Journal: Translational Cancer Research

Article Title: In vitro characterization of a novel anti-GD2/ROR1 bispecific antibody exhibiting tumor cell killing efficacy in neuroblastoma cross-cancer activity in ROR1-positive triple-negative breast cancer

doi: 10.21037/tcr-2026-0723

Figure Lengend Snippet: Molecular design schematic and purification gel image of bispecific antibody G/R-001. (A) Molecular design diagram of the light chain and heavy chain of bispecific antibody G/R-001. (B) Schematic diagram of GD2/ROR1 bispecific antibody G/R-001. (C) SDS-PAGE gel image of the bispecific antibody under reducing or non-reducing conditions. The left shows the SDS-PAGE gel image under reducing conditions, with two bands in the lane-the upper band representing the heavy chain and the lower band representing the light chain; the right shows the SDS-PAGE gel image under non-reducing conditions, with one band in the lane representing the intact antibody. CMV, cytomegalovirus; G/R-001, anti-GD2/ROR1 bispecific antibody; GD2, disialoganglioside 2; H, heavy chain; IgG, immunoglobulin G; L, light chain; ROR1, receptor tyrosine kinase-like orphan receptor 1; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; VH, variable domain of heavy chain; VK, variable domain of light chain.

Article Snippet: Isotype IgG (Cat#: HY-P99001, MedChemExpress, Princeton, NJ, USA), naxitamab (anti-GD2) (Cat#: HY-P99206, MedChemExpress), zilovertamab (anti-ROR1) (Cat#: HY-P99201, MedChemExpress), and GD2 × ROR1 BsAb were transiently expressed in HEK293 cells using the OPM-293 system (Cat#: 91070-010, OPM, Shanghai, China).

Techniques: Purification, SDS Page, Polyacrylamide Gel Electrophoresis

Journal: Translational Cancer Research

Article Title: In vitro characterization of a novel anti-GD2/ROR1 bispecific antibody exhibiting tumor cell killing efficacy in neuroblastoma cross-cancer activity in ROR1-positive triple-negative breast cancer

doi: 10.21037/tcr-2026-0723

Figure Lengend Snippet: ELISA detection of the binding activity of bispecific antibody G/R-001 to GD2 and ROR1. GD2 or ROR1 antigens were coated, and G/R-001, zilovertamab, naxitamab, and isotype IgG antibodies were added to the ELISA plate at concentrations of 0–150 nM with 2-fold serial dilutions. After secondary antibody incubation, absorbance values were detected at 450 nM wavelength by a microplate reader, curves were plotted, and EC 50 values were calculated. (A) Binding affinity of G/R-001 and naxitamab to GD2, with EC 50 values of 12.1 and 11.9 nM, respectively; (B) Binding affinity of G/R-001 and zilovertamab to ROR1, with EC 50 values of 3.5 and 3.1 nM, respectively. EC 50 , half maximal effective concentration; ELISA, enzyme-linked immunosorbent assay; G/R-001, anti-GD2/ROR1 bispecific antibody; GD2, disialoganglioside 2; IgG, immunoglobulin G; OD, optical density; ROR1, receptor tyrosine kinase-like orphan receptor 1.

Article Snippet: Isotype IgG (Cat#: HY-P99001, MedChemExpress, Princeton, NJ, USA), naxitamab (anti-GD2) (Cat#: HY-P99206, MedChemExpress), zilovertamab (anti-ROR1) (Cat#: HY-P99201, MedChemExpress), and GD2 × ROR1 BsAb were transiently expressed in HEK293 cells using the OPM-293 system (Cat#: 91070-010, OPM, Shanghai, China).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Activity Assay, Incubation, Concentration Assay

Journal: Translational Cancer Research

Article Title: In vitro characterization of a novel anti-GD2/ROR1 bispecific antibody exhibiting tumor cell killing efficacy in neuroblastoma cross-cancer activity in ROR1-positive triple-negative breast cancer

doi: 10.21037/tcr-2026-0723

Figure Lengend Snippet: Flow cytometry detection of the binding activity of bispecific antibody G/R-001 to GD2/ROR1 positive cells. GD2 − /ROR1 − HEK293 cells were used as a negative control. G/R-001, zilovertamab, naxitamab, and isotype IgG at 10 nM concentration showed significant binding activity to GD2 − /ROR1 + MDA-MB-468 cells, GD2 + /ROR1 − LAN-1 cells, and GD2 + /ROR1 + MHH-NB-11 cells. FITC, fluorescein isothiocyanate; G/R-001, anti-GD2/ROR1 bispecific antibody; GD2, disialoganglioside 2; IgG, immunoglobulin G; ROR1, receptor tyrosine kinase-like orphan receptor 1.

Article Snippet: Isotype IgG (Cat#: HY-P99001, MedChemExpress, Princeton, NJ, USA), naxitamab (anti-GD2) (Cat#: HY-P99206, MedChemExpress), zilovertamab (anti-ROR1) (Cat#: HY-P99201, MedChemExpress), and GD2 × ROR1 BsAb were transiently expressed in HEK293 cells using the OPM-293 system (Cat#: 91070-010, OPM, Shanghai, China).

Techniques: Flow Cytometry, Binding Assay, Activity Assay, Negative Control, Concentration Assay

Journal: Translational Cancer Research

Article Title: In vitro characterization of a novel anti-GD2/ROR1 bispecific antibody exhibiting tumor cell killing efficacy in neuroblastoma cross-cancer activity in ROR1-positive triple-negative breast cancer

doi: 10.21037/tcr-2026-0723

Figure Lengend Snippet: LDH assay detection of the in vitro killing activity of bispecific antibody G/R-001 against GD2/ROR1 positive neuroblastoma and other cells. Isotype IgG, naxitamab, zilovertamab, naxitamab + zilovertamab, and G/R-001 antibodies (50 µL) at different concentrations (0–100.0 nM) were added to the experimental wells and cultured for 48 h. Following the LDH kit procedure, absorbance was read at 490 nm using a microplate reader. (A) HEK293 cells (GD2 − /ROR1 − ), (B) LAN-1 cells (GD2 + /ROR1 − ), (C) MDA-MB-468 cells (GD2 − /ROR1 + ), and (D) MHH-NB-11 cells (GD2 + /ROR1 + ). With increasing concentrations, Naxitamab, Zilovertamab, Naxitamab+Zilovertamab, and G/R-001 antibodies showed significantly increased toxicity against target-positive cells, with G/R-001 demonstrating the most significant cytotoxicity. G/R-001, anti-GD2/ROR1 bispecific antibody; GD2, disialoganglioside 2; IgG, immunoglobulin G; LDH, lactate dehydrogenase; ROR1, receptor tyrosine kinase-like orphan receptor 1.

Article Snippet: Isotype IgG (Cat#: HY-P99001, MedChemExpress, Princeton, NJ, USA), naxitamab (anti-GD2) (Cat#: HY-P99206, MedChemExpress), zilovertamab (anti-ROR1) (Cat#: HY-P99201, MedChemExpress), and GD2 × ROR1 BsAb were transiently expressed in HEK293 cells using the OPM-293 system (Cat#: 91070-010, OPM, Shanghai, China).

Techniques: Lactate Dehydrogenase Assay, In Vitro, Activity Assay, Cell Culture

Journal: Biomedicines

Article Title: Variant Transcript of ROR1 ENST00000545203 Does Not Encode ROR1 Protein

doi: 10.3390/biomedicines12071573

Figure Lengend Snippet: ( A ) ROR1 lentiviral plasmid map containing full-length ROR1 or ROR v3 upstream of an IRES and gene encoding RFP, namely dTomato. MEC1, MEC1-ROR1, or MEC1-ROR1 v3 were examined for RFP fluorescence by ( B ) flow cytometry cells or ( C ) fluorescence microscopy.

Article Snippet: Western blot analysis was performed using primary mAbs specific for ROR1 (1:1000 dil, catalog 4102, Cell Signaling Technology, Danvers, MA, USA), ROR1 (1:500 dil, catalog ROR1-101AP, FabGennix, Frisco, TX, USA), ERK1/2 (1:1000 dil, catalog 9102, Cell Signaling Technology), phosphoERK1/2 (1:1000 dil, catalog 9101, Cell Signaling Technology), or β-actin (1:1000 catalog 4967, Cell Signaling Technology), which were detected using anti-rabbit IgG HRP-linked secondary antibody (1:2000 dil, catalog 7074, Cell Signaling Technology).

Techniques: Plasmid Preparation, Fluorescence, Flow Cytometry, Microscopy

Journal: Biomedicines

Article Title: Variant Transcript of ROR1 ENST00000545203 Does Not Encode ROR1 Protein

doi: 10.3390/biomedicines12071573

Figure Lengend Snippet: ROR1 mRNA and protein detection. ( A ) ROR1 mRNA expression analysis by RT-PCR. Two-way ANOVA was used to determine statistical significance for mRNA expression. Bar graphs represent data summarized as mean ± S.E.M. and were analyzed by unpaired two-sided Mann–Whitney test. Data shown are representative of three experiments. NS (non-significant). ( B ) ROR1 cell surface staining was assessed via flow cytometry using fluorochrome labeled anti-ROR1 mAb 4A5 or UC-961. ( C ) Fixed and permeabilized cells were stained with an Alexa-488-conjugated mAb specific for ERK1/2 and an Alexa-647-conjugated mAb specific for phosphorylated ERK1/2 and then examined via flow cytometry. ( D ) Fixed and permeabilized cells were stained with Alexa-647-conjugated anti-ROR1 mAbs, 4A5 or UC961, and then analyzed via flow cytometry, as indicated in the figure. ( E ) Immunoblot of cell lysates of MEC1, MEC1-ROR1, or MEC1-ROR v3 probed for ROR1 using CST antibody (CST ROR1 Cat No: #4102, left) or FabGennix antibody (FabGennix Cat No: ROR1-101AP, right). The uncropped blots are shown in .

Article Snippet: Western blot analysis was performed using primary mAbs specific for ROR1 (1:1000 dil, catalog 4102, Cell Signaling Technology, Danvers, MA, USA), ROR1 (1:500 dil, catalog ROR1-101AP, FabGennix, Frisco, TX, USA), ERK1/2 (1:1000 dil, catalog 9102, Cell Signaling Technology), phosphoERK1/2 (1:1000 dil, catalog 9101, Cell Signaling Technology), or β-actin (1:1000 catalog 4967, Cell Signaling Technology), which were detected using anti-rabbit IgG HRP-linked secondary antibody (1:2000 dil, catalog 7074, Cell Signaling Technology).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY, Staining, Flow Cytometry, Labeling, Western Blot

Journal: Clinical and Translational Medicine

Article Title: WNT5B drives osteosarcoma stemness, chemoresistance and metastasis

doi: 10.1002/ctm2.1670

Figure Lengend Snippet: ROR1 is a therapeutic target for osteosarcoma stem cells. (A) Expression array correlation data showing a significantly positive correlation between WNT5B and ROR1 in osteosarcoma samples. Data were analysed using the R2 genomics analysis and visualization platform, and data generated by Kuijjer et al. ¹⁸ n = 127, p < 3.5 × 10 −14 . (B) Representative images of immunohistochemistry on an osteosarcoma tissue microarray stained for ROR1 and WNT5B. Matched images from two samples, one low for both WNT5B and ROR1 and one high for both WNT5B and ROR1. n = 40 cores in duplicate, scale bar = 400 µm. (C) Correlation graph of osteosarcoma tissue microarray depicted in (B. Average immune reactive scoring of all WNT5B and ROR1 cores indicates a positive correlation between WNT5B and ROR1 in osteosarcoma patient samples. n = 40 cores, p = .0007. (D) Immunofluorescence of 143B adherent cells and spheres. 10× microscopy images, WNT5B = green, ROR1 = red, DAPI nuclear stain = blue. White arrows indicate areas of WNT5B/ROR1 colocalisation (yellow), scale bar = 400 µm. (E) 143B spheres treated with siRNA to ROR1 or scrambled siRNA for 72 h. Sphere size quantified using ImageJ. n = > 380 spheres per group, **** p < .0001. (F) 143B spheres treated with 100 µg/mL D10 and/or 50 ng/mL rWNT5B for 48 h. The sphere area was quantified using ImageJ. n ≥ 80 spheres per group, * p < .05. (G) 143B spheres treated with a low dose of (40 µg/mL) D10 and/or high dose (10 µg/mL) MTX for 48 h to assess reduction in chemoresistance. The sphere area was quantified using ImageJ. n ≥ 110 spheres per group, * p < .05, **** p < .0001. (H) PDX‐derived spheres treated with increasing doses (0, 10, 40, 100 µg/mL) of D10. Sphere area quantified using ImageJ. n ≥ 210 spheres per group, ** p < .01. (I) SOX2 western blot of 143B spheres control, spheres treated with 40 µg/mL D10 for 48 h, and 143B WNT5B KO spheres. (J) ImageJ quantification of the western blot in (I). * p < .05, ** p < .01, ns = not significant. Normalised to β‐Actin, n = 3 experimental replicates. PDX, Patient‐derived xenograft; MTX, methotrexate.

Article Snippet: Cells were treated with 100 nM of either a scrambled control or ROR1 (Santa Cruz: sc‐76424) siRNA combined with Lipofectamine Reagent for 15 min and then added to the cells for transfection.

Techniques: Expressing, Generated, Immunohistochemistry, Microarray, Staining, Immunofluorescence, Microscopy, Derivative Assay, Western Blot, Control

Journal: Pharmaceutics

Article Title: A Small Molecule Targeting the Intracellular Tyrosine Kinase Domain of ROR1 (KAN0441571C) Induced Significant Apoptosis of Non-Small Cell Lung Cancer (NSCLC) Cells.

doi: 10.3390/pharmaceutics15041148

Figure Lengend Snippet: Figure 1. (A) Surface staining for ROR1 (flow cytometry) (red line: isotype control; blue line: anti- ROR1 antibody); (B) Western blot for ROR1 and pROR1 expression (130 KDa) in 5 lung cancer cell lines. Actin was used as internal control.

Article Snippet: The following primary antibodies were used for protein staining: phospho (p) ROR1 (against amino acid residues Ser 652, Tyr 641, 646) [46], anti-ROR1 antibody (R&D Systems, Minneapolis, MN, USA), anti-EGFR and anti-pEGFR (Y1173) antibodies, anti-SRC and anti-p-SRC (Tyr 416) antibodies, anti-AKT and anti-p-AKT (Ser 473) antibodies, anti-mTOR and anti-p-mTOR (Ser 2448) antibodies, anti-cAMP response element-binding protein (CREB) and anti-p-CREB (Ser 133) antibodies, anti-cleaved poly ADP ribose polymerase (PARP) antibody, anti-B-cell lymphoma (BCL)-2 antibody, anti-BAX antibody, anti-myeloid cell leukemia (MCL)-1 antibody, anti-cleaved caspases 3, 8, 9 antibodies (Cell Signaling Technology, Danvers, MA, USA), anti-phosphoinositide 3-kinase (PI3K) p110δ and anti-p-PI3Kp110δ (Tyr 485) antibodies (Santa Cruz), and antiβ-actin antibody (Sigma-Aldrich).

Techniques: Staining, Cytometry, Control, Western Blot, Expressing