flt3 wt  (Carna Inc)

Journal: Nature Cancer

Article Title: A T cell receptor targeting a recurrent driver mutation in FLT3 mediates elimination of primary human acute myeloid leukemia in vivo

doi: 10.1038/s43018-023-00642-8

Figure Lengend Snippet: a , Schematic illustration of FLT3. TKD, tyrosine kinase domain. b , Naive CD8 + T cells co-cultured with autologous HLA-A2 + mRNA-transfected moDCs stained with FLT3 D/Y pMHC multimers. c , CD8 + T cells transduced to express TCR FLT3 D/Y stained with FLT3 D/Y pMHC multimers (Gating strategy in Extended Data Fig. ). d , Parallel reaction-monitoring analysis, targeting the FLT3 D835Y peptide ( m / z = 1,091.4389 1+ ) in primary AML cells from two patient samples and the B721.221 cell line transduced to express FLT3 D835Y and HLA-A2. NL = normalization level. e , Off-rates for FLT3 WT or FLT3 D/Y peptide binding to HLA-A2 measured by flow cytometry. Vertical lines indicate calculated half-lives in each experiment. Dots represent mean fluorescence intensity (MFI) values of intact pMHC complexes on fluorescent particles at the indicated time points (h) (one replicate per experiment, n = 3 independent experiments). f , Activation of TCR FLT3D/Y cells (CD137 + ) co-incubated with peptide-pulsed K562 cells. Data points are from n = 4 donors transduced to express TCR in n = 3 independent experiments, with each circle representing the mean of three technical replicates per donor, shown as mean ± s.e.m. g , Activation of CD8 + TCR FLT3D/Y cells co-incubated with HLA-A2 + cell lines with or without FLT3 D/Y peptide. Results are from one experiment representative of n = 4 (BV173, CHP-212, EBV-LCL, K562, Daoy, RS4;11), n = 3 (HaCaT, U-2 OS, FM6, U-87 MG, HeLa, MV-4-11, EoL-1, MOLM-13) or, for the remaining cell lines, n = 2 independent experiments using different T cell donors; data points represent n = 3 technical replicates. The suffix + A2 denotes that cell lines were transduced with HLA-A*02:01, whereas remaining cell lines naturally express it. Connecting lines in f and bars in g show mean. The dashed line in g shows the highest level of activation by cell lines alone. h – j , IFN-γ produced by TCR FLT3D/Y cells co-incubated with K562 cells loaded with peptides from the mimotope library ( h ) or pulsed with the peptides that were predicted as potentially cross-reactive from the in silico search ( i ) or transfected with mRNA constructs encoding 30–32-mer peptides with the candidate cross-reactive peptide inducing reactivity (shown in i ) in the middle, flanked by its naturally occurring sequence, or transfected with mRNA encoding the FLT3 D/Y epitope or FLT3 WT ( j ). White circles in h , amino acids of the FLT3 D/Y peptide. Positive reaction for IFN-γ, 5,000–35,000 pg ml −1 . LZTR1, leucine zipper-like post-translational regulator 1; MED1, mediator complex subunit 1; PRADC1, protease-associated domain-containing protein 1. Data in h – j are from one of n = 2 independent experiments, and individual data points represent one ( h , i ) or three ( j ) technical replicates.

Article Snippet: When indicated, target cells were pulsed with FLT3 D/Y or FLT3 WT peptide (purities >90%) or 161 single-amino acid-substituted variants of the FLT3 D/Y peptide (purity >70%) (GenScript Biotech) for 1–2 h or electroporated with mRNA encoding either FLT3 WT or the FLT3 D/Y minigene.

Techniques: Cell Culture, Transfection, Staining, Targeted Proteomics, Binding Assay, Flow Cytometry, Fluorescence, Activation Assay, Incubation, Transduction, Produced, In Silico, Construct, Sequencing

Journal: Nature Cancer

Article Title: A T cell receptor targeting a recurrent driver mutation in FLT3 mediates elimination of primary human acute myeloid leukemia in vivo

doi: 10.1038/s43018-023-00642-8

Figure Lengend Snippet: (a) Staining of TCR FLT3D/Y cells with pMHC multimers complexed with either the FLT3 D/Y or the FLT3WT peptide (each multimer conjugated to both APC and PE, gating strategy shown in Extended Data Fig. . Data shown is from one representative donor out of two stained in one experiment. (b) Gating strategy for TCR FLT3D/Y cells. Panels show gating on: CD8 + cells labeled with the APC pMHC multimer and antibodies reactive to mouse TCR-β or human TCR-α (middle panels), and CD8 + cells staining positively for APC and PE-labeled pMHC multimers complexed with the FLT3 D/Y peptide (right plot). (c) Percentage of mTCR-β + cells or pMHC multimer + cells among CD8 + cells following transduction with the TCR FLT3D/Y . Each data point represents a different HLA-A2 pos donor ( n = 7 donors from 3 independent experiments). Data are analyzed by unpaired, two-tailed Student’s t-test and p < 0.05 was considered statistically significant. (d) Gating strategy to identify differentiation stages of TCR-transduced T cells three days after spinoculation. Top panels show gating on FSC/SSC hi , Live/Dead Fixable Near-IR neg , CD3 + events. Bottom panels show gating on CD4 + or CD8 + populations and subsequent identification of naïve, central memory (CM), effector (E) and effector memory (EM) cells as defined by expression of CD45RA and CD62L analyzed in two donors in one experiment. (e) Expansion of HLA-A2 pos PB T cells from two donors transduced in parallel with the TCR FLT3D/Y or TCR 1G4 following indicated days after retroviral transduction. Data are from one experiment and dots represent one technical replicate for each HLA-A2 pos PB T cell donor. (f) Activation of TCR 1G4 cells measured as upregulation of CD137 after co-incubation with peptide-pulsed (NY-ESO-1 peptide SLLMWITQC) BV173 cells (K562 cells were not used, in contrast to Fig. , as they express NY-ESO-1). EC 50 = half maximal effective concentration (linear curve fitting). Data shown are from one experiment with one (out of two) T cell donors with individual data points representing technical replicates ( n = 3).

Article Snippet: When indicated, target cells were pulsed with FLT3 D/Y or FLT3 WT peptide (purities >90%) or 161 single-amino acid-substituted variants of the FLT3 D/Y peptide (purity >70%) (GenScript Biotech) for 1–2 h or electroporated with mRNA encoding either FLT3 WT or the FLT3 D/Y minigene.

Techniques: Staining, Labeling, Transduction, Two Tailed Test, Expressing, Retroviral, Activation Assay, Incubation, Concentration Assay

Journal: Nature Cancer

Article Title: A T cell receptor targeting a recurrent driver mutation in FLT3 mediates elimination of primary human acute myeloid leukemia in vivo

doi: 10.1038/s43018-023-00642-8

Figure Lengend Snippet: (a) Predicted binding affinity of FLT3 D/Y and FLT3 WT peptides using the NetMHC-4.0 algorithm. Peptides with affinity <50 nM (EL%Rank <0.500) classify as strong binders, 50 nM500 nM as non- binders . (b) Gating strategy to identify CD8 + T cells staining as double positive events for pMHC multimers (APC- and PE-conjugated) complexed with the FLT3 D/Y peptide. The three left plots show gates used for FSC/SSC, singlets and Live/Dead Fixable Near- IR neg /CD8 + events. The plot to the right shows multimer positive events in co-culture of naïve healthy donor CD8 + T cells with autologous moDCs transfected with target mRNA. (c) The TCR FLT3D/Y sequence. (d) Staining of samples from AML patients taken at point of diagnosis (top row, left three panels) and post allo-HSCT (bottom row) with pMHC multimers. TCR FLT3D/Y transduced T cells were used as a positive control for multimer staining (top right). (e) Staining of samples obtained from patient 2 after allo-HSCT following 5 days of in vitro expansion in absence (left) and presence (right) of the FLT3 D/Y peptide (top panels). As positive control for in vitro expansion of memory T cells, TCR FLT3D/Y T cells were spiked into autologous healthy PBMCs under the same conditions (bottom panels). Inset numbers represent the percentage of pMHC multimer + cells out of CD8 + cells. The graph to the right shows data for all three patients and positive controls. Data shown are from one experiment with each dot representing a technical replicate ( n = 7 for pt 1, n = 12 for pt 2, n = 8 for pt 12 and n = 3 for PBMC healthy donor).

Article Snippet: When indicated, target cells were pulsed with FLT3 D/Y or FLT3 WT peptide (purities >90%) or 161 single-amino acid-substituted variants of the FLT3 D/Y peptide (purity >70%) (GenScript Biotech) for 1–2 h or electroporated with mRNA encoding either FLT3 WT or the FLT3 D/Y minigene.

Techniques: Binding Assay, Staining, Co-Culture Assay, Transfection, Sequencing, Biomarker Discovery, Positive Control, In Vitro

Journal: Nature Cancer

Article Title: A T cell receptor targeting a recurrent driver mutation in FLT3 mediates elimination of primary human acute myeloid leukemia in vivo

doi: 10.1038/s43018-023-00642-8

Figure Lengend Snippet: Mirror image plots comparing the fragmentation spectra of endogenous FLT3 D/Y peptide to its isotopically labelled counterpart YI(13C6,15 N)MSDSNYV. Spectra obtained from immunoprecipitated HLA derived from the B721.221 cell line retrovirally transduced with a minigene encoding the D835Y mutation, and from patient 1 and patient 3, are displayed.

Article Snippet: When indicated, target cells were pulsed with FLT3 D/Y or FLT3 WT peptide (purities >90%) or 161 single-amino acid-substituted variants of the FLT3 D/Y peptide (purity >70%) (GenScript Biotech) for 1–2 h or electroporated with mRNA encoding either FLT3 WT or the FLT3 D/Y minigene.

Techniques: Immunoprecipitation, Derivative Assay, Transduction, Mutagenesis

Journal: Nature Cancer

Article Title: A T cell receptor targeting a recurrent driver mutation in FLT3 mediates elimination of primary human acute myeloid leukemia in vivo

doi: 10.1038/s43018-023-00642-8

Figure Lengend Snippet: (a) Graphs depicting IFN-γ response of TCR FLT3D/Y cells to K562 cells loaded with individual peptides from mimotope library containing a total of 161 nine-mers for the FLT3 D/Y peptide, at a concentration of 10 −8 M. Purple dots in each graph represent response to the FLT3 D/Y peptide. Substituted amino acid in the original peptide is highlighted. IFN-γ concentration range for positive reactions was 5000–35000 pg/mL (cut-off indicated by horizontal lines). Graphs show results from two independent experiments that were performed, one technical replicate (dot) per peptide and experiment. (b) Peptide reactivity motifs for FLT3 D/Y that were queried in the ScanProsite search tool against human proteome databases. Amino acids in square brackets [] indicate alternatives that are allowed for the given position in the peptide motifs. (c) mRNA-encoded amino acid sequences(30-32mers) derived from the sequence of the human proteins LZTR1, MED1 and PRADC1, with the candidate cross-reactive peptide identified in the ScanProsite database underlined. (d) Expression of mRNA encoding LZTR1, MED1 and PRADC1 sequences following electroporation of HLA-A2 pos K562 cells as measured by GFP-reporter fluorescence using flow cytometry.

Article Snippet: When indicated, target cells were pulsed with FLT3 D/Y or FLT3 WT peptide (purities >90%) or 161 single-amino acid-substituted variants of the FLT3 D/Y peptide (purity >70%) (GenScript Biotech) for 1–2 h or electroporated with mRNA encoding either FLT3 WT or the FLT3 D/Y minigene.

Techniques: Concentration Assay, Derivative Assay, Sequencing, Expressing, Electroporation, Fluorescence, Flow Cytometry

Journal: Nature Cancer

Article Title: A T cell receptor targeting a recurrent driver mutation in FLT3 mediates elimination of primary human acute myeloid leukemia in vivo

doi: 10.1038/s43018-023-00642-8

Figure Lengend Snippet: (a) Mutation data (SNVs and indels) from AML patients reported in Papaemmanuil et al , 2016 NEJM were downloaded from https://www.cbioportal.org/ . VAF was estimated from reported alternative allele reads divided by sequencing depth for the position. Patients harboring a FLT3 D835Y mutation were selected for in-depth analysis and displayed here. Patients are sorted from right to left within each subsection in descending order of FLT3 D835Y VAF. The following genes were considered as initiating events in clonal hematopoiesis (CH): DNMT3A, TET2, ASXL1, PPM1D, JAK2, SF3B1, SRSF2, TP53 , GNA S and GNB1 . (b) Mutation data from FLT3 D835Y positive AML patients ( n = 9) reported in Morita et al , 2020 Nat Com were downloaded. The fraction of cells with mutations (mutant cell fraction) in each AML patient with mutations are plotted in indicated colors. Patients were categorized into 3 groups; FLT3 D835Y largest, only preceded by mutations in DNMT3A, TET2 , and/or ASXL1 (DTA) and/or splicing factor mutations ( SF3B1, SRAF2 , and U2AF1 ) which are closely related with clonal hematopoiesis ‘CH’, and the rest of cases with FLT3 D835Y mutation according to the cell fraction of mutations. Interquartile range (IQR) and median values are shown. The dashed lines indicate 1.5xIQR and the dots indicate outliers.

Article Snippet: When indicated, target cells were pulsed with FLT3 D/Y or FLT3 WT peptide (purities >90%) or 161 single-amino acid-substituted variants of the FLT3 D/Y peptide (purity >70%) (GenScript Biotech) for 1–2 h or electroporated with mRNA encoding either FLT3 WT or the FLT3 D/Y minigene.

Techniques: Mutagenesis, Sequencing

Journal: Nature Cancer

Article Title: A T cell receptor targeting a recurrent driver mutation in FLT3 mediates elimination of primary human acute myeloid leukemia in vivo

doi: 10.1038/s43018-023-00642-8

Figure Lengend Snippet: a , Percentage myeloid cells of live leukocytes for patients (Pt.) 1–8 with AML; gating strategy is shown in Extended Data Fig. . Dots represents technical replicates from one representative experiment as described in d . b , PB or BM FLT3 D/Y VAF for patients 1–8 as determined by next-generation sequencing. c , Representative t -distributed stochastic neighbor embedding ( t -SNE) plots showing live primary myeloid cells (CD3 − CD19 − CD20 − events) in red, T cells (CD3 + ) in blue, B cells (CD19 + CD20 + ) in orange and normal CD34 + lin − progenitor cells in green from n = 3 representative HLA-A2 + FLT3 D/Y patients (patients 2, 3 and 6) with AML and one HLA-A2 − FLT3 D/Y patient (patient 8) following 72 h of co-culture with TCR 1G4 (negative control, top) or TCR FLT3D/Y cells (E:T ratio, 1:2) as quantified by flow cytometry. Cells transduced to express TCR were excluded from analysis as CellTrace Violet (CTV)-positive events. d , Diagnostic samples from 11 patients with AML and the FLT3 D/Y (patients 1–8), FLT3 D/E (patient 9) or FLT3 D/H (patient 10) mutation or FLT3 WT (patient 11) (all HLA-A2 + except patient 8), analyzed as described in c . Each dot represents the fraction of live myeloid cells, B cells or T cells after co-culture with TCR FLT3D/Y cells (purple) in percent mean of the corresponding numbers in cultures treated with TCR 1G4 cells (blue). Data points represent n = 3 technical replicates, and horizontal lines show means. Data shown are from one experiment representative of two to four experiments performed for each patient sample ( n = 1 only for patient 7). e , t -SNE plots of PB diagnostic samples from patients 2 and 3 with AML showing live myeloid, T and B cells (color coded as in Fig. 2c) after 72 h of co-culture with autologous T cells transduced to express TCR FLT3D/Y or the mock control. Inset numbers in c , e denote absolute event counts of the indicated cell populations. The gating strategy is shown in Extended Data Fig. .

Article Snippet: When indicated, target cells were pulsed with FLT3 D/Y or FLT3 WT peptide (purities >90%) or 161 single-amino acid-substituted variants of the FLT3 D/Y peptide (purity >70%) (GenScript Biotech) for 1–2 h or electroporated with mRNA encoding either FLT3 WT or the FLT3 D/Y minigene.

Techniques: Next-Generation Sequencing, Co-Culture Assay, Negative Control, Flow Cytometry, Diagnostic Assay, Mutagenesis, Control

Journal: Nature Cancer

Article Title: A T cell receptor targeting a recurrent driver mutation in FLT3 mediates elimination of primary human acute myeloid leukemia in vivo

doi: 10.1038/s43018-023-00642-8

Figure Lengend Snippet: (a) Flow cytometry plots showing gating strategy to identify cell subsets in AML patient samples from PB (patient 2-6) or BM (patient 1). Cells are gated on FSC/SSC hi , single, Live/Dead Fixable Near-IR events (top row), showing the fractions of CD3 + T cells, CD19 + CD20 + B cells and myeloid cells, defined as CD3 − CD19 − CD20 − events in the bottom row. (b) Populations were overlaid on a t - SNE plot (patient 1) with designated colors as indicated. Inset numbers show event counts for myeloid cells after co-culture with TCR 1G4 or TCR FLT3D/Y cells, quantified as shown in e. (c) Quantification of normal CD19 + B cells isolated from n = 3 healthy blood donors (Buffy coat (BC) 1 - BC 3) and tumor cells from patient 1 (positive control) after performing the flow cytometry-based cytotoxicity assay for 72 h. Data points represent n = 3 technical replicates from one experiment and horizontal lines show mean. (d) Bar graph showing IFN-γ response of Mock (gray) and TCR FLT3D/Y cells (purple) after 24 h co-culture with HLA-A2 pos patient cells expressing the FLT3 D835Y (Pt.1-6), FLT3 D835E (Pt.9) or FLT3 D835H (Pt. 10), or Pt.8 cells expressing FLT3 D835Y but being HLA-A2 neg . Data points represent n = 3 technical replicates and bars show mean. Data shown are from one experiment representative of at least two performed for each patient sample. (e) Gating strategy for flow cytometry cytotoxicity assay to quantify viable cells in subpopulations from AML patients 1-3 after 72 h of co-culture with autologous T cells either expressing TCR FLT3D/Y (bottom row) or mock-transduced. Transduced T cells were labeled with cell-trace violet (shown in upper right plot) prior to co-culture with AML cells to distinguish them from T cells in the AML samples. Patient cell subsets are gated on FSC/SSC, singlets, Live/Dead Fixable Near-IR neg , CTV neg events. Numbers indicate absolute counts for CD3 + , CD19 + /CD20 + and myeloid cells after co-culture, as determined by addition of fluorescent beads (10,000) into each well and where 3,500 beads were acquired for flow cytometry analysis (shown in upper left plot).

Article Snippet: When indicated, target cells were pulsed with FLT3 D/Y or FLT3 WT peptide (purities >90%) or 161 single-amino acid-substituted variants of the FLT3 D/Y peptide (purity >70%) (GenScript Biotech) for 1–2 h or electroporated with mRNA encoding either FLT3 WT or the FLT3 D/Y minigene.

Techniques: Flow Cytometry, Co-Culture Assay, Isolation, Positive Control, Cytotoxicity Assay, Expressing, Labeling

Journal: Nature Cancer

Article Title: A T cell receptor targeting a recurrent driver mutation in FLT3 mediates elimination of primary human acute myeloid leukemia in vivo

doi: 10.1038/s43018-023-00642-8

Figure Lengend Snippet: (a) Flow cytometry histograms showing expression of FLT3 D/Y as measured by GFP- reporter fluorescence in transduced AML and B-ALL cell lines. Negative control (non-transduced BV173 cells) in top histogram. (b) Remaining viable FLT3 D/Y -transduced, HLA-A2+ cells (purple dots) after 24 h co-culture with TCR FLT3D/Y cells (E:T ratio of 1:2), in percent of corresponding numbers following treatment with mock-transduced T cells (grey dots), quantified by flow cytometry. +A2 denotes that HLA-A2 was introduced by transduction. Data points are from n = 3 independent experiments with each dot representing the mean of n = 3 technical replicates in each experiment and are shown as mean ± s.e.m. Gating strategy and quantification as shown in Extended Data Fig. . (c) Schematic overview of the BV173 D835Y in vivo model. (d) Bioluminescence imaging (BLI) analysis of NSG mice day 13 after BV173 D835Y cell injection, one day prior to T-cell therapy. Data shown are from one experiment, with mice grouped into untreated ( n = 3 mice), TCR 1G4 ( n = 3 mice) and TCR FLT3D/Y ( n = 4 mice) cell treated. (e) BLI of BV173 D835Y engrafted leukemic cells in mice on indicated days relative to treatment with TCR 1G4 or TCR FLT3D/Y cells, or no treatment. (f) Quantification of BV173 D835Y engrafted leukemic cells in mice, 21 days after treatment with TCR 1G4 or TCR FLT3D/Y cells or left untreated. (g) Flow cytometry plots showing BM tumor burden in two untreated and two TCR 1G4 cell-treated mice at time of sacrifice (d 21), as well as two TCR FLT3D/Y cell treated mice sacrificed at end of experiment (d 53). (h) Percentage of bone marrow BV173 D835Y leukemic cells in mice treated with TCR 1G4 or TCR FLT3D/Y cells, or left untreated, out of total mouse and human CD45 + cells (21-53 days after treatment start). (i) Percentage of TCR-β + cells out of human CD8 + cells in the BM of mice analyzed at point of sacrifice. Data in d, f, h, i are presented as mean ± s.e.m, are from one experiment with each dot representing an individual mouse, and are analyzed by unpaired, two-tailed Student’s t-test. P values are shown and p < 0.05 was considered statistically significant.

Article Snippet: When indicated, target cells were pulsed with FLT3 D/Y or FLT3 WT peptide (purities >90%) or 161 single-amino acid-substituted variants of the FLT3 D/Y peptide (purity >70%) (GenScript Biotech) for 1–2 h or electroporated with mRNA encoding either FLT3 WT or the FLT3 D/Y minigene.

Techniques: Flow Cytometry, Expressing, Fluorescence, Negative Control, Co-Culture Assay, Transduction, In Vivo, Imaging, Injection, Two Tailed Test

Journal: Nature Cancer

Article Title: A T cell receptor targeting a recurrent driver mutation in FLT3 mediates elimination of primary human acute myeloid leukemia in vivo

doi: 10.1038/s43018-023-00642-8

Figure Lengend Snippet: a , Schematic overview of the PDX in vivo model with FLT3 D835Y -mutated primary AML cells from patient 7. b , Percentage of human hCD45 + CD33 + cells in PB at baseline (1 d before T cell infusion) and on the indicated days after infusion with TCR 1G4 ( n = 6 mice) or TCR FLT3D/Y ( n = 7 mice) cells. Numbers were adjusted for hCD3 + T cells. c , Representative flow cytometry plots of viable single BM mononuclear cells (MNCs) from TCR 1G4 (top) and TCR FLT3D/Y (bottom) cell-treated NSG-SGM3 mice stably engrafted with primary AML FLT3 D/Y cells from patient 7. d , Percentage of hCD45 + CD33 + cells in the BM and spleen at terminal analysis 15 d after T cell infusion. Numbers were adjusted for hCD3 + T cells. e , Number of FLT3 D835Y -mutated BM hCD45 + CD3 − cells determined by ddPCR. f , g , Number of mouse (m)CD45 + cells in BM ( f ) and mTCR-β + CD8 + cells in the BM and spleen ( g ) at the endpoint. All data are presented as mean ± s.e.m. and were generated from one experiment including six mice treated with TCR 1G4 cells and seven mice treated with TCR FLT3D/Y cells. Each dot represents one mouse, and statistical analysis was performed with two-tailed Mann–Whitney test. P values are shown, and P < 0.05 was considered statistically significant.

Article Snippet: When indicated, target cells were pulsed with FLT3 D/Y or FLT3 WT peptide (purities >90%) or 161 single-amino acid-substituted variants of the FLT3 D/Y peptide (purity >70%) (GenScript Biotech) for 1–2 h or electroporated with mRNA encoding either FLT3 WT or the FLT3 D/Y minigene.

Techniques: In Vivo, Flow Cytometry, Stable Transfection, Generated, Two Tailed Test, MANN-WHITNEY

Journal: Nature Cancer

Article Title: A T cell receptor targeting a recurrent driver mutation in FLT3 mediates elimination of primary human acute myeloid leukemia in vivo

doi: 10.1038/s43018-023-00642-8

Figure Lengend Snippet: (a) Representative FACS plots of viable single PB MNCs from TCR 1G4 (top) and TCR FLT3D/Y (bottom) T cell-treated NSG-SGM3 mice stably engrafted with primary AML FLT3 D835Y cells from patient 7 at 14 days post T cell infusion. Equivalent gating as shown was also used for spleen. (b) Number of total MNCs in BM and spleen of TCR T cell treated mice. (c) Number of mouse MNCs (mCD45+ cells) in spleen. (d) Number of total hCD3+ cells in BM and spleen. (e-f) Percentage of mTCR-β + CD8 + cells of hCD3 + cells (e) and percentage of mTCR-β + cells of hCD8+ cells (f) in T cell samples used for injection and at indicated days after T cell injection, in PB . (g) Distribution (%) of CD4 and CD8 cells within the human CD3 + T cell samples used for injection, and in PB, BM and spleen 14-15 days after injection into mice. All data are from terminal analysis 15 days post T-cell infusion if not otherwise stated. The data are presented as mean ± s.e.m. of n = 6 individual mice treated with TCR 1G4 cells and of n = 7 individual mice treated with TCR FLT3D/Y cells, measured in one experiment. Each dot represents one mouse and statistical analysis was performed with two-tailed Mann-Whitney test. P values are shown and p < 0.05 was considered statistically significant.

Article Snippet: When indicated, target cells were pulsed with FLT3 D/Y or FLT3 WT peptide (purities >90%) or 161 single-amino acid-substituted variants of the FLT3 D/Y peptide (purity >70%) (GenScript Biotech) for 1–2 h or electroporated with mRNA encoding either FLT3 WT or the FLT3 D/Y minigene.

Techniques: Stable Transfection, Injection, Two Tailed Test, MANN-WHITNEY

Journal: Nature Cancer

Article Title: A T cell receptor targeting a recurrent driver mutation in FLT3 mediates elimination of primary human acute myeloid leukemia in vivo

doi: 10.1038/s43018-023-00642-8

Figure Lengend Snippet: a , Schematic overview of the PDX in vivo model with FLT3 D835Y -mutated primary AML cells from patient 1. b , Representative flow cytometry plots of BM from TCR 1G4 (top) and TCR FLT3D/Y (bottom) cell-treated NSG-SGM3 mice stably engrafted with primary AML FLT3 D/Y cells from patient 1. Equivalent gating was also used for PB and the spleen. c , Percentage of hCD45 + CD33 + CD34 + and hCD45 + CD33 + CD34 − cells in the BM, PB and spleen at the endpoint (day 34 after T cell infusion) of TCR 1G4 ( n = 4 mice) or TCR FLT3D/Y ( n = 4 mice) cell-treated mice. Numbers were adjusted for hCD3 + T cells. NS, not significant. d , Percentage VAF determined by ddPCR of FLT3 D835Y and WT1 H507P driver mutations in primary BM cells from patient 1 (top) and hCD45 + CD33 + CD34 + (middle) and hCD45 + CD33 + CD34 − (bottom) cells from TCR cell-treated mice. N/D, not analyzed due to insufficient hCD45 + CD33 + CD34 + cells. Numbers show VAF and 95% confidence intervals. The dashed line at 50% indicates 100% clonality unless loss of heterozygosity. No significant differences in VAFs of the FLT3 D835Y and WT1 H507P mutations were observed. e , Number of FLT3 D835Y -mutated hCD45 + CD33 + CD34 + and hCD45 + CD33 + CD34 − cells in the BM as determined by ddPCR. N/D, not detected due to lack of hCD45 + CD33 + CD34 + cells. f , Number of mTCR-β + CD8 + cells in the BM and spleen at the endpoint. All data are presented as mean ± s.e.m. from terminal analysis 34 d after T cell infusion from one experiment including four mice treated with TCR 1G4 cells and another four mice treated with TCR FLT3D/Y cells. Each dot represents one mouse, and statistical analysis was performed with two-tailed Mann–Whitney test. P values are shown, and P < 0.05 was considered statistically significant.

Article Snippet: When indicated, target cells were pulsed with FLT3 D/Y or FLT3 WT peptide (purities >90%) or 161 single-amino acid-substituted variants of the FLT3 D/Y peptide (purity >70%) (GenScript Biotech) for 1–2 h or electroporated with mRNA encoding either FLT3 WT or the FLT3 D/Y minigene.

Techniques: In Vivo, Flow Cytometry, Stable Transfection, Two Tailed Test, MANN-WHITNEY

Journal: Nature Cancer

Article Title: A T cell receptor targeting a recurrent driver mutation in FLT3 mediates elimination of primary human acute myeloid leukemia in vivo

doi: 10.1038/s43018-023-00642-8

Figure Lengend Snippet: (a) Percentage of CD33 + cells in BM and PB at endpoint (day 34 post T cell infusion) of TCR 1G4 cell ( n = 4) or TCR FLT3D/Y cell ( n = 4) treated mice. Numbers adjusted for hCD3 + T cells. (b) Number of total MNCs in BM and spleen of TCR T cell treated mice. (c) Representative ddPCR plots of BM from TCR cell-treated mice. Numbers in quadrants represent %VAF (mean ± s.e.m.) of the FLT3 D/Y in BM among hCD45 + CD33 + CD34 + , hCD45 + CD33 + CD34 − and hCD45 + CD19 + cells from TCR FLT3D/Y cell-treated (left) and TCR 1G4 cell-treated (right) mice. ‘No remaining cells’ due to elimination of hCD45 + CD33 + CD34 + cells in TCR FLT3D/Y cell-treated mice. (d) Number of total hCD3 + cells in BM and spleen. (e-g) Percentage of mTCR-β + CD8 + cells of hCD3 + cells (e) and percentage of mTCR-β + cells of hCD8 + cells (f) in sample used for injection and at indicated days after T cell infusion in PB. (g) Distribution (%) of CD4 and CD8 cells within the human CD3 + T cell samples analyzed prior to injection, and in PB, BM and spleen of individual mice at endpoint. All data are from terminal analysis 34 days post T-cell infusion unless otherwise stated. The data are presented as mean ± s.e.m. of n = 4 individual mice treated with TCR 1G4 cells and of n = 4 mice treated with TCR FLT3D/Y cells, measured in one experiment. Each dot represents one mouse and statistical analysis was performed with two-tailed Mann-Whitney test. P values are shown and p < 0.05 was considered statistically significant.

Article Snippet: When indicated, target cells were pulsed with FLT3 D/Y or FLT3 WT peptide (purities >90%) or 161 single-amino acid-substituted variants of the FLT3 D/Y peptide (purity >70%) (GenScript Biotech) for 1–2 h or electroporated with mRNA encoding either FLT3 WT or the FLT3 D/Y minigene.

Techniques: Injection, Two Tailed Test, MANN-WHITNEY

Journal: Nature Cancer

Article Title: A T cell receptor targeting a recurrent driver mutation in FLT3 mediates elimination of primary human acute myeloid leukemia in vivo

doi: 10.1038/s43018-023-00642-8

Figure Lengend Snippet: a , Schematic overview of the MRD PDX in vivo model with FLT3 D835Y -mutated primary AML cells from patient 1. b , Percentage of hCD45 + CD33 + cells in the BM of NSG mice engrafted with low levels of AML after treatment with TCR 1G4 ( n = 4 mice) or TCR FLT3D/Y ( n = 4 mice) cells 11 d after T cell infusion. Numbers were adjusted for hCD3 + T cells. Data are presented as mean ± s.e.m. and were generated from one experiment. Each dot represents one mouse, and statistical analysis was performed with two-tailed Mann–Whitney test. c , Schematic overview of the PDX in vivo model with FLT3 D835Y -mutated primary AML cells from patient 1 after in vitro targeting with TCR 1G4 or TCR FLT3D/Y cells. d , Percentage of hCD45 + CD33 + cells in PB at the indicated time after transplantation of primary AML cells from patient 1 following 48 h of co-culture without T cells ( n = 3 mice) or with TCR 1G4 ( n = 3 mice) or TCR FLT3D/Y ( n = 5 mice) cells. Data are presented as mean ± s.e.m. and were generated from two independent experiments. Each dot represents one mouse, and statistical analysis was performed by multilevel linear regression using the R package ‘lmerTest’ (further described in the ). P values are shown, and P < 0.05 was considered statistically significant.

Article Snippet: When indicated, target cells were pulsed with FLT3 D/Y or FLT3 WT peptide (purities >90%) or 161 single-amino acid-substituted variants of the FLT3 D/Y peptide (purity >70%) (GenScript Biotech) for 1–2 h or electroporated with mRNA encoding either FLT3 WT or the FLT3 D/Y minigene.

Techniques: In Vivo, Generated, Two Tailed Test, MANN-WHITNEY, In Vitro, Transplantation Assay, Co-Culture Assay

Journal: Nature Cancer

Article Title: A T cell receptor targeting a recurrent driver mutation in FLT3 mediates elimination of primary human acute myeloid leukemia in vivo

doi: 10.1038/s43018-023-00642-8

Figure Lengend Snippet: a , Schematic illustration of FLT3. TKD, tyrosine kinase domain. b , Naive CD8 + T cells co-cultured with autologous HLA-A2 + mRNA-transfected moDCs stained with FLT3 D/Y pMHC multimers. c , CD8 + T cells transduced to express TCR FLT3 D/Y stained with FLT3 D/Y pMHC multimers (Gating strategy in Extended Data Fig. ). d , Parallel reaction-monitoring analysis, targeting the FLT3 D835Y peptide ( m / z = 1,091.4389 1+ ) in primary AML cells from two patient samples and the B721.221 cell line transduced to express FLT3 D835Y and HLA-A2. NL = normalization level. e , Off-rates for FLT3 WT or FLT3 D/Y peptide binding to HLA-A2 measured by flow cytometry. Vertical lines indicate calculated half-lives in each experiment. Dots represent mean fluorescence intensity (MFI) values of intact pMHC complexes on fluorescent particles at the indicated time points (h) (one replicate per experiment, n = 3 independent experiments). f , Activation of TCR FLT3D/Y cells (CD137 + ) co-incubated with peptide-pulsed K562 cells. Data points are from n = 4 donors transduced to express TCR in n = 3 independent experiments, with each circle representing the mean of three technical replicates per donor, shown as mean ± s.e.m. g , Activation of CD8 + TCR FLT3D/Y cells co-incubated with HLA-A2 + cell lines with or without FLT3 D/Y peptide. Results are from one experiment representative of n = 4 (BV173, CHP-212, EBV-LCL, K562, Daoy, RS4;11), n = 3 (HaCaT, U-2 OS, FM6, U-87 MG, HeLa, MV-4-11, EoL-1, MOLM-13) or, for the remaining cell lines, n = 2 independent experiments using different T cell donors; data points represent n = 3 technical replicates. The suffix + A2 denotes that cell lines were transduced with HLA-A*02:01, whereas remaining cell lines naturally express it. Connecting lines in f and bars in g show mean. The dashed line in g shows the highest level of activation by cell lines alone. h – j , IFN-γ produced by TCR FLT3D/Y cells co-incubated with K562 cells loaded with peptides from the mimotope library ( h ) or pulsed with the peptides that were predicted as potentially cross-reactive from the in silico search ( i ) or transfected with mRNA constructs encoding 30–32-mer peptides with the candidate cross-reactive peptide inducing reactivity (shown in i ) in the middle, flanked by its naturally occurring sequence, or transfected with mRNA encoding the FLT3 D/Y epitope or FLT3 WT ( j ). White circles in h , amino acids of the FLT3 D/Y peptide. Positive reaction for IFN-γ, 5,000–35,000 pg ml −1 . LZTR1, leucine zipper-like post-translational regulator 1; MED1, mediator complex subunit 1; PRADC1, protease-associated domain-containing protein 1. Data in h – j are from one of n = 2 independent experiments, and individual data points represent one ( h , i ) or three ( j ) technical replicates.

Article Snippet: When indicated, target cells were pulsed with FLT3 D/Y or FLT3 WT peptide (purities >90%) or 161 single-amino acid-substituted variants of the FLT3 D/Y peptide (purity >70%) (GenScript Biotech) for 1–2 h or electroporated with mRNA encoding either FLT3 WT or the FLT3 D/Y minigene.

Techniques: Cell Culture, Transfection, Staining, Targeted Proteomics, Binding Assay, Flow Cytometry, Fluorescence, Activation Assay, Incubation, Transduction, Produced, In Silico, Construct, Sequencing

Journal: Nature Cancer

Article Title: A T cell receptor targeting a recurrent driver mutation in FLT3 mediates elimination of primary human acute myeloid leukemia in vivo

doi: 10.1038/s43018-023-00642-8

Figure Lengend Snippet: (a) Staining of TCR FLT3D/Y cells with pMHC multimers complexed with either the FLT3 D/Y or the FLT3WT peptide (each multimer conjugated to both APC and PE, gating strategy shown in Extended Data Fig. . Data shown is from one representative donor out of two stained in one experiment. (b) Gating strategy for TCR FLT3D/Y cells. Panels show gating on: CD8 + cells labeled with the APC pMHC multimer and antibodies reactive to mouse TCR-β or human TCR-α (middle panels), and CD8 + cells staining positively for APC and PE-labeled pMHC multimers complexed with the FLT3 D/Y peptide (right plot). (c) Percentage of mTCR-β + cells or pMHC multimer + cells among CD8 + cells following transduction with the TCR FLT3D/Y . Each data point represents a different HLA-A2 pos donor ( n = 7 donors from 3 independent experiments). Data are analyzed by unpaired, two-tailed Student’s t-test and p < 0.05 was considered statistically significant. (d) Gating strategy to identify differentiation stages of TCR-transduced T cells three days after spinoculation. Top panels show gating on FSC/SSC hi , Live/Dead Fixable Near-IR neg , CD3 + events. Bottom panels show gating on CD4 + or CD8 + populations and subsequent identification of naïve, central memory (CM), effector (E) and effector memory (EM) cells as defined by expression of CD45RA and CD62L analyzed in two donors in one experiment. (e) Expansion of HLA-A2 pos PB T cells from two donors transduced in parallel with the TCR FLT3D/Y or TCR 1G4 following indicated days after retroviral transduction. Data are from one experiment and dots represent one technical replicate for each HLA-A2 pos PB T cell donor. (f) Activation of TCR 1G4 cells measured as upregulation of CD137 after co-incubation with peptide-pulsed (NY-ESO-1 peptide SLLMWITQC) BV173 cells (K562 cells were not used, in contrast to Fig. , as they express NY-ESO-1). EC 50 = half maximal effective concentration (linear curve fitting). Data shown are from one experiment with one (out of two) T cell donors with individual data points representing technical replicates ( n = 3).

Article Snippet: When indicated, target cells were pulsed with FLT3 D/Y or FLT3 WT peptide (purities >90%) or 161 single-amino acid-substituted variants of the FLT3 D/Y peptide (purity >70%) (GenScript Biotech) for 1–2 h or electroporated with mRNA encoding either FLT3 WT or the FLT3 D/Y minigene.

Techniques: Staining, Labeling, Transduction, Two Tailed Test, Expressing, Retroviral, Activation Assay, Incubation, Concentration Assay

Journal: Nature Cancer

Article Title: A T cell receptor targeting a recurrent driver mutation in FLT3 mediates elimination of primary human acute myeloid leukemia in vivo

doi: 10.1038/s43018-023-00642-8

Figure Lengend Snippet: (a) Predicted binding affinity of FLT3 D/Y and FLT3 WT peptides using the NetMHC-4.0 algorithm. Peptides with affinity <50 nM (EL%Rank <0.500) classify as strong binders, 50 nM500 nM as non- binders . (b) Gating strategy to identify CD8 + T cells staining as double positive events for pMHC multimers (APC- and PE-conjugated) complexed with the FLT3 D/Y peptide. The three left plots show gates used for FSC/SSC, singlets and Live/Dead Fixable Near- IR neg /CD8 + events. The plot to the right shows multimer positive events in co-culture of naïve healthy donor CD8 + T cells with autologous moDCs transfected with target mRNA. (c) The TCR FLT3D/Y sequence. (d) Staining of samples from AML patients taken at point of diagnosis (top row, left three panels) and post allo-HSCT (bottom row) with pMHC multimers. TCR FLT3D/Y transduced T cells were used as a positive control for multimer staining (top right). (e) Staining of samples obtained from patient 2 after allo-HSCT following 5 days of in vitro expansion in absence (left) and presence (right) of the FLT3 D/Y peptide (top panels). As positive control for in vitro expansion of memory T cells, TCR FLT3D/Y T cells were spiked into autologous healthy PBMCs under the same conditions (bottom panels). Inset numbers represent the percentage of pMHC multimer + cells out of CD8 + cells. The graph to the right shows data for all three patients and positive controls. Data shown are from one experiment with each dot representing a technical replicate ( n = 7 for pt 1, n = 12 for pt 2, n = 8 for pt 12 and n = 3 for PBMC healthy donor).

Article Snippet: When indicated, target cells were pulsed with FLT3 D/Y or FLT3 WT peptide (purities >90%) or 161 single-amino acid-substituted variants of the FLT3 D/Y peptide (purity >70%) (GenScript Biotech) for 1–2 h or electroporated with mRNA encoding either FLT3 WT or the FLT3 D/Y minigene.

Techniques: Binding Assay, Staining, Co-Culture Assay, Transfection, Sequencing, Biomarker Discovery, Positive Control, In Vitro

Journal: Nature Cancer

Article Title: A T cell receptor targeting a recurrent driver mutation in FLT3 mediates elimination of primary human acute myeloid leukemia in vivo

doi: 10.1038/s43018-023-00642-8

Figure Lengend Snippet: Mirror image plots comparing the fragmentation spectra of endogenous FLT3 D/Y peptide to its isotopically labelled counterpart YI(13C6,15 N)MSDSNYV. Spectra obtained from immunoprecipitated HLA derived from the B721.221 cell line retrovirally transduced with a minigene encoding the D835Y mutation, and from patient 1 and patient 3, are displayed.

Article Snippet: When indicated, target cells were pulsed with FLT3 D/Y or FLT3 WT peptide (purities >90%) or 161 single-amino acid-substituted variants of the FLT3 D/Y peptide (purity >70%) (GenScript Biotech) for 1–2 h or electroporated with mRNA encoding either FLT3 WT or the FLT3 D/Y minigene.

Techniques: Immunoprecipitation, Derivative Assay, Transduction, Mutagenesis

Journal: Nature Cancer

Article Title: A T cell receptor targeting a recurrent driver mutation in FLT3 mediates elimination of primary human acute myeloid leukemia in vivo

doi: 10.1038/s43018-023-00642-8

Figure Lengend Snippet: (a) Graphs depicting IFN-γ response of TCR FLT3D/Y cells to K562 cells loaded with individual peptides from mimotope library containing a total of 161 nine-mers for the FLT3 D/Y peptide, at a concentration of 10 −8 M. Purple dots in each graph represent response to the FLT3 D/Y peptide. Substituted amino acid in the original peptide is highlighted. IFN-γ concentration range for positive reactions was 5000–35000 pg/mL (cut-off indicated by horizontal lines). Graphs show results from two independent experiments that were performed, one technical replicate (dot) per peptide and experiment. (b) Peptide reactivity motifs for FLT3 D/Y that were queried in the ScanProsite search tool against human proteome databases. Amino acids in square brackets [] indicate alternatives that are allowed for the given position in the peptide motifs. (c) mRNA-encoded amino acid sequences(30-32mers) derived from the sequence of the human proteins LZTR1, MED1 and PRADC1, with the candidate cross-reactive peptide identified in the ScanProsite database underlined. (d) Expression of mRNA encoding LZTR1, MED1 and PRADC1 sequences following electroporation of HLA-A2 pos K562 cells as measured by GFP-reporter fluorescence using flow cytometry.

Article Snippet: When indicated, target cells were pulsed with FLT3 D/Y or FLT3 WT peptide (purities >90%) or 161 single-amino acid-substituted variants of the FLT3 D/Y peptide (purity >70%) (GenScript Biotech) for 1–2 h or electroporated with mRNA encoding either FLT3 WT or the FLT3 D/Y minigene.

Techniques: Concentration Assay, Derivative Assay, Sequencing, Expressing, Electroporation, Fluorescence, Flow Cytometry

Journal: Nature Cancer

Article Title: A T cell receptor targeting a recurrent driver mutation in FLT3 mediates elimination of primary human acute myeloid leukemia in vivo

doi: 10.1038/s43018-023-00642-8

Figure Lengend Snippet: (a) Mutation data (SNVs and indels) from AML patients reported in Papaemmanuil et al , 2016 NEJM were downloaded from https://www.cbioportal.org/ . VAF was estimated from reported alternative allele reads divided by sequencing depth for the position. Patients harboring a FLT3 D835Y mutation were selected for in-depth analysis and displayed here. Patients are sorted from right to left within each subsection in descending order of FLT3 D835Y VAF. The following genes were considered as initiating events in clonal hematopoiesis (CH): DNMT3A, TET2, ASXL1, PPM1D, JAK2, SF3B1, SRSF2, TP53 , GNA S and GNB1 . (b) Mutation data from FLT3 D835Y positive AML patients ( n = 9) reported in Morita et al , 2020 Nat Com were downloaded. The fraction of cells with mutations (mutant cell fraction) in each AML patient with mutations are plotted in indicated colors. Patients were categorized into 3 groups; FLT3 D835Y largest, only preceded by mutations in DNMT3A, TET2 , and/or ASXL1 (DTA) and/or splicing factor mutations ( SF3B1, SRAF2 , and U2AF1 ) which are closely related with clonal hematopoiesis ‘CH’, and the rest of cases with FLT3 D835Y mutation according to the cell fraction of mutations. Interquartile range (IQR) and median values are shown. The dashed lines indicate 1.5xIQR and the dots indicate outliers.

Article Snippet: When indicated, target cells were pulsed with FLT3 D/Y or FLT3 WT peptide (purities >90%) or 161 single-amino acid-substituted variants of the FLT3 D/Y peptide (purity >70%) (GenScript Biotech) for 1–2 h or electroporated with mRNA encoding either FLT3 WT or the FLT3 D/Y minigene.

Techniques: Mutagenesis, Sequencing

Journal: Nature Cancer

Article Title: A T cell receptor targeting a recurrent driver mutation in FLT3 mediates elimination of primary human acute myeloid leukemia in vivo

doi: 10.1038/s43018-023-00642-8

Figure Lengend Snippet: a , Percentage myeloid cells of live leukocytes for patients (Pt.) 1–8 with AML; gating strategy is shown in Extended Data Fig. . Dots represents technical replicates from one representative experiment as described in d . b , PB or BM FLT3 D/Y VAF for patients 1–8 as determined by next-generation sequencing. c , Representative t -distributed stochastic neighbor embedding ( t -SNE) plots showing live primary myeloid cells (CD3 − CD19 − CD20 − events) in red, T cells (CD3 + ) in blue, B cells (CD19 + CD20 + ) in orange and normal CD34 + lin − progenitor cells in green from n = 3 representative HLA-A2 + FLT3 D/Y patients (patients 2, 3 and 6) with AML and one HLA-A2 − FLT3 D/Y patient (patient 8) following 72 h of co-culture with TCR 1G4 (negative control, top) or TCR FLT3D/Y cells (E:T ratio, 1:2) as quantified by flow cytometry. Cells transduced to express TCR were excluded from analysis as CellTrace Violet (CTV)-positive events. d , Diagnostic samples from 11 patients with AML and the FLT3 D/Y (patients 1–8), FLT3 D/E (patient 9) or FLT3 D/H (patient 10) mutation or FLT3 WT (patient 11) (all HLA-A2 + except patient 8), analyzed as described in c . Each dot represents the fraction of live myeloid cells, B cells or T cells after co-culture with TCR FLT3D/Y cells (purple) in percent mean of the corresponding numbers in cultures treated with TCR 1G4 cells (blue). Data points represent n = 3 technical replicates, and horizontal lines show means. Data shown are from one experiment representative of two to four experiments performed for each patient sample ( n = 1 only for patient 7). e , t -SNE plots of PB diagnostic samples from patients 2 and 3 with AML showing live myeloid, T and B cells (color coded as in Fig. 2c) after 72 h of co-culture with autologous T cells transduced to express TCR FLT3D/Y or the mock control. Inset numbers in c , e denote absolute event counts of the indicated cell populations. The gating strategy is shown in Extended Data Fig. .

Article Snippet: When indicated, target cells were pulsed with FLT3 D/Y or FLT3 WT peptide (purities >90%) or 161 single-amino acid-substituted variants of the FLT3 D/Y peptide (purity >70%) (GenScript Biotech) for 1–2 h or electroporated with mRNA encoding either FLT3 WT or the FLT3 D/Y minigene.

Techniques: Next-Generation Sequencing, Co-Culture Assay, Negative Control, Flow Cytometry, Diagnostic Assay, Mutagenesis, Control

Journal: Nature Cancer

Article Title: A T cell receptor targeting a recurrent driver mutation in FLT3 mediates elimination of primary human acute myeloid leukemia in vivo

doi: 10.1038/s43018-023-00642-8

Figure Lengend Snippet: (a) Flow cytometry plots showing gating strategy to identify cell subsets in AML patient samples from PB (patient 2-6) or BM (patient 1). Cells are gated on FSC/SSC hi , single, Live/Dead Fixable Near-IR events (top row), showing the fractions of CD3 + T cells, CD19 + CD20 + B cells and myeloid cells, defined as CD3 − CD19 − CD20 − events in the bottom row. (b) Populations were overlaid on a t - SNE plot (patient 1) with designated colors as indicated. Inset numbers show event counts for myeloid cells after co-culture with TCR 1G4 or TCR FLT3D/Y cells, quantified as shown in e. (c) Quantification of normal CD19 + B cells isolated from n = 3 healthy blood donors (Buffy coat (BC) 1 - BC 3) and tumor cells from patient 1 (positive control) after performing the flow cytometry-based cytotoxicity assay for 72 h. Data points represent n = 3 technical replicates from one experiment and horizontal lines show mean. (d) Bar graph showing IFN-γ response of Mock (gray) and TCR FLT3D/Y cells (purple) after 24 h co-culture with HLA-A2 pos patient cells expressing the FLT3 D835Y (Pt.1-6), FLT3 D835E (Pt.9) or FLT3 D835H (Pt. 10), or Pt.8 cells expressing FLT3 D835Y but being HLA-A2 neg . Data points represent n = 3 technical replicates and bars show mean. Data shown are from one experiment representative of at least two performed for each patient sample. (e) Gating strategy for flow cytometry cytotoxicity assay to quantify viable cells in subpopulations from AML patients 1-3 after 72 h of co-culture with autologous T cells either expressing TCR FLT3D/Y (bottom row) or mock-transduced. Transduced T cells were labeled with cell-trace violet (shown in upper right plot) prior to co-culture with AML cells to distinguish them from T cells in the AML samples. Patient cell subsets are gated on FSC/SSC, singlets, Live/Dead Fixable Near-IR neg , CTV neg events. Numbers indicate absolute counts for CD3 + , CD19 + /CD20 + and myeloid cells after co-culture, as determined by addition of fluorescent beads (10,000) into each well and where 3,500 beads were acquired for flow cytometry analysis (shown in upper left plot).

Article Snippet: When indicated, target cells were pulsed with FLT3 D/Y or FLT3 WT peptide (purities >90%) or 161 single-amino acid-substituted variants of the FLT3 D/Y peptide (purity >70%) (GenScript Biotech) for 1–2 h or electroporated with mRNA encoding either FLT3 WT or the FLT3 D/Y minigene.

Techniques: Flow Cytometry, Co-Culture Assay, Isolation, Positive Control, Cytotoxicity Assay, Expressing, Labeling

Journal: Nature Cancer

Article Title: A T cell receptor targeting a recurrent driver mutation in FLT3 mediates elimination of primary human acute myeloid leukemia in vivo

doi: 10.1038/s43018-023-00642-8

Figure Lengend Snippet: (a) Flow cytometry histograms showing expression of FLT3 D/Y as measured by GFP- reporter fluorescence in transduced AML and B-ALL cell lines. Negative control (non-transduced BV173 cells) in top histogram. (b) Remaining viable FLT3 D/Y -transduced, HLA-A2+ cells (purple dots) after 24 h co-culture with TCR FLT3D/Y cells (E:T ratio of 1:2), in percent of corresponding numbers following treatment with mock-transduced T cells (grey dots), quantified by flow cytometry. +A2 denotes that HLA-A2 was introduced by transduction. Data points are from n = 3 independent experiments with each dot representing the mean of n = 3 technical replicates in each experiment and are shown as mean ± s.e.m. Gating strategy and quantification as shown in Extended Data Fig. . (c) Schematic overview of the BV173 D835Y in vivo model. (d) Bioluminescence imaging (BLI) analysis of NSG mice day 13 after BV173 D835Y cell injection, one day prior to T-cell therapy. Data shown are from one experiment, with mice grouped into untreated ( n = 3 mice), TCR 1G4 ( n = 3 mice) and TCR FLT3D/Y ( n = 4 mice) cell treated. (e) BLI of BV173 D835Y engrafted leukemic cells in mice on indicated days relative to treatment with TCR 1G4 or TCR FLT3D/Y cells, or no treatment. (f) Quantification of BV173 D835Y engrafted leukemic cells in mice, 21 days after treatment with TCR 1G4 or TCR FLT3D/Y cells or left untreated. (g) Flow cytometry plots showing BM tumor burden in two untreated and two TCR 1G4 cell-treated mice at time of sacrifice (d 21), as well as two TCR FLT3D/Y cell treated mice sacrificed at end of experiment (d 53). (h) Percentage of bone marrow BV173 D835Y leukemic cells in mice treated with TCR 1G4 or TCR FLT3D/Y cells, or left untreated, out of total mouse and human CD45 + cells (21-53 days after treatment start). (i) Percentage of TCR-β + cells out of human CD8 + cells in the BM of mice analyzed at point of sacrifice. Data in d, f, h, i are presented as mean ± s.e.m, are from one experiment with each dot representing an individual mouse, and are analyzed by unpaired, two-tailed Student’s t-test. P values are shown and p < 0.05 was considered statistically significant.

Article Snippet: When indicated, target cells were pulsed with FLT3 D/Y or FLT3 WT peptide (purities >90%) or 161 single-amino acid-substituted variants of the FLT3 D/Y peptide (purity >70%) (GenScript Biotech) for 1–2 h or electroporated with mRNA encoding either FLT3 WT or the FLT3 D/Y minigene.

Techniques: Flow Cytometry, Expressing, Fluorescence, Negative Control, Co-Culture Assay, Transduction, In Vivo, Imaging, Injection, Two Tailed Test

Journal: Nature Cancer

Article Title: A T cell receptor targeting a recurrent driver mutation in FLT3 mediates elimination of primary human acute myeloid leukemia in vivo

doi: 10.1038/s43018-023-00642-8

Figure Lengend Snippet: a , Schematic overview of the PDX in vivo model with FLT3 D835Y -mutated primary AML cells from patient 7. b , Percentage of human hCD45 + CD33 + cells in PB at baseline (1 d before T cell infusion) and on the indicated days after infusion with TCR 1G4 ( n = 6 mice) or TCR FLT3D/Y ( n = 7 mice) cells. Numbers were adjusted for hCD3 + T cells. c , Representative flow cytometry plots of viable single BM mononuclear cells (MNCs) from TCR 1G4 (top) and TCR FLT3D/Y (bottom) cell-treated NSG-SGM3 mice stably engrafted with primary AML FLT3 D/Y cells from patient 7. d , Percentage of hCD45 + CD33 + cells in the BM and spleen at terminal analysis 15 d after T cell infusion. Numbers were adjusted for hCD3 + T cells. e , Number of FLT3 D835Y -mutated BM hCD45 + CD3 − cells determined by ddPCR. f , g , Number of mouse (m)CD45 + cells in BM ( f ) and mTCR-β + CD8 + cells in the BM and spleen ( g ) at the endpoint. All data are presented as mean ± s.e.m. and were generated from one experiment including six mice treated with TCR 1G4 cells and seven mice treated with TCR FLT3D/Y cells. Each dot represents one mouse, and statistical analysis was performed with two-tailed Mann–Whitney test. P values are shown, and P < 0.05 was considered statistically significant.

Article Snippet: When indicated, target cells were pulsed with FLT3 D/Y or FLT3 WT peptide (purities >90%) or 161 single-amino acid-substituted variants of the FLT3 D/Y peptide (purity >70%) (GenScript Biotech) for 1–2 h or electroporated with mRNA encoding either FLT3 WT or the FLT3 D/Y minigene.

Techniques: In Vivo, Flow Cytometry, Stable Transfection, Generated, Two Tailed Test, MANN-WHITNEY

Journal: Nature Cancer

Article Title: A T cell receptor targeting a recurrent driver mutation in FLT3 mediates elimination of primary human acute myeloid leukemia in vivo

doi: 10.1038/s43018-023-00642-8

Figure Lengend Snippet: (a) Representative FACS plots of viable single PB MNCs from TCR 1G4 (top) and TCR FLT3D/Y (bottom) T cell-treated NSG-SGM3 mice stably engrafted with primary AML FLT3 D835Y cells from patient 7 at 14 days post T cell infusion. Equivalent gating as shown was also used for spleen. (b) Number of total MNCs in BM and spleen of TCR T cell treated mice. (c) Number of mouse MNCs (mCD45+ cells) in spleen. (d) Number of total hCD3+ cells in BM and spleen. (e-f) Percentage of mTCR-β + CD8 + cells of hCD3 + cells (e) and percentage of mTCR-β + cells of hCD8+ cells (f) in T cell samples used for injection and at indicated days after T cell injection, in PB . (g) Distribution (%) of CD4 and CD8 cells within the human CD3 + T cell samples used for injection, and in PB, BM and spleen 14-15 days after injection into mice. All data are from terminal analysis 15 days post T-cell infusion if not otherwise stated. The data are presented as mean ± s.e.m. of n = 6 individual mice treated with TCR 1G4 cells and of n = 7 individual mice treated with TCR FLT3D/Y cells, measured in one experiment. Each dot represents one mouse and statistical analysis was performed with two-tailed Mann-Whitney test. P values are shown and p < 0.05 was considered statistically significant.

Article Snippet: When indicated, target cells were pulsed with FLT3 D/Y or FLT3 WT peptide (purities >90%) or 161 single-amino acid-substituted variants of the FLT3 D/Y peptide (purity >70%) (GenScript Biotech) for 1–2 h or electroporated with mRNA encoding either FLT3 WT or the FLT3 D/Y minigene.

Techniques: Stable Transfection, Injection, Two Tailed Test, MANN-WHITNEY

Journal: Nature Cancer

Article Title: A T cell receptor targeting a recurrent driver mutation in FLT3 mediates elimination of primary human acute myeloid leukemia in vivo

doi: 10.1038/s43018-023-00642-8

Figure Lengend Snippet: a , Schematic overview of the PDX in vivo model with FLT3 D835Y -mutated primary AML cells from patient 1. b , Representative flow cytometry plots of BM from TCR 1G4 (top) and TCR FLT3D/Y (bottom) cell-treated NSG-SGM3 mice stably engrafted with primary AML FLT3 D/Y cells from patient 1. Equivalent gating was also used for PB and the spleen. c , Percentage of hCD45 + CD33 + CD34 + and hCD45 + CD33 + CD34 − cells in the BM, PB and spleen at the endpoint (day 34 after T cell infusion) of TCR 1G4 ( n = 4 mice) or TCR FLT3D/Y ( n = 4 mice) cell-treated mice. Numbers were adjusted for hCD3 + T cells. NS, not significant. d , Percentage VAF determined by ddPCR of FLT3 D835Y and WT1 H507P driver mutations in primary BM cells from patient 1 (top) and hCD45 + CD33 + CD34 + (middle) and hCD45 + CD33 + CD34 − (bottom) cells from TCR cell-treated mice. N/D, not analyzed due to insufficient hCD45 + CD33 + CD34 + cells. Numbers show VAF and 95% confidence intervals. The dashed line at 50% indicates 100% clonality unless loss of heterozygosity. No significant differences in VAFs of the FLT3 D835Y and WT1 H507P mutations were observed. e , Number of FLT3 D835Y -mutated hCD45 + CD33 + CD34 + and hCD45 + CD33 + CD34 − cells in the BM as determined by ddPCR. N/D, not detected due to lack of hCD45 + CD33 + CD34 + cells. f , Number of mTCR-β + CD8 + cells in the BM and spleen at the endpoint. All data are presented as mean ± s.e.m. from terminal analysis 34 d after T cell infusion from one experiment including four mice treated with TCR 1G4 cells and another four mice treated with TCR FLT3D/Y cells. Each dot represents one mouse, and statistical analysis was performed with two-tailed Mann–Whitney test. P values are shown, and P < 0.05 was considered statistically significant.

Article Snippet: When indicated, target cells were pulsed with FLT3 D/Y or FLT3 WT peptide (purities >90%) or 161 single-amino acid-substituted variants of the FLT3 D/Y peptide (purity >70%) (GenScript Biotech) for 1–2 h or electroporated with mRNA encoding either FLT3 WT or the FLT3 D/Y minigene.

Techniques: In Vivo, Flow Cytometry, Stable Transfection, Two Tailed Test, MANN-WHITNEY

Journal: Nature Cancer

Article Title: A T cell receptor targeting a recurrent driver mutation in FLT3 mediates elimination of primary human acute myeloid leukemia in vivo

doi: 10.1038/s43018-023-00642-8

Figure Lengend Snippet: (a) Percentage of CD33 + cells in BM and PB at endpoint (day 34 post T cell infusion) of TCR 1G4 cell ( n = 4) or TCR FLT3D/Y cell ( n = 4) treated mice. Numbers adjusted for hCD3 + T cells. (b) Number of total MNCs in BM and spleen of TCR T cell treated mice. (c) Representative ddPCR plots of BM from TCR cell-treated mice. Numbers in quadrants represent %VAF (mean ± s.e.m.) of the FLT3 D/Y in BM among hCD45 + CD33 + CD34 + , hCD45 + CD33 + CD34 − and hCD45 + CD19 + cells from TCR FLT3D/Y cell-treated (left) and TCR 1G4 cell-treated (right) mice. ‘No remaining cells’ due to elimination of hCD45 + CD33 + CD34 + cells in TCR FLT3D/Y cell-treated mice. (d) Number of total hCD3 + cells in BM and spleen. (e-g) Percentage of mTCR-β + CD8 + cells of hCD3 + cells (e) and percentage of mTCR-β + cells of hCD8 + cells (f) in sample used for injection and at indicated days after T cell infusion in PB. (g) Distribution (%) of CD4 and CD8 cells within the human CD3 + T cell samples analyzed prior to injection, and in PB, BM and spleen of individual mice at endpoint. All data are from terminal analysis 34 days post T-cell infusion unless otherwise stated. The data are presented as mean ± s.e.m. of n = 4 individual mice treated with TCR 1G4 cells and of n = 4 mice treated with TCR FLT3D/Y cells, measured in one experiment. Each dot represents one mouse and statistical analysis was performed with two-tailed Mann-Whitney test. P values are shown and p < 0.05 was considered statistically significant.

Article Snippet: When indicated, target cells were pulsed with FLT3 D/Y or FLT3 WT peptide (purities >90%) or 161 single-amino acid-substituted variants of the FLT3 D/Y peptide (purity >70%) (GenScript Biotech) for 1–2 h or electroporated with mRNA encoding either FLT3 WT or the FLT3 D/Y minigene.

Techniques: Injection, Two Tailed Test, MANN-WHITNEY

Journal: Nature Cancer

Article Title: A T cell receptor targeting a recurrent driver mutation in FLT3 mediates elimination of primary human acute myeloid leukemia in vivo

doi: 10.1038/s43018-023-00642-8

Figure Lengend Snippet: a , Schematic overview of the MRD PDX in vivo model with FLT3 D835Y -mutated primary AML cells from patient 1. b , Percentage of hCD45 + CD33 + cells in the BM of NSG mice engrafted with low levels of AML after treatment with TCR 1G4 ( n = 4 mice) or TCR FLT3D/Y ( n = 4 mice) cells 11 d after T cell infusion. Numbers were adjusted for hCD3 + T cells. Data are presented as mean ± s.e.m. and were generated from one experiment. Each dot represents one mouse, and statistical analysis was performed with two-tailed Mann–Whitney test. c , Schematic overview of the PDX in vivo model with FLT3 D835Y -mutated primary AML cells from patient 1 after in vitro targeting with TCR 1G4 or TCR FLT3D/Y cells. d , Percentage of hCD45 + CD33 + cells in PB at the indicated time after transplantation of primary AML cells from patient 1 following 48 h of co-culture without T cells ( n = 3 mice) or with TCR 1G4 ( n = 3 mice) or TCR FLT3D/Y ( n = 5 mice) cells. Data are presented as mean ± s.e.m. and were generated from two independent experiments. Each dot represents one mouse, and statistical analysis was performed by multilevel linear regression using the R package ‘lmerTest’ (further described in the ). P values are shown, and P < 0.05 was considered statistically significant.

Article Snippet: When indicated, target cells were pulsed with FLT3 D/Y or FLT3 WT peptide (purities >90%) or 161 single-amino acid-substituted variants of the FLT3 D/Y peptide (purity >70%) (GenScript Biotech) for 1–2 h or electroporated with mRNA encoding either FLT3 WT or the FLT3 D/Y minigene.

Techniques: In Vivo, Generated, Two Tailed Test, MANN-WHITNEY, In Vitro, Transplantation Assay, Co-Culture Assay