Journal: Molecular Therapy Oncolytics

Article Title: Anti-CD117 CAR T cells incorporating a safety switch eradicate human acute myeloid leukemia and hematopoietic stem cells

doi: 10.1016/j.omto.2023.07.003

Figure Lengend Snippet: Transiently expressed anti-CD117 CAR T cells demonstrate efficient killing in vitro and in vivo (A) Schematic representation of the method to generate mRNA CAR T cells. (B) CAR expression of T cells electroporated with 10 μg of CAR mRNA over time. One representative histogram of results from five donors is shown. (C) Percentage of CAR+ cells at different time points after electroporation as determined by flow cytometry with protein A. Two-way ANOVA with Turkey multiple comparison. (D) Percentage of CD3+CAR+ cells at different time points after electroporation as determined by flow cytometry with the recombinant c-Kit protein. Two-way ANOVA with Turkey multiple comparison. (E) CAR expression level (geometric mean) at different time points after electroporation as determined by flow cytometry with the recombinant c-Kit protein. Data illustrate the mean ± SD from five different donors for 10 μg, LV and NT cells, and three donors for 1.5 μg. (F) Viability of T cells electroporated with 10 μg of CAR mRNA over time as determined by Annexin V and 7AAD staining. Data illustrate the mean ± SD from three different donors. (G) Cytotoxicity of mRNA CAR T cells at different time points after electroporation compared with NT and LV CAR T cells against CD117+ HLA-60 at different E:T ratios and WT HL-60 (E:T = 1:1). Data illustrate the mean ± SD from three different donors. Two-way ANOVA with Turkey multiple comparison (∗compared with NT cells). (H) Schematic outline of the co-culture experiment with CFSE-stained T cells. Representative flow cytometric immunophenotyping showing elimination of target cells (cytotoxicity, gated in live cells), and proliferation of CAR T cells (gated in CD3+ cells). One representative donor of results from three donors is shown. (I) Schematic outline of the in vivo experiment. (J) Percentage of CD3+ cells in PB of all treated groups at endpoint. (K) Percentage of BM CD33+CD117+ cells of all treated groups at endpoint. Data illustrate the mean ± SD from three different mice for mRNA CAR T cells. One-way ANOVA with Turkey multiple comparison.

Article Snippet: Surface expression of the anti-CD117 CAR was detected with the Recombinant human c-Kit Protein (His Tag, SinoBiological), followed by the anti-CD117 antibody.

Techniques: In Vitro, In Vivo, Expressing, Electroporation, Flow Cytometry, Recombinant, Staining, Co-Culture Assay

Journal: Molecular Therapy Oncolytics

Article Title: Anti-CD117 CAR T cells incorporating a safety switch eradicate human acute myeloid leukemia and hematopoietic stem cells

doi: 10.1016/j.omto.2023.07.003

Figure Lengend Snippet: Generation and characterization of anti-CD117 CAR T cells incorporating iC9 by SB vector (A) Schematic representation of the SB vector containing a bicistronic construct with the iC9 and anti-CD117 CAR transgenes under the regulation of hEF-1α. The transgenes are linked by a T2A peptide. The expression cassette is enclosed in the PT4 left/right inverted repeats/directed repeats (LIR/RIR). (B) Schematic representation of the method to generate SB CAR T cells. CAR T cells were produced by electroporation with the SB vector and the SB100X transposase. (C) Cell viability at the end of the manufacturing process. (D) Fold expansion calculated by dividing the number of total viable cells at the end of the expansion phase by the respective number on day 0. (E) CAR expression as percentage of CD3 cells and expression level (geometric mean) as determined by flow cytometry with the recombinant c-Kit protein. (F) Flow cytometric immunophenotyping by dual-density plots in one representative batch (n = 6). (G) Percentage of CD3+CD45RA+CD62L+CD95+ naïve-like (T N ) and stem cell memory (T SCM ), CD3+CD45RA−CD62L+ central memory (T CM ), CD3+CD45RA−CD62L− effector memory (T EM ), and CD3+CD45RA+CD62L− terminal effector (T TE ). (H) Percentage of CD3+CD8+ and CD3+CD4+. (I) Percentage of CD3+PD-1+, CD3+TIM-3+ and CD3+LAG-3+. Data illustrate the mean ± SD from six different donors. Two-way ANOVA with Turkey multiple comparison (∗compared with NT cells).

Article Snippet: Surface expression of the anti-CD117 CAR was detected with the Recombinant human c-Kit Protein (His Tag, SinoBiological), followed by the anti-CD117 antibody.

Techniques: Plasmid Preparation, Construct, Expressing, Produced, Electroporation, Flow Cytometry, Recombinant

Journal: Molecular Therapy Oncolytics

Article Title: Anti-CD117 CAR T cells incorporating a safety switch eradicate human acute myeloid leukemia and hematopoietic stem cells

doi: 10.1016/j.omto.2023.07.003

Figure Lengend Snippet: Anti-CD117.iC9 CAR T cells exhibit potent functionality and are rapidly terminated by iC9 activation (A) Killing activity of CAR T cells after 24 hours of co-culture with CD117+ Molm-14 at an E:T ratio of 1:1. (B) IFN-γ, TNF-α, and IL-2 secretion of CAR T cells after 24 hours of co-culture with CD117+ Molm-14. (C) Killing activity of sorted CAR T cells at different E:T ratios. One representative experiment of results from three donors is shown. (D) Schematic outline of the co-culture experiment with CID. Apoptosis assessed in CAR T cells in the presence of 200 nM CID. (E) Apoptosis assessed in sorted CAR T cells at different CID concentrations. One representative experiment of results from three donors is shown. (F) Killing activity of sorted CAR T cells after 24 and 48 hours of co-culture with CD117+ Molm-14 expressing high, medium, and low levels of CD117 at an E:T ratio of 1:1. Data illustrate the mean ± SD from three different donors. One-way ANOVA with Turkey multiple comparison (∗compared with NT cells).

Article Snippet: Surface expression of the anti-CD117 CAR was detected with the Recombinant human c-Kit Protein (His Tag, SinoBiological), followed by the anti-CD117 antibody.

Techniques: Activation Assay, Activity Assay, Co-Culture Assay, Expressing

Journal: Molecular Therapy Oncolytics

Article Title: Anti-CD117 CAR T cells incorporating a safety switch eradicate human acute myeloid leukemia and hematopoietic stem cells

doi: 10.1016/j.omto.2023.07.003

Figure Lengend Snippet: SB-generated anti-CD117 CAR T cells show potent anti-leukemic activity in vivo (A) Schematic representation of the in vivo experiment. NSG mice were sub-lethally irradiated and injected with MOLM-14 CD117high Luciferase+ cells. At day 9, mice were injected with 2 × 10 6 CAR T cells, generated with SB or LV. (B) IVIS images of mice injected with CAR T cells, NT cells, or left untreated. (C) Bioluminescence intensity (total flux per second) over time in mice receiving the indicated treatment. Two-way ANOVA with Turkey multiple comparison (∗compared with untreated). (D) Kaplan-Meier survival curves over time. Statistical differences are calculated by Mantel-Cox test. (E) Flow cytometry analyses of BM at euthanization, corresponding to day 20–23 for control groups (humane endpoints), and day 30 for groups treated with CAR T cells. (F) Flow cytometry analyses of spleen at euthanization. (G) Flow cytometry analyses of PB at euthanization. Data illustrate the mean ± SD from four different mice per group. One-way ANOVA with Turkey multiple comparison.

Article Snippet: Surface expression of the anti-CD117 CAR was detected with the Recombinant human c-Kit Protein (His Tag, SinoBiological), followed by the anti-CD117 antibody.

Techniques: Generated, Activity Assay, In Vivo, Irradiation, Injection, Luciferase, Flow Cytometry

Journal: Molecular Therapy Oncolytics

Article Title: Anti-CD117 CAR T cells incorporating a safety switch eradicate human acute myeloid leukemia and hematopoietic stem cells

doi: 10.1016/j.omto.2023.07.003

Figure Lengend Snippet: SB-generated CAR T cells are able to deplete human CD117+ HSPCs and are terminated by iC9 activation and ATG (A) Newborn mice were sub-lethally irradiated and injected intrahepatically with human CB-derived CD34+ cells. Following engraftment at day 40, mice were injected with 2 × 10 6 CAR T cells generated with SB. (B) Representative flow cytometry plots of CD117+ and CD3+ cells in BM of animals from each treatment group after 2 weeks of treatment. (C) Percentage of CD117+ of hCD45+ cells in BM. (D) Percentage of CD3+ cells of hCD45+ cells in BM. (E) Newborn mice were sub-lethally irradiated and injected intrahepatically with human CB-derived CD34+ cells. Following engraftment at day 60, mice were injected with 2 × 10 6 CAR T cells generated with SB or LV. Two weeks after treatment, PB and BM analysis was performed by bleeding and BM puncture. Mice were then treated with a single dose of CID (50 μg/mouse) and three subsequent doses of ATG (500 μg/mouse) from day 75 to day 77. (F) Percentage of CD117+ cells of hCD45+ cells in BM after 1 or 2 weeks of treatment. (G) Representative flow cytometry plots of hCD3+ cells in PB of animals from each treatment group before and after CAR T termination. (H) Percentage of CD3+ cells of hCD45+ cells and (I) CD3+ cells/mL in PB before and after CAR T termination.

Article Snippet: Surface expression of the anti-CD117 CAR was detected with the Recombinant human c-Kit Protein (His Tag, SinoBiological), followed by the anti-CD117 antibody.

Techniques: Generated, Activation Assay, Irradiation, Injection, Derivative Assay, Flow Cytometry

Journal: International journal of molecular sciences

Article Title: Characterization of a Human Gastrointestinal Stromal Tumor Cell Line Established by SV40LT-Mediated Immortalization.

doi: 10.3390/ijms241713640

Figure Lengend Snippet: Figure 1. Pathological assessment of GIST tissue. (A) Hematoxylin-eosin (HE) staining of tumor tissue. The GIST tissue positively stained for (B) DOG-1, (C) CD117, and (D) CD34. Magnification = 200×.

Article Snippet: After blocking with 5% BSA, the membranes were incubated overnight at 4 ◦C with primary antibodies against CD117 (1:1000 dilution; Proteintech, Wuhan, China), SV40 (1:1000 dilution; Proteintech), P53 (1:1000 dilution; Proteintech), and GAPDH (1:10,000 dilution; Proteintech).

Techniques: Staining

Journal: International journal of molecular sciences

Article Title: Characterization of a Human Gastrointestinal Stromal Tumor Cell Line Established by SV40LT-Mediated Immortalization.

doi: 10.3390/ijms241713640

Figure Lengend Snippet: Figure 2. Protein expression and cell morphology of PriGIST and ImGIST cells. (A) Isolation of PriGIST cells (100×). (B) Morphological observations of PriGIST cells after 24 h of adherence (100×). (C) Mor- phological observations of PriGIST cells at P12 (100×). Immunofluorescence staining showed PriGIST cells positively expressed (D) DOG-1, (E) CD117, and (F) CD34 (100×). Morphological observation of (G) PriGIST cells (P0), (H) PriGIST cells during transfection, and (I) ImGIST cells (P15) (100×). PriGIST cells: primary GIST cells; ImGIST cells: immortalized GIST cells; P: passage.

Article Snippet: After blocking with 5% BSA, the membranes were incubated overnight at 4 ◦C with primary antibodies against CD117 (1:1000 dilution; Proteintech, Wuhan, China), SV40 (1:1000 dilution; Proteintech), P53 (1:1000 dilution; Proteintech), and GAPDH (1:10,000 dilution; Proteintech).

Techniques: Expressing, Isolation, Immunofluorescence, Staining, Transfection

Journal: International journal of molecular sciences

Article Title: Characterization of a Human Gastrointestinal Stromal Tumor Cell Line Established by SV40LT-Mediated Immortalization.

doi: 10.3390/ijms241713640

Figure Lengend Snippet: Figure 6. Expression of SV40, P53, CD117, and GAPDH in PriGIST and ImGIST cells was measured by real-time PCR and western blotting. (A,B) Real-time PCR was used to detect the mRNA levels of SV40 and P53 in PriGIST and ImGIST cells normalized to GAPDH (n = 4). (C) Representative image of a western blot showing bands for SV40, P53, CD117, and GAPDH. (D–F) Relative optical density was normalized to that of GAPDH (n = 4). All data are presented as means ± SD. *** p < 0.001, vs. PriGIST P0 group; N.S., no significant difference when compared to PriGIST P0 in panel F. SV40: Simian virus 40; P: passage.

Article Snippet: After blocking with 5% BSA, the membranes were incubated overnight at 4 ◦C with primary antibodies against CD117 (1:1000 dilution; Proteintech, Wuhan, China), SV40 (1:1000 dilution; Proteintech), P53 (1:1000 dilution; Proteintech), and GAPDH (1:10,000 dilution; Proteintech).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Virus