Journal: Cancer cell

Article Title: Logic-gated ROR1 chimeric antigen receptor expression rescues T cell-mediated toxicity to normal tissues and enables selective tumor targeting

doi: 10.1016/j.ccell.2019.02.003

Figure Lengend Snippet: (A) Map of retroviral constructs used to generate ROR1 CAR and control T cells. TM = transmembrane. hIgG4 = human immunoglobulin G4. scFv = single chain variable fragment. (B) Percent change in body weight in BALB/c mice (left) and representative flow cytometric plots showing frequency of CD8+CD45.1+ donor T cells of live cells and frequency of CD8+tCD19+ cells of CD45.1+ donor T cells 7 days post-transfer (right). (C) Percent change in body weight in BALB/c mice. n=4 mice per group. Two-way ANOVA with Tukey’s post-test (ROR1 CAR-T vs control T at Day 7, 9, 11: p<0.00001). (D) Representative flow cytometric plots showing expression of tCD19 transduction marker and PD-1 on CD8+CD45.1+ donor T cells 4 days post-transfer (left) and summary of absolute number and PD-1 median fluorescence intensity (MFI) on CD45.1+CD8+tCD19+ control or ROR1 CAR-T cells 4 days post-transfer (right). n=4 mice per group. Unpaired Student’s two-way t-test (Abs #: spleen, p=0.0004; BM, p=0.0002; PD-1: spleen, p=0.0007; BM, p=0.0007; blood, p=0.000008). (E) White blood cell (WBC), lymphocyte, red blood cell (RBC), and platelet (PLT) counts in peripheral blood. n=4 mice per group. Two-way ANOVA with Tukey’s post-test (ROR1 CAR-T vs control T: Lymphocytes Day 14, p<0.005; RBC Day 10,14, p<0.00001; PLT Day 14, p<0.005). (F) Representative H&E stains of spleen and femur (BM) 14 days post-transfer (left) and histopathology scoring of control or ROR1 CAR-T cell-treated tissues (right). n=4 mice per group. Two-way ANOVA with Sidak post-test (Spleen Day 8,10,14, p<0.00001; BM Day 10, p=0.004; BM Day 14, p<0.00001). Data are representative of 5 independent experiments. All data are presented as the mean values ± SEM. See also Table S1 and Figures S1-S2.

Article Snippet: Anti-human ROR1 APC , Miltenyi Biotec , Cat# 130–117-942, RRID:AB_2733449.

Techniques: Retroviral, Construct, Control, Expressing, Transduction, Marker, Fluorescence, Histopathology

Journal: Cancer cell

Article Title: Logic-gated ROR1 chimeric antigen receptor expression rescues T cell-mediated toxicity to normal tissues and enables selective tumor targeting

doi: 10.1016/j.ccell.2019.02.003

Figure Lengend Snippet: (A) Percent change in body weight in BALB/c mice lymphodepleted (LD) as indicated. n=4 mice per group. Two-way ANOVA with Tukey’s post-test (ROR1 CAR-T vs control T: 500 R Day 7, 9, p<0.00001; 200 mg/kg cyclophosphamide (Cy) Day 7, p=0.0004; 200 mg/kg Cy Day 9, p<0.00001). (B) Frequency of CD8+CD45.1+tCD19+ ROR1 CAR-T cells in peripheral blood of BALB/c mice receiving 100 R, 500 R, 100 mg/kg Cy, or 200 mg/kg Cy for LD. n=4 mice per group. (C-E) Percent change in body weight (C), frequency (D) and PD-1 and TIM3 expression (E) on CD8+CD45.1+tCD19+ donor T cells in peripheral blood of BALB/c mice irradiated 500 R and treated as indicated. n=4 mice per group. Two-way ANOVA with Tukey’s post-test (1 M CAR-T vs control T: Day 7, p=0.0003; Day 9,18, p<0.00001; 0.5 M CAR-T vs control T: Day 20, p<0.00001; Day 24, p=0.0005; Day 27, p=0.0044). Data are representative of 2 independent experiments. All data are presented as the mean values ± SEM. See also Figure S3.

Article Snippet: Anti-human ROR1 APC , Miltenyi Biotec , Cat# 130–117-942, RRID:AB_2733449.

Techniques: Control, Expressing, Irradiation

Journal: Cancer cell

Article Title: Logic-gated ROR1 chimeric antigen receptor expression rescues T cell-mediated toxicity to normal tissues and enables selective tumor targeting

doi: 10.1016/j.ccell.2019.02.003

Figure Lengend Snippet: (A) Percent change in body weight (left), RBC count in peripheral blood (middle), and representative pictures of spleens 9 days post-transfer (right) from B6 Ror1fl/fl (WT) or EIIα-Cre+Ror1fl/fl (KO) mice treated with 500 R and control or ROR1 CAR-T cells. n=3 mice per group. Left: two-way ANOVA with Tukey post-test (WT+CAR-T vs. KO+CAR-T: Day 7,8,9, p<0.00001). Middle: one-way ANOVA with Tukey post-test (p=0.0003). (B) Percent change in body weight (left), RBC count in peripheral blood (middle), and representative pictures of spleens 15 days post-transfer (right) from WT>WT or ROR1-KO>WT BM chimeric mice treated with 500 R and control or ROR1 CAR-T cells. n=3 mice per group. Left: two-way ANOVA with Tukey post-test (WT>WT CAR-T vs. KO>WT CAR-T: n.s. = not significant). Middle: one-way ANOVA with Tukey post-test. (C) Percent change in body weight (left), RBC count in peripheral blood (middle), and representative pictures of spleens 40 days post-transfer (right) from WT>WT or WT>ROR1-KO BM chimeric mice treated with 500 R and control or ROR1 CAR-T cells. n=3 mice per group. Left: two-way ANOVA with Tukey post-test (WT>WT CAR-T vs. WT>KO CAR-T: Day 7, p=0.00013; Day 9, p=0.0045; Day 10,11, p<0.00001). Middle: one-way ANOVA with Tukey post-test (p=0.0044). Data are representative of 2 independent experiments. All data are presented as the mean values ± SEM.

Article Snippet: Anti-human ROR1 APC , Miltenyi Biotec , Cat# 130–117-942, RRID:AB_2733449.

Techniques: Control

Journal: Cancer cell

Article Title: Logic-gated ROR1 chimeric antigen receptor expression rescues T cell-mediated toxicity to normal tissues and enables selective tumor targeting

doi: 10.1016/j.ccell.2019.02.003

Figure Lengend Snippet: (A) qPCR analysis of Ror1 expression in sorted BM progenitors (left) and BM stromal cells (right) from BALB/c mice left untreated or irradiated 500 R 48 hr prior to euthanasia. Ror1 expression was normalized to Actb expression and expressed relative to Ror1 expression in E10.5 embryos. OBL = osteoblasts. MSC = mesenchymal stem cells. EC = endothelial cells. n=3 mice per group. (B) ELISA analysis of IFNγ production by CD8+ control or ROR1 CAR-T cells co-cultured for 48 hr with primary MSC derived from WT or ROR1-KO femurs and expanded in vitro for 14 days. Data are summarized from 3 independent experiments. Unpaired two-way Student’s t-test (p=0.0041). (C) Absolute number of cells in BM flushed from femurs and tibia of control or ROR1 CAR-T cell-treated mice 9 days post-transfer (left) and number of MSC, pre-B cell, granulocyte/macrophage, erythrocyte, and megakaryocyte colonies formed after culture of BM cells from femurs of control or ROR1 CAR-T cell-treated BALB/c mice collected 9 days post-transfer (right). n=3 mice per group. Unpaired two-way Student’s t-test (# BM cells, p=0.0004; MSC, p=0.0004; Pre-B, p=0.0023). (D) ELISA analysis of IFNγ production by CD8+ control or ROR1 CAR-T cells co-cultured for 48 hr with CD45+ or CD45−splenic cells. Data are summarized from 3 independent experiments. Unpaired two-way Student’s t-test (p=0.0049). (E) qPCR analysis of Ror1 expression in splenic subpopulations sorted from BALB/c mice left untreated or irradiated 500 R 48 hr prior to euthanasia. n.d. = not detected. Data are summarized from 3 independent experiments. All data are presented as the mean values ± SEM.

Article Snippet: Anti-human ROR1 APC , Miltenyi Biotec , Cat# 130–117-942, RRID:AB_2733449.

Techniques: Expressing, Irradiation, Enzyme-linked Immunosorbent Assay, Control, Cell Culture, Derivative Assay, In Vitro

Journal: Cancer cell

Article Title: Logic-gated ROR1 chimeric antigen receptor expression rescues T cell-mediated toxicity to normal tissues and enables selective tumor targeting

doi: 10.1016/j.ccell.2019.02.003

Figure Lengend Snippet: (A) Representative flow cytometric analysis of EpCAM expression on 4T1 tumor cells, MSC, osteoblasts (OBL), CD45−Ter119−PDGFRβ+ spleen cells, and CD45−Ter119−VE-cadherin+ spleen cells from BALB/c mice. Percent lysis of the indicated 51Cr-labeled tumor cells after 6 hr (top) or 24 hr (bottom) of co-culture with CD8+ untransduced (black), ROR1 CAR-T cells (gray), or EpCAM-inducible ROR1 CAR-T cells (open circles). (C) Intracellular cytokine analysis of tCD19 CAR marker and IFNγ expression in the indicated T cells co-cultured with tumor cells in the presence of Brefeldin A for the last 6 hr of culture. Data are representative of two independent experiments. See also Figures S4 and S5.

Article Snippet: Anti-human ROR1 APC , Miltenyi Biotec , Cat# 130–117-942, RRID:AB_2733449.

Techniques: Expressing, Lysis, Labeling, Co-Culture Assay, Marker, Cell Culture

Journal: Cancer cell

Article Title: Logic-gated ROR1 chimeric antigen receptor expression rescues T cell-mediated toxicity to normal tissues and enables selective tumor targeting

doi: 10.1016/j.ccell.2019.02.003

Figure Lengend Snippet: (A) Percent change in body weight in BALB/c mice inoculated with 4T1-mROR1 tumors and treated as indicated. n=6–12 mice per group. Two-way ANOVA with Tukey post-test (ROR1-CAR vs. EpCAM-inducible ROR1 CAR: Day 4, p<0.0001; Day 7, p<0.0001; Day 10, p=0.0002). (B) RBC and PLT counts from peripheral blood of tumor-bearing mice treated as indicated. n=6–12 mice per group. Two-way ANOVA with Tukey post-test (ROR1-CAR vs. EpCAM-inducible ROR1 CAR: RBC Day 4,8,15,22:, p<0.0001; PLT Day 8 p=0.0114; PLT Day 15, 22, p<0.0001). (C) Representative pictures of spleens 10 days post-transfer from tumor-bearing BALB/c mice treated as indicated. (D) Tumor volume in BALB/c mice treated as indicated. n=6–12 mice per group. Two-way ANOVA with Tukey post-test (ROR1-CAR vs. UT: Day 14, p<0.0001; Day 17, p<0.0001; EpCAM-inducible ROR1 CAR vs. UT: Day 14, p<0.0001; Day 17, p<0.0001; ROR1-CAR vs EpCAM-inducible ROR1 CAR: n.s). (E) Survival of mice treated as indicated. n=6–12. Log-rank Mantel-Cox test (UT vs. ROR1 CAR, p=0.0003; UT vs. EpCAM-inducible ROR1 CAR, p=0.0016; ROR1 CAR vs. EpCAM-inducible ROR1 CAR, p=0.0001). (F) Representative flow cytometric analysis of ROR1 CAR and PD-1 expression on CD8+CD45.1+ donor T cells from mice treated with Cy and the indicated T cells 10 days post-transfer. (G) Summary of frequency of ROR1 CAR+, CAR+Ki67+, and CAR+PD-1+ T cells of CD8+CD45.1+ donor T cells from mice treated with Cy and the indicated T cells 10 days post-transfer. n=6–12 mice per group. Unpaired two-way Student’s t-test (Frequency: spleen, p<0.0001; BM, p<0.0001; PD-1: spleen, p=0.0004; BM, p=0.0003; Ki67: spleen, p<0.0001; BM, p<0.0001). Data are summarized from 2 independent experiments. All data are presented as the mean values ± SEM.

Article Snippet: Anti-human ROR1 APC , Miltenyi Biotec , Cat# 130–117-942, RRID:AB_2733449.

Techniques: Expressing

Journal: Cancer cell

Article Title: Logic-gated ROR1 chimeric antigen receptor expression rescues T cell-mediated toxicity to normal tissues and enables selective tumor targeting

doi: 10.1016/j.ccell.2019.02.003

Figure Lengend Snippet: (A) Map of lentiviral constructs encoding CD19-Gal4VP64 synNotch Receptor, UAS inducible R11 ROR1 CAR, and constitutive R11 ROR1 CAR. (B) Raji tumor bioluminescence over time in NSG mice irradiated 250 R and treated with the indicated primary human T cells. n=6–7 per group. Two-way ANOVA with Tukey post-test (UT vs. ROR1 CAR-T: Day 13, 20, p<0.0001; UT vs. CD19-inducible ROR1 CAR-T: Day 13, 20, p<0.0001). (C) Summary of tEGFR CAR marker and PD-1 expression on the indicated donor CD8+CD45+ T cells in peripheral blood. n=4–7 per group. Two-way ANOVA with Tukey post-test (CAR: UT vs. ROR1 CAR, Day 3, 7, p<0.0001; ROR1 CAR vs. CD19-inducible ROR1 CAR, Day 3, p<0.0001; Day 7, n.s. PD-1: UT vs. ROR1 CAR, Day 3, 7, p<0.0001; ROR1 CAR vs. CD19-inducible ROR1 CAR, Day 3, p<0.0001; Day 7, n.s.). (D) Survival of tumor-bearing mice irradiated 250 R and treated with the indicated T cell groups. n=6–7 per group. Log-rank Mantel-Cox test (UT vs. ROR1 CAR, p=0.0007; UT vs. CD19-inducible ROR1 CAR, p=0.0007; ROR1 CAR vs. CD19-inducible ROR1 CAR, n.s.). Data are representative of 2 independent experiments. All data are presented as the mean values ± SEM. See also Figure S6.

Article Snippet: Anti-human ROR1 APC , Miltenyi Biotec , Cat# 130–117-942, RRID:AB_2733449.

Techniques: Construct, Irradiation, Marker, Expressing

Journal: Cancer cell

Article Title: Logic-gated ROR1 chimeric antigen receptor expression rescues T cell-mediated toxicity to normal tissues and enables selective tumor targeting

doi: 10.1016/j.ccell.2019.02.003

Figure Lengend Snippet: (A) Percent change in body weight in NSG mice implanted with MDA-MB-231 tumors, irradiated 250 R and treated with the indicated primary human T cells. n=4 mice per group. Two-way ANOVA with Tukey post-test (ROR1-CAR vs. EpCAM-inducible ROR1 CAR: Day 13, 14, 19, p<0.0001). (B) RBC and PLT counts from peripheral blood of tumor-bearing mice treated as indicated 20 days post-T cell transfer. n=4 mice per group. Two-way ANOVA with Tukey post-test (RBC: ROR1 CAR vs. EpCAM-inducible ROR1 CAR, p=0.0426; UT vs. ROR1 CAR, p=0.0418. PLT: ROR1 CAR vs. EpCAM-inducible ROR1 CAR, p=0.0117; UT vs. ROR1 CAR, p=0.0442). (C and D) Tumor bioluminescence (C) and survival (D) of NSG mice treated as indicated. n=4 mice per group. Two-way ANOVA with Tukey post-test (ROR1 CAR vs. UT: Day 13, p=0.0167; Day 20, p<0.0001. EpCAM-inducible ROR1 CAR vs. UT: Day 13, p=0.0029; Day 20, p<0.0001. ROR1 CAR vs EpCAM-inducible ROR1 CAR: n.s). (E) Representative flow cytometric analysis of ROR1 CAR and Ki67 expression on CD8+CD45+ donor T cells from NSG mice irradiated 250 R and given indicated T cells 22 days post-transfer. (F) Summary of frequency of ROR1 CAR+, Ki67+, and PD-1+ T cells of CD8+CD45+ donor T cells from mice irradiated 250 R and given indicated T cells 22 days post-transfer. n=4 mice per group. Unpaired two-way Student’s t-test (Frequency: spleen, p<0.00001; BM, p<0.00001; tumor, n.s. Ki67: spleen, p<0.0001; BM, p<0.0001; tumor, n.s. PD-1: spleen, p<0.0001; BM, p<0.0001; tumor, n.s.). Data are representative of 2 independent experiments. All data are presented as the mean values ± SEM. See also Figures S7 and S8.

Article Snippet: Anti-human ROR1 APC , Miltenyi Biotec , Cat# 130–117-942, RRID:AB_2733449.

Techniques: Irradiation, Expressing

Journal: Cancer cell

Article Title: Logic-gated ROR1 chimeric antigen receptor expression rescues T cell-mediated toxicity to normal tissues and enables selective tumor targeting

doi: 10.1016/j.ccell.2019.02.003

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-human ROR1 APC , Miltenyi Biotec , Cat# 130–117-942, RRID:AB_2733449.

Techniques: Control, Histopathology, Recombinant, Cell Isolation, cDNA Synthesis, Plasmid Preparation, Software, SYBR Green Assay

Journal: Haematologica

Article Title: Expression of COBLL1 encoding novel ROR1 binding partner is robust predictor of survival in chronic lymphocytic leukemia

doi: 10.3324/haematol.2017.178699

Figure Lengend Snippet: COBLL1 is an ROR1-interaction partner. (A) (Left) COBLL1-ROR1 complex was efficiently immunoprecipitated in HEK293 cells transfected with plasmids encoding FLAG-tagged COBLL1 and V5-tagged ROR1. (Right). Endogenous COBLL1 was pulled down with endogenous ROR1 in MAVER-1 cells; unspecific IgG was used as a negative control. Immunoprecipitation input is loaded on the right. Protein levels were determined using western blotting and anti-FLAG, anti-V5, anti-ROR1 and anti-COBLL1 antibodies. IP: immunoprecipitation. (B–D) Immunofluorescence of HEK293 cells transfected with plasmids encoding FLAG-COBLL1 (B–D) and V5-ROR1 (C and D). COBLL1 over-expressed in HEK293 cells shows mostly cytoplasmic localization (B), but co-localizes with ROR1 in the membrane when ROR1 is co-expressed (C). The most efficient ROR1 and COBLL1 co-localization is observed in filopodia formed as a consequence of ROR1 overexpression (D, indicated by arrows). Protein expression was visualized using anti-FLAG, anti-V5 and corresponding secondary fluorescein-conjugated antibodies. Nuclei were visualized using DAPI staining.

Article Snippet: In order to confirm the interaction on an endogenous level in lymphoid cells, we also co-immunoprecipitated COBLL1 in endogenous ROR1 pulldown using anti-ROR1 antibody from protein lysates of MAVER-1 cells, a mantle cell lymphoma cell line expressing both COBLL1 and ROR1 ( right).

Techniques: Immunoprecipitation, Transfection, Negative Control, Western Blot, Immunofluorescence, Membrane, Over Expression, Expressing, Staining