspended in D-PBS were injected into mice via the tail vein. Three and six days later, 7.5 × 106 FLT3 CAR T, dual CAR T, CD19 CAR T cells, or D-PBS was infused via the tail vein, and tumor burdens were monitored using the IVIS Lumina Series III system (PerkinElmer, Inc., Waltham, MA, USA), and regions of interest on the displayed images were quantified in photons per second (ph/s) using Living Image v2 (PerkinElmer, Inc.) as described previously [30]. Bone marrow (BM) cells were obtained by sequential BM aspiration from tibias at several time points. The BM cells were stained with an APC-conjugated anti-human CD3 antibody (BioLegend), and the long-term persistence of human T cells was evaluated using flow cytometry. The mice were euthanized at predefined endpoints under conditions that met the euthanasia criteria stipulated by the Center for Comparative Medicine at the Kyoto Prefectural University of Medicine.

Statistical analysis

Statistical comparisons between groups were determined using two-tailed parametric or non-parametric tests (Mann–Whitney U-test) for unpaired data or two-tailed paired Student's t-tests for matched samples. All data are presented as the mean ± standard deviation. The log-rank test was used to compare survival curves obtained using the Kaplan–Meier method. Statistical significance was set at P < 0.05. All statistical analyses were performed using Prism 9 software (GraphPad Software, San Diego, USA).

Results

Generation and characterization of FLT3‐specific CAR T cells via PB transposon‐based gene transfer

First, to identify optimal anti-FLT3 specific CAR T cells, we generated two types of antigen recognition sites for FLT3, the EB10 single-chain variable fragments (scFv) and the natural ligand of FLT3 (FLT3LG), and two types of spacer sites (an IgG1 CH3 region and a G4S linker) (Supplementary Fig. 2a). After 14 days of CAR T cells culture, the total cell number and CAR expression on T cells of these FLT3 CAR T cells were assessed using flow cytometry (Supplementary Fig. 2b, c). FLT3 scFv CH3 CAR T cells showed superior expansion capacity and highest CAR expression after expansion for 14 days; therefore, FLT3 scFv CH3 CAR T cells were selected as the lead CARs for further development (Fig. 1a). PB-FLT3 scFv CH3 CAR T cells were successfully generated with a positivity rate of 47.7% ± 2.6% for FLT3-CAR at day 14 after transfection. PD-1 was scarcely expressed on PB- FLT3 scFv CH3 CAR T cells (1.4% ± 0.7% in CAR-positive T cells), and PB-FLT3 scFv CH3 CAR T cells predominantly comprised the naïve/stem cell memory fraction (CD45RA+CCR7+ fraction; 83.4% ± 4.8%; Fig. 1b).

Fig. 1.

Fig. 1

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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

Cytotoxic effects of PB-FLT3 CAR T cells in FLT3-positive leukemia cells in vitro

We evaluated the cytotoxic effects of PB-FLT3 CAR T cells on leukemic cells using co-culture assays. First, we assessed the expression of FLT3 in leukemic cell lines using flow cytometry; FLT3 was positive in KOCL44, KOCL58, REH, THP-1, and MV4-11 cells and negative in JURKAT cells (Fig. 2a, Supplementary Fig. 3a). PB-FLT3 CAR T and control CAR T cells were co-cultured with these cell lines at various E:T ratios, and the survival of tumor cells was measured using flow cytometry. PB-FLT3 CAR T cells exhibited antigen-specific cytotoxicity against FLT3 positive leukemic cells, in proportion to the E:T ratio (Fig. 2b, Supplementary Fig. 3b). We also evaluated the production of inflammatory cytokines in the cell culture supernatant in response to co-culture with leukemia cells; production of IFN-γ, TNF, and IL-2 was observed only in PB-FLT3 CAR T cells with FLT3-positive leukemia cells (Fig. 2c, Supplementary Fig. 3c).

Fig. 2.

Fig. 2

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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

In vitro therapeutic effects of PB-FLT3 and CD19-dual CAR T cells in CD19-KO infant lymphoblastic leukemia cell lines with KMT2A-r

CD19-negative immune escape is frequently observed in relapsed KMT2A-r B-ALL cells after CD19-targeted therapy [16]. Therefore, we modeled CD19-negative KMT2A-r B-ALL cells via CRISPR-induced CD19 gene-knockout (KO) (KOCL44 CD19-KO and KOCL58 CD19-KO) and confirmed stable and complete CD19-KO cells using flow cytometry (Fig. 3a). To evaluate the efficacy of FLT3-targeted cell therapy against the CD19-negative immune escape model of KMT2A-r B-ALL cells, we introduced the FLT3 and CD19 CAR plasmids into T cells to target both CD19 and FLT3 (dual CAR T). Dual CAR T cells were generated with a positivity rate of 23.0% ± 0.5% for both FLT3 and CD19 CAR, 14 days after transfection (Fig. 3b). We then evaluated the cytotoxicity of either dual CAR T or CD19 CAR T cells against wild-type (WT), CD19-KO KMT2A-r B-ALL, and JURKAT (control) cells in vitro. CD19 CAR T cells showed cytotoxic effects only in WT KMT2A-r B-ALL cells, whereas dual CAR T cells showed cytotoxic effects in both WT and CD19-KO KMT2A-r B-ALL cells, in proportion to the E:T ratio (Fig. 3c). In addition, IFN-γ, TNF, and IL-2 production were observed only in dual CAR T cells with CD19-KO KMT2A-r B-ALL cells (Fig. 3d). These data indicate that FLT3-targeted CAR T cells have a cytotoxic effect in CD19-negative immune escape models of KMT2A-r B-ALL cells.

Fig. 3.

Fig. 3

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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

FLT3-targeted CAR T cells prolong the survival of CD19-KO KMT2A-r B-ALL bearing mice

To evaluate the antitumor efficacy of FLT3-targeted CAR T cells in vivo, KOCL44 CD19-KO-FFLuc bearing NSG female mice were divided into four groups (n = 5 each) and were treated with intravenous injections of FLT3 CAR T, dual CAR T, CD19 CAR T cells, or PBS. FLT3 CAR T cells induced more significant tumor reduction and prolonged median survival compared to CD19 CAR T cells (Fig. 4a–c). Furthermore, in two of the long-lived mice in the FLT3 CAR group, human CD3+ T cells were expanded, and leukemic cells were controlled even after 48 days of treatment, as confirmed by sequential BM studies (Fig. 4d), which indicated the antigen-induced proliferation and long-term functionality of FLT3 CAR T cells in vivo. In contrast, dual CAR T cells did not demonstrate beneficial antitumor efficacy over CD19-CAR T cells or FLT3-CAR T cells in vivo (Fig. 4a–c).

Fig. 4.

Fig. 4

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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

Discussion

In this study, we generated PB transposon-mediated CAR T cells targeting FLT3, which optimized the antigen recognition portion of FLT3 and the spacer region. PB-FLT3 scFv CH3 CAR was selected as the lead CAR, and these CAR T cells showed antitumor efficacy against the CD19-negative immune escape model of KMT2A-r B-ALL cells, both in vitro and in vivo. Furthermore, we generated CAR T cells with dual targeting of CD19 and FLT3, which showed cytotoxic effects against both WT and CD19-KO KMT2A-r B-ALL cells, whereas CD19 CAR T cells demonstrated cytotoxic effects only against WT KMT2A-r B-ALL cells in vitro. Although dual CAR T cells should be further optimized to increase their in vivo cytotoxicity, targeting FLT3-specific CAR T cells would be a promising strategy for KMT2A-r B-ALL cells even with a CD19-negative relapsed model.

KMT2A-r B-ALL is clearly distinguished from both conventional ALL and AML by gene expression profiles and has the feature of constitutive wild-type FLT3 overexpression [20, 21]. In primary KMT2A-r infantile B-ALL samples, high-level FLT3 expression is associated with FLT3 ligand-independent FLT3 activation [31] and is associated with worse outcomes [32]. Even though FLT3 is an optimal target for KMT2A-r B-ALL and FLT3 inhibitor (FLT3i) has been investigated for the treatment of KMT2A-r B-ALL, there was no difference in 3-year event-free survival (EFS) between the KMT2A-r B-ALL patients treated with chemotherapy plus FLT3i lestaurtinib compared to chemotherapy alone [33]. The effectiveness of FLT3i against KMT2A-r B-ALL patients correlated with the pharmacodynamics (PD) profiles and ex vivo sensitivity (EVS) of FLT3i which would be associated with the activation or mutation status of FLT3 on the leukemic blasts. Since FLT3 is highly expressed in KMT2A-r B-ALL regardless of mutations of FLT3 gene [20, 21], and CAR T cells directly recognize FLT3 regardless of its activation or mutation status, FLT3 is an optimal target of CAR T cells for KMT2A-r B-ALL.

Immune escape due to loss of antigen surface expression is a major concern in CAR T cell therapy [34]. In recent clinical trials, relapse after antigen-based immunotherapy or CAR T cell therapy due to loss or diminished expression of the targeted antigen has been observed [1–6], which indicates the importance of targeting multiple antigens on tumor cells to reduce antigen loss-induced relapse. There are several approaches to overcome this issue, such as targeting two different antigens on tumor cells. The most straightforward approach is cocktail therapy with a mixture of varying CAR T cells (pooled CAR) [8], which requires two different CAR T cells products for manufacturing. Another approach is the co-transduction of two other CAR vectors or a bicistronic CAR vector to produce T cells expressing two CARs targeting distinct antigens [35, 36]. However, expressing both CARs at a similar density level is challenging [37], and this dual transduction strategy usually results in a mixture of cells expressing both single and dual transduced CAR T cells. The transduction strategy of a bicistronic CAR vector allows both CARs to be expressed in all transduced cells at the same density level; however, the expression of two different CARs in one vector may require codon optimization, and the combination of co-stimulatory domains in bicistronic CAR T cells also requires optimization [38]. Another alternative approach is to design a receptor with two distinct antigen recognition domains and a single co-stimulatory domain in a tandem CAR [6, 39, 40]. Compared with bicistronic CARs, tandem CARs have the advantage of a smaller transgene size but require optimization of the spacer length and linker sequence between the two antigen recognition domains. Hegde et al. revealed that compared to pooled CAR T cells, bicistronic CAR T cells induced more significant tumor reduction and prolonged median survival, and compared to bicistronic CAR T cells, tandem CAR T cells induced greater tumor reduction and prolonged median survival in vivo [35, 39]. Furthermore, in a recent clinical trial, CAR T cells with dual targeting of CD19 and CD22 treatment in pediatric and young adult patients with relapsed or refractory B-ALL were not found superior to the standard approved CD19 CAR T cell therapy [1, 7]. Therefore, the function of dual-targeted CAR T cells should be improved. In this study, we aimed to demonstrate the importance of dual-targeted CAR T cells in a CD19-KO relapsed model of KMT2A-r B-ALL cells, thus generating dual CAR T cells using a dual transduction strategy. Although dual CAR T cells showed cytotoxicity against CD19-KO KMT2A-r B-ALL cells in vitro, they did not show beneficial antitumor efficacy over CD19 CAR T or FLT3 CAR T cells in vivo, which is a limitation of our study. One of the conceivable factors of inefficiency of dual CAR T cells in vivo is low CAR expression in T cells for both FLT3 and CD19 CAR; only positivity rate of 23.0% ± 0.5% (Fig. 3b). These differences of FLT3 CAR expression in T cells between FLT3 CAR T and dual CAR T may cause differences of in vivo efficacy, so we need to improve the manufacturing of dual CAR T cells to achieve high levels of CAR expression as dual CAR T. Furthermore, a recent study revealed that the combination of a spacer region and transmembrane and co-stimulatory domains should be customized to produce an effective multivalent CAR. Hirabayashi et al. [38] demonstrated that the optimal configuration of dual CAR, in GD2/B7-H3 dual CAR T cells, comprised CD8-derived transmembrane domain with CD28 co-stimulatory domain and CD3ζ chain for one CAR construct, and CD8-derived transmembrane domain with only 4-1BB co-stimulatory domain (no CD3ζ chain) for another CAR construct. Therefore, by exploring the different combinations of each CAR component, the in vivo efficacy of our CD19/FLT3 dual CAR T cells can be further improved.

Although several studies have reported the efficacy of FLT3-targeted CAR T cells in the treatment of AML [41–43], its efficacy in ALL has not been well-studied. Niswander et al. have recently reported that targeting FLT3 by CAR T cells was highly effective against FLT3-mutant AML and KMT2A-r B-ALL. They demonstrated the anti-tumor potency of FLT3 CAR T cells for KMT2A-r B-ALL, including a post-tisagenlecleucel ALL-to-AML lineage switch patient-derived xenograft model [44]. Furthermore, they also demonstrated significant in vitro and in vivo anti-tumor efficacy of bispecific CD19xFLT3 CAR T against KMT2A-r B-ALL. They utilized anti-FLT3-scFv (NC7) as an antigen binding domain with a lower affinity than EB10 anti-FLT3-scFv we used in this study [25]. Moreover, they included CD28 for CD19 CAR and 4-1BB for FLT3 CAR as a co-stimulatory domain. All of these components are strongly associated with the function of CAR T cells, which could be one of the explanations for the differences in the anti-tumor efficacy of CD19xFLT3 CAR T cells between their study and ours. Therefore, the binding affinity of scFv, the co-stimulatory domain for each CAR construct, and the expression vector configuration should be optimized for the better function of dual-targeting CAR T cells.

Since FLT3 is expressed on normal hematopoietic stem and progenitor cells (HSPCs) [45], the off-target cytotoxicity of FLT3 CAR T therapies against HSPCs must be considered. FLT3 CAR T cells have minor cytotoxic effects on normal HSPCs [41, 42]. Furthermore, Sommer et al. [43] revealed that FLT3 CAR T cells showed sensitivity and on-target off-tumor toxicity against HSPCs in vitro, and a correlation between the magnitude of on-tumor activity and the off-tumor effects in vivo. Although we did not estimate the off-target cytotoxicity of FLT3 CAR T cells against normal HSPCs, we suggest the use of suicide switches that inactivate CAR T cell function or rescue hematopoietic stem cell transplantation following FLT3 CAR T cell therapy.

In conclusion, we demonstrated the utility of FLT3 CAR T cells in the treatment of KMT2A-r B-ALL. Targeting myeloid-lineage marker by CAR-T cells is a promising strategy for KMT2A-r B-ALL and would be a potent therapeutic option even for CD19-negative, lineage-switch relapsed cases.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (TIFF 12692 kb) (12.4MB, tiff)
Supplementary file2 (TIFF 12692 kb) (12.4MB, tiff)
Supplementary file3 (TIFF 12692 kb) (12.4MB, tiff)

Acknowledgements

The authors acknowledge Ms. Kumiko Yamashima and Ms. Mami Kotoura for their valuable technical assistance and Ms. Mika Tanimura, Ms. Ryoko Murata, and Ms. Yasuko Hashimoto for their secretarial assistance. The authors also thank Editage (http://www.editage.com) for proofreading, editing, and reviewing this manuscript. This work was supported by the Japan Agency for Medical Research and Development (AMED) (18ck0106413h0001), JSPS KAKENHI (19K08326) and Kawano Masanori Memorial Public Interest Incorporated Foundation for Promotion of Pediatrics.

Author contributions

Conceptualization, SY, YN, SO, TI, and TI; Methodology, MS, SY, HY, and YN; Investigation, MS, SY; Writing—Original Draft, MS, and SY; Writing—Review & Editing, MS, SY, SO, HY, TI, YN, TI, and TI; Funding Acquisition, SY and SO; Resources, SY, TI, and TI; Supervision, SY, TI, and TI.

Declarations

Conflict of interest

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