goat anti human ror1 polyclonal ab  (R&D Systems)

Journal: PLoS ONE

Article Title: IGF1R- and ROR1-Specific CAR T Cells as a Potential Therapy for High Risk Sarcomas

doi: 10.1371/journal.pone.0133152

Figure Lengend Snippet: (A) Schematic representation of the Sleeping Beauty (SB) transposons encoding IGF1R CAR and GFP (pKT2-CaIG), IGF1R and GFP:Zeocin (pKT2-CaIG:Z), ROR1 CAR and GFP (pKT2-CaRG), and ROR1 CAR and GFP:Zeocin (pKT2-CaRG:Z). The CAR contains the 4-1BB signaling domain (not shown). A Caggs promoter and a “self-cleaving” 2A peptide flanking the CAR sequence were used to regulate co-expression of CARs and GFP or GFP-Zeocin fusion in the SB transposon trans vectors, namely pKT2-CaIG, pKT2-CaRG, pKT2-CaIG:Z, and pKT2-CaRG:Z. (B) Expression of CAR and GFP in T cells derived from a sarcoma patient (Patient 1) after transfection of PBMCs with pKT2-CaIG:Z/pCMV-SB100X or pKT2-CaRG:Z/pCMV-SB100X or no DNA (as mock) and selection with zeocin. Similar data were obtained in T cells from other two patients with sarcomas and two healthy donors (data not shown). (C) Flow cytometric analysis of IGF1R expression in sarcoma cell lines including Ewing sarcoma (EWS), alveolar or embryonal rhabdomyosarcoma (ARMS or ERMS), osteosarcoma (OS), and fibrosarcoma (FS). K562 (erythroleukemia), Daudi (B-cell Burkitt lymphoma), MCF7 (breast cancer) cell lines were used as control. (D) Flow cytometric analysis of ROR1 expression in sarcoma cell lines. RPMI8226 (multiple myeloma) cell line was used as control. Similar data shown in (C) and (D) were obtained in at least three independent assays.

Article Snippet: Goat anti-human ROR1 polyclonal ab (catalog #AF2000), normal goat IgG control (AB-108-C) and allophycocyamin conjugated donkey anti-goat IgG (F0108) were purchased from RnD Systems.

Techniques: Sequencing, Expressing, Derivative Assay, Transfection, Selection, Control

Journal: PLoS ONE

Article Title: IGF1R- and ROR1-Specific CAR T Cells as a Potential Therapy for High Risk Sarcomas

doi: 10.1371/journal.pone.0133152

Figure Lengend Snippet: (A) Cytotoxicity against IGF1R + target cells including sarcoma cell lines by SB modified IGF1R CAR T cells derived from two healthy donors. PBL1-IGZ, PBL1-IG, PBL2-IGZ and PBL2-mock T cells were generated by transfection of PBMCs derived from two healthy donors (PBL1 and PBL2) using pKT2-CaIG:Z or pKT2-CaIG plus pCMV-SB100X plasmids or without DNA. PBL-CD19CAR T cells were used as control. PBL1-IGZ: Rh1 vs K562 and OS17 vs K562, p < 0.001 at all 3 E/T ratios. PBL1-IG: Rh1 vs K562 and OS17 vs K562, p < 0.01. PBL2-IGZ: Rh1 vs K562, p < 0.001, OS17 vs K562, p < 0.01. Killing of Rh1 and OS17 in each CAR T cell group was also significant compared to the corresponding tumor cells in mock T cell group ( p < 0.05). (B) Cytotoxicity of IGF1R CAR T cells against a panel of sarcoma cell lines. Comparisons in PBL1-IGZ and PBL2-IGZ were conducted between each sarcoma line and K562, P = 0.0001 at all E/T ratios. (C) Cytotoxicity against sarcoma cells by IGF1R CAR T cells derived from four more healthy donors but not by mock T cells. Comparisons in PBL3-IGZ, PBL4-IGZ, PBL5-IGZ and PBL6-IGZ were conducted between Rh30 vs K562 and SaOS2 vs K562, P = 0.0001 at all E/T ratios.(D) Expression of human IGF1R in R- transfected cell line confirmed by flow cytometry. (E) Specific cytotoxicity against human IGF1R transfected cell line by IGF1R CAR T cells. Comparisons in PBL3-IGZ, PBL4-IGZ, PBL5-IGZ and PBL6-IGZ were conducted between R- and R-/IGF1R, P = 0.0001 at all E/T ratios. (F) Expression of ROR1 in DB and RBV-LCL cell lines. ROR1 - K562 and ROR1 + RPMI8226 were used as control (data not shown). (G) Cytotoxicity against ROR1 + target cells including a sarcoma cell line by ROR1 CAR T cells derived from two healthy donors. Comparisons in PBL7-RGZ, PBL7-GZ, PBL8-RGZ and PBL8-GZ were conducted between each target cell and K562 at all E/T ratios. PBL7-RGZ: SaOS2 vs K562, p = 0.0001, 0.0008, 0.0001 (E/T of 60:1, 20:1 and 6:1). PBL7-GZ: SaOS2 vs K562, p = 0.0221, 0.0762, 0.1121. PBL8-RGZ: SaOS2 vs K562, p = 0.0001, 0.0009, 0.0041. PBL8-GZ: SAOS2 vs K562, p = 0.2585, 0.8439, 0.9298. P values for DB vs K562 and EBV-LCL vs K562 were not shown. All cytotoxicity data shown are mean ± S.E. of triplicates.

Article Snippet: Goat anti-human ROR1 polyclonal ab (catalog #AF2000), normal goat IgG control (AB-108-C) and allophycocyamin conjugated donkey anti-goat IgG (F0108) were purchased from RnD Systems.

Techniques: Modification, Derivative Assay, Generated, Transfection, Control, Expressing, Flow Cytometry

Journal: PLoS ONE

Article Title: IGF1R- and ROR1-Specific CAR T Cells as a Potential Therapy for High Risk Sarcomas

doi: 10.1371/journal.pone.0133152

Figure Lengend Snippet: (A) Significant production of IFN-γ, TNF-α, and IL-13 by IGF1R and ROR1 CAR T cells derived from a healthy donor. Data shown are mean ± S.E. of duplicates. One out of two representative data is shown. (B) Antigen-specific production of IFN-γ by IGF1R and ROR1 CAR T cells derived from three sarcoma patients. Data shown are mean ± S.E. of duplicates.

Article Snippet: Goat anti-human ROR1 polyclonal ab (catalog #AF2000), normal goat IgG control (AB-108-C) and allophycocyamin conjugated donkey anti-goat IgG (F0108) were purchased from RnD Systems.

Techniques: Derivative Assay

Journal: PLoS ONE

Article Title: IGF1R- and ROR1-Specific CAR T Cells as a Potential Therapy for High Risk Sarcomas

doi: 10.1371/journal.pone.0133152

Figure Lengend Snippet: (A) The experimental schedule of tumor cell injection, CAR T cell infusion and BLI monitoring. Prior to testing, all mice displayed normal healthy status. B) Bioluminescent imaging (BLI) of tumor growth in NSG mice (three groups, n = 6–8 each) treated with a sarcoma patient derived T cells expressing IGF1R CAR (IGZ), ROR1 CAR (RGZ) or mock T cells. SaOS2-fflucN cells were transduced with a lentiviral vector expressing humanized firefly luciferase and truncated nerve growth factor receptor (NGFR). Two mice in the mock group died of tumor progression on day 8. Four mice in IGZ group died of unknown causes on day 8, 9, 16 and 24, probably due to cytokine storms. (C) Bioluminescent intensity of the mice treated with the T cells. All p values were shown in the right panel table and were verified independently. (D) Animal survival after T-cell therapy. All p values were determined using Mantel-Haenszel logrank test and shown in the right panel table. The p values were independently confirmed. Note that p > 0.05 between mock vs IGZ was likely due to a small sample size.

Article Snippet: Goat anti-human ROR1 polyclonal ab (catalog #AF2000), normal goat IgG control (AB-108-C) and allophycocyamin conjugated donkey anti-goat IgG (F0108) were purchased from RnD Systems.

Techniques: Injection, Imaging, Derivative Assay, Expressing, Transduction, Plasmid Preparation, Luciferase

Journal: PLoS ONE

Article Title: IGF1R- and ROR1-Specific CAR T Cells as a Potential Therapy for High Risk Sarcomas

doi: 10.1371/journal.pone.0133152

Figure Lengend Snippet: (A) The experimental schedule of tumor cell injection, CAR T cell infusion and BLI monitoring. Prior to testing, all mice displayed normal healthy status. (B) Bioluminescent imaging (BLI) of tumor growth in NOD/SCID mice (four groups, n = 12–13 each) treated with a sarcoma patient derived T cells expressing IGF1R CAR (IGZ), ROR1 CAR (RGZ) or mock T cells. One group mice were untreated. Two mice in the untreated group died of tumor progression on day 13 and 20 and the other two mice died of unknown causes on day 12 and 13. Three mice in the mock group died of tumor progression on day 7, 12 and 19 andone died of unknown causes on day 13. Three mice in the IGZ group died of unknown causes on day 10 and two died of unknown courses on day 17 and 18. Two mice in the RZG group died of tumor progression on day 12 and 50 andone died of unknown causes on day 13. (C) Bioluminescent intensity of the mice treated with the T cells. All p values were shown in the right panel table and were verified independently. (D) Animal survival after T-cell therapy. All p values were determined by the Mantel-Haenszel logrank test and are shown in the right panel table.

Article Snippet: Goat anti-human ROR1 polyclonal ab (catalog #AF2000), normal goat IgG control (AB-108-C) and allophycocyamin conjugated donkey anti-goat IgG (F0108) were purchased from RnD Systems.

Techniques: Injection, Imaging, Derivative Assay, Expressing

Journal: PLoS ONE

Article Title: The Tyrosine Kinase Receptor ROR1 Is Constitutively Phosphorylated in Chronic Lymphocytic Leukemia (CLL) Cells

doi: 10.1371/journal.pone.0078339

Figure Lengend Snippet: Western blot analysis of CLL lysates from 3 CLL patients (5072, 5194, 5002) treated with a lambda phosphatase enzyme. - : enzyme-untreated sample, + : enzyme-treated sample. PVDF blot was stripped and reprobed with a goat anti-ROR1 polyclonal Ab.

Article Snippet: Filters were incubated with an anti-pROR1 mAb (against the intracellular tyrosine kinase domain of ROR1) (a gift from Monoclonal Antibody Research Center, Avicenna Research Institute, Tehran, Iran), anti-phospho-tyrosine mAb (clone 4G10), anti-phospho-serine mAb (clone 4A4) (Millipore) and a polyclonal goat anti-human ROR1 Ab (R&D) for 2 h at room temperature.

Techniques: Western Blot

Journal: PLoS ONE

Article Title: The Tyrosine Kinase Receptor ROR1 Is Constitutively Phosphorylated in Chronic Lymphocytic Leukemia (CLL) Cells

doi: 10.1371/journal.pone.0078339

Figure Lengend Snippet: Representative experiments using non-immunoprecipitated CLL cell lysates from 5 CLL patients showing phosphorylated dimerized ROR1 (260 kDa), fully glycosylated (130 kDa) and non-glycosylated (105 kDa) ROR1 molecules (A). NP indicates non-progressive disease and P progressive disease. As controls, CLL cell lysates immunoprecipitated with a non-relevant mAb (mouse IgG1 isotype) were used. No bands could be seen. Furthermore, in PBMC of healthy donors, no bands were detected (data not shown). Representative experiments of PBMC using surface staining for ROR1 (left panel) and intracytoplasmic staining for pROR1 (right panel) in three CLL patients (B). Expression of ROR1 isoforms in the cytoplasm (C), nucleus (N) and total cell lysate (TCL) of leukemic CLL cells (C). Confirmation of protein localization was done using antibodies against α/β –tubulin, histone H3 and nucleolin before analysing the expression pattern of ROR1.

Article Snippet: Filters were incubated with an anti-pROR1 mAb (against the intracellular tyrosine kinase domain of ROR1) (a gift from Monoclonal Antibody Research Center, Avicenna Research Institute, Tehran, Iran), anti-phospho-tyrosine mAb (clone 4G10), anti-phospho-serine mAb (clone 4A4) (Millipore) and a polyclonal goat anti-human ROR1 Ab (R&D) for 2 h at room temperature.

Techniques: Immunoprecipitation, Staining, Expressing

Journal: PLoS ONE

Article Title: The Tyrosine Kinase Receptor ROR1 Is Constitutively Phosphorylated in Chronic Lymphocytic Leukemia (CLL) Cells

doi: 10.1371/journal.pone.0078339

Figure Lengend Snippet: pROR1/ROR1 intensity (mean ± SEM) of the 105, 130 and combined 105+130 kDa bands in progressive (■) (n=21) and non-progressive (☐) CLL patients (n=17). *** p= 0.001.

Article Snippet: Filters were incubated with an anti-pROR1 mAb (against the intracellular tyrosine kinase domain of ROR1) (a gift from Monoclonal Antibody Research Center, Avicenna Research Institute, Tehran, Iran), anti-phospho-tyrosine mAb (clone 4G10), anti-phospho-serine mAb (clone 4A4) (Millipore) and a polyclonal goat anti-human ROR1 Ab (R&D) for 2 h at room temperature.

Techniques:

Journal: PLoS ONE

Article Title: The Tyrosine Kinase Receptor ROR1 Is Constitutively Phosphorylated in Chronic Lymphocytic Leukemia (CLL) Cells

doi: 10.1371/journal.pone.0078339

Figure Lengend Snippet: Representative experiments of time-dependent ROR1 dephosphorylation and apoptosis in vitro incubated with an anti-KNG (4A7) (A and C) and anti-CRD (1D8) (B and D) anti-ROR1 mAb respectively. Representative experiments from two progressive (CLL 5102 and CLL 5116) CLL patients are shown. Leukemic cells were incubated for 20 min, 1, 4 hours with a non-relevant IgG1 isotype control mAb (-) and the ROR1 mAbs (+). Cells were harvested and lysed for western blot experiments (A and B). The non-relevant isotype control mAb did not induce dephosphorylation of ROR1 (130 kDa) while the anti-ROR1 mAb induced dephosphorylation already after 20 min, which increased by time. The intensity of pROR1 was measured by ImageJ software. The ratio of pROR1/ROR1 intensity of treated sample to pROR1/ROR1 intensity of untreated sample (relative intensity) is shown in a histogram. A value <1 indicates dephosphorylation of ROR1 after treatment with the anti-ROR1 mAb compared to the non-relevant isotype control mAb. Apoptosis of CLL cells (Annexin V + /PI + ) after 0, 12 and 18 h incubation with the anti ROR1 mAbs without the addition of immune effector cells or complement (C and D). An anti-CD20 mAb (ofatumumab) did not induce dephosphorylation of ROR1 after 4h incubation in two CLL patients. A representative experiment is shown for one of the CLL patients (E).

Article Snippet: Filters were incubated with an anti-pROR1 mAb (against the intracellular tyrosine kinase domain of ROR1) (a gift from Monoclonal Antibody Research Center, Avicenna Research Institute, Tehran, Iran), anti-phospho-tyrosine mAb (clone 4G10), anti-phospho-serine mAb (clone 4A4) (Millipore) and a polyclonal goat anti-human ROR1 Ab (R&D) for 2 h at room temperature.

Techniques: De-Phosphorylation Assay, In Vitro, Incubation, Control, Western Blot, Software