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

Introduction

Neuroblastoma (NB) is a malignant tumor originating from the sympathetic nervous system, primarily affecting children aged 0–14 years and accounting for 8–10% of all pediatric tumors (1,2). Approximately 40–50% of cases are diagnosed within the first year of life, and about 90% occur before the age of 10 (2). According to the World Health Organization (WHO), there are an estimated 5,901–39,340 cases of NB globally, with 744–4,960 cases in China. NB is characterized by high invasiveness and rapid proliferation, making its treatment extremely challenging and requiring comprehensive therapeutic strategies to improve patient survival rates (3-5). Despite current treatment modalities including surgery, chemotherapy, radiotherapy, and immunotherapy, the prognosis for NB remains unfavorable, with many patients still at risk of recurrence and drug resistance (6). Therefore, developing novel antibody therapies to improve treatment efficacy, especially bispecific antibodies targeting tumor-specific antigens, has become a research hotspot.

Disialoganglioside 2 (GD2) is a glycosphingolipid highly expressed in multiple neuroectodermal tumors. Approximately 98% of NB patients showing high GD2 expression, and its high expression on NB cell surfaces is closely associated with tumor invasiveness and poor prognosis (7-9). Additionally, GD2 is highly expressed in 80% of glioma and melanoma patients, as well as in tumors such as small cell lung cancer (8). Consequently, GD2-targeted therapy has become particularly important. Since receiving U.S. Food and Drug Administration (FDA) approval in 2020, Naxitamab has achieved significant clinical efficacy, effectively improving the prognosis of patients with NB, small cell lung cancer, and other conditions (10-13). However, serious adverse events have been observed during treatment with GD2 monoclonal antibodies like Naxitamab (14). Therefore, developing novel therapeutic approaches to address this clinical challenge is imperative.

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a tyrosine protein kinase receptor of which its abnormal expression in various tumor cells is associated with tumor proliferation, survival, and drug resistance (15). An increasing body of literature has established ROR1 as a marker for cancers, such as in chronic lymphocytic leukemia (CLL), NB, and other malignancies (16). Studies have shown that anti-ROR1 chimeric antigen receptor-natural killer (CAR-NK) cells significantly enhanced cytotoxicity against ROR1-positive NB in both in vitro and in vivo models, and prolonged the survival of tumor-bearing mice (17). Furthermore, Zilovertamab vedotin (MK-2140) has demonstrated promising efficacy in phase 3 clinical trials for NB, offering new hope for the treatment of NB.

In recent years, bispecific antibodies (BsAb) have gained widespread attention as a novel immunotherapeutic approach. Bispecific antibodies can simultaneously target two different antigens, bridging immune cells and tumor cells to enhance the recognition and killing capability of immune cells against tumor cells. This dual-targeting strategy not only improves the specificity and efficacy of treatment, but also reduces damage to normal tissues and decreases treatment side effects. This study aims to develop a novel anti-GD2/ROR1 bispecific antibody (BsAb) G/R-001 and characterize its tumor cell killing effect in NB in vitro. This provides a new effective approach for NB treatment and contributes to improving patient prognosis and quality of life. We present this article in accordance with the MDAR reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-0723/rc).

Methods

Cell lines

HEK293 cell line (Cat#IM-H371, IMMOCELL, Xiamen, China) was cultured in MEM medium (Cat#IMC-304, IMMOCELL) supplemented with 10% fetal bovine serum (Cat#A5256701, Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin (Cat#C0222, Beyotime, Shanghai, China). MDA-MB-468 (Cat#IM-H316, IMMOCELL) was cultured in MDA-MB-468 cell-specific medium (Cat#IM-H316-1, IMMOCELL). LAN-1 cells (Cat#ACC655, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Deutschland) were cultured in RPMI-1640 medium (Cat#IMC-202, IMMOCELL) supplemented with 20% fetal bovine serum (Cat#A5256701, Gibco) and 1% penicillin-streptomycin (Cat#C0222, Beyotime). MHH-NB-11 cells (Cat#ACC157, Deutsche Sammlung von Mikroorganismen und Zellkulturen) were cultured in RPMI-1640 medium (Cat#IMC-202, IMMOCELL) supplemented with 15% fetal bovine serum (Cat#A5256701, Gibco) and 1% penicillin-streptomycin (Cat#C0222, Beyotime). All cells were cultured at 37 ℃ with 95% O₂ and 5% CO₂.

Design and molecular construction of BsAb

To obtain anti-GD2 and anti-ROR1 antibodies, this study identified the light and heavy chain sequences of naxitamab (anti-GD2) and zilovertamab (anti-ROR1) through the international ImMunoGeneTics information system (IMGT) (Table S1). The genes were synthesized (with Human/CHO codon optimization) by Anhui Universal Biotechnology Co., Ltd. to construct expression vectors for the light and heavy chains of naxitamab and zilovertamab. The Chain H expression vector for G/R-001 was constructed through polymerase chain reaction (PCR) amplification (using the light chain and heavy chain expression vectors of zilovertamab and the heavy chain expression vector of naxitamab as templates) and Gibson assembly (Table S2). All correctly cloned expression plasmids were verified by first-generation sequencing (Qingke Biology, Beijing, China).

Expression and purification of BsAb

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). After 6–7 days of transient transfection, cell culture supernatants were collected from each sample (cell viability >60%), and antibodies were purified using gravity column-Protein A affinity chromatography. The samples were concentrated and buffer-exchanged to phosphate-buffered saline (PBS) (pH 6–7) by ultrafiltration, filtered through a 0.22-µm sterile membrane, aliquoted, and stored at −80 ℃ for future use. Non-reducing and reducing SDS-PAGE were used to detect antibody purity; Nanodrop A280/A260 was used to determine antibody concentration.

Enzyme-linked immunosorbent assay (ELISA) detection of G/R-001 affinity

ELISA was used to detect the binding affinity of G/R-001 antibody to GD2 and ROR1. Each well of an ELISA 96-well plate was coated with 100 µL of Disialoganglioside GD2 or human ROR1-ECD (1 µg/mL, diluted in carbonate buffer pH 9.6) overnight at 4 ℃. The following day, plates were washed three times with PBS-Tween 20 (0.05%). Bovine serum albumin (BSA) (200 µL of 1% solution) was added to each well and incubated at 37 ℃ for 1–2 h, followed by three washes with PBS-Tween 20 (0.05%). Subsequently, isotype IgG, naxitamab (anti-GD2), zilovertamab (anti-ROR1), and GD2 × ROR1 BsAb G/R-001 were serially diluted to concentrations of 0, 2.344, 4.688, 9.375, 18.75, 37.5, 75, and 150 nM, and 100 µL was added to each well. After incubation at 37 ℃ for 1–2 h, plates were washed three times with PBS-Tween 20. HRP-labeled goat anti-human IgG secondary antibody (Cat#: 2040-05, SouthernBiotech, Birmingham, AL, USA) (1:10,000 dilution) was then added (100 µL/well) and incubated at 37 ℃ for 1 h, followed by three washes with PBS-Tween 20 (0.05%). 3,3',5,5'-Tetramethylbenzidine (TMB) substrate solution was added, incubated at room temperature for 15 min, and the reaction was stopped with 2 M sulfuric acid. Absorbance was measured at 450 nm within 15 min using a microplate reader. Four-parameter logistic regression model curves were plotted to calculate the affinity of the tested antibodies.

Flow cytometry detection of G/R-001 binding activity

Flow cytometry was used to detect the binding of G/R-001 to HEK293 cells, MDA-MB-468 cells, LAN-1 cells, and MHH-NB-11 cells. When cells reached 80–90% confluence, they were collected by trypsin digestion and resuspended in PBS to prepare single-cell suspensions. Antibodies (G/R-001, zilovertamab, naxitamab, IgG isotype) were serially diluted in PBS containing 0.5% BSA. Cells were incubated with antibodies at a concentration of 10 nM at 4 ℃ for 45 min. Subsequently, free antibodies were washed away with PBS containing 0.5% BSA. The cells were then incubated with FITC-labeled anti-human IgG Fc secondary antibody (Cat#: F9512, Sigma, St. Louis, MO, USA) (1:200 dilution) in the dark at 4 ℃ for 45 min. After washing, FITC fluorescence signals of the cells were analyzed using a NovoCyte 2060R flow cytometer (Agilent,, Santa Clara, CA, USA). The acquired data were analyzed using FlowJo X software.

Cell killing-antitumor activity detection by lactate dehydrogenase (LDH) assay

HEK293 cells, MDA-MB-468 cells, LAN-1 cells, and MHH-NB-11 cells in logarithmic growth phase (1×10⁴ cells/well) were co-cultured with NK92MI-hCD16-V158 cells (at an E:T ratio of 5:1) in 96-well plates. Isotype IgG, naxitamab (anti-GD2), zilovertamab (anti-ROR1), and G/R-001 antibodies (50 µL) at different concentrations (0, 0.1, 1.0, 10.0, and 100.0 nM) were added [a combination treatment group of naxitamab (anti-GD2) and zilovertamab (anti-ROR1) was also included]. Controls included spontaneous release wells (tumor cells + medium) and maximum release wells (tumor cells + 1% Triton™ X-100). After incubation at 37 ℃ with 5% CO₂ for 4–6 h, plates were centrifuged (250 ×g, 5 min), and 50 µL of cell supernatant was transferred to a new 96-well assay plate. LDH reaction (50 µL) solution (Cat#: 37291S, CST, Danvers, MA, USA) was added to each well and incubated in the dark for 30 min. Absorbance was measured at 490 nm using a microplate reader. The killing rate (%) was calculated as: (test well OD − spontaneous release well OD)/(maximum release well OD − spontaneous release well OD) ×100%.

Statistical analysis

All experiments were performed with at least three biological replicates, and all data are presented as mean ± standard error of the mean (SEM). GraphPad Prism 10.0 software (GraphPad, La Jolla, CA, USA) was used for statistical calculations in this study. When the sample reached three or more groups, one-way analysis of variance was used for inter-group comparisons and followed by Tukey’s post hoc test. Statistical significance was defined as P<0.05.

Results

Preparation and characterization of G/R-001

G/R-001 is a novel BsAb antibody that specifically binds to GD2 and ROR1. This antibody was constructed using naxitamab (anti-GD2) developed by Memorial Sloan Kettering Cancer Center and zilovertamab (anti-ROR1) developed by the University of California. The BsAb produced in this study is based on the human IgG1 subtype, with a heavy chain consisting of one variable VH domain and three constant domains CH1, CH2, and CH3, while the corresponding light chain consists of one variable VK domain and one constant Kc domain (Figure 1A). As a recombinant fusion protein, the structural characteristic of G/R-001 Chain H is that Anti-ROR1 scFv is fused to the C-terminus of the Anti-GD2 heavy chain via a (G4S)₄ linker (“GGGGSGGGSGGGSGGGS”) (Figure 1B). The VH and VK of the Anti-ROR1 scFv are also connected and stabilized through a (G4S)₄ linker. Molecular weight prediction of G/R-001 showed that the complete G/R-001 has a molecular weight of 198.82 kDa (the glycosylated intact antibody showed an estimated molecular weight of 248.68 kDa on non-reducing electrophoresis), with its heavy and light chains having molecular weights of 76.3 and 23.1 kDa, respectively. Therefore, under reducing conditions, SDS-PAGE results showed two distinct bands corresponding to the heavy and light chains; while under non-reducing conditions, SDS-PAGE results showed a single band representing the intact antibody (Figure 1C). The single band also indicated high purity and good stability.

Figure 1 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.

G/R-001 demonstrated binding affinity to GD2 and ROR1

To verify the binding affinity of G/R-001 to GD2 and ROR1 antigens, the study employed two methods: ELISA and flow cytometry. ELISA results showed that G/R-001 and naxitamab exhibited binding affinity to hGD2, while zilovertamab and isotype IgG did not demonstrate binding affinity to hGD2. The half maximal effective concentration (EC₅₀) values of G/R-001 and naxitamab were 12.1 nM and 11.9 nM, respectively (Figure 2A). Simultaneously, regarding binding affinity to hROR1, G/R-001 demonstrated binding strength comparable to zilovertamab, while naxitamab and isotype IgG showed no binding affinity to hROR1. The EC₅₀ values of G/R-001 and zilovertamab were 3.5 and 3.1 nM, respectively (Figure 2B).

Figure 2 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 EC₅₀ values were calculated. (A) Binding affinity of G/R-001 and naxitamab to GD2, with EC₅₀ values of 12.1 and 11.9 nM, respectively; (B) Binding affinity of G/R-001 and zilovertamab to ROR1, with EC₅₀ values of 3.5 and 3.1 nM, respectively. EC₅₀, 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.

Additionally, flow cytometry results showed that in GD2⁻/ROR1⁻ HEK293 cells, G/R-001, zilovertamab, naxitamab, and isotype IgG exhibited consistent cell binding numbers; in GD2⁻/ROR1⁺ MDA-MB-468 cells, G/R-001 and zilovertamab showed significantly increased numbers of positive binding cells; in GD2⁺/ROR1⁻ LAN-1 cells, G/R-001 and naxitamab demonstrated significantly increased numbers of positive binding cells; in GD2⁺/ROR1⁺ MHH-NB-11 cells, G/R-001, zilovertamab, and naxitamab all showed significantly increased numbers of positive binding cells (Figure 3). In summary, the G/R-001 BsAb can simultaneously bind to both GD2 and ROR1.

Figure 3 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.

G/R-001 BsAb demonstrated in vitro killing activity against NB and other cells

To further understand the toxicity of G/R-001 against GD2/ROR1 positive cells, the study evaluated the in vitro killing activity of G/R-001 against GD2/ROR1 positive NB cells and other related cancer cell lines using the LDH assay. The results showed that isotype IgG, naxitamab, zilovertamab, naxitamab + zilovertamab, and G/R-001 antibodies at different concentrations from 0 to 100.0 nM (in 10-fold serial dilutions) did not exhibit significant cytotoxicity against GD2⁻/ROR1⁻ HEK293 cells (Figure 4A). Compared to the isotype IgG control, naxitamab significantly reduced the viability of GD2⁺/ROR1⁻ LAN-1 cells (P<0.05 in a dose-dependent manner, with cell death rates reaching 15.83% at 100 nM concentration (Figure 4B). Zilovertamab significantly reduced the viability of GD2⁻/ROR1⁺ MDA-MB-468 cells (P<0.05), with cell death rates reaching 52.56% and 62.02%, respectively, at 100 nM concentration (Figure 4C). Additionally, naxitamab and zilovertamab significantly reduced the viability of GD2⁺/ROR1⁺ MHH-NB-11 cells (P<0.05), with cell death rates reaching 45.60% and 62.02%, respectively, at 100 nM concentration (Figure 4D). After treatment with naxitamab + zilovertamab and G/R-001 antibody, the viability of GD2⁺/ROR1⁻ LAN-1 cells, GD2^-/ROR1⁺ MDA-MB-468 cells, and GD2⁺/ROR1⁺ MHH-NB-11 cells all decreased significantly (P<0.05) in a dose-dependent manner. At 100 nM concentration, naxitamab + zilovertamab achieved cell death rates of 55.85%, 59.25%, and 69.73%, respectively, while G/R-001 antibody achieved cell death rates of 59.03%, 67.58%, and 84.50%, respectively (Figure 4B-4D). In conclusion, G/R-001 antibody demonstrated significant tumor-killing effects with dose dependency, providing strong experimental evidence for a novel anti-tumor drug.

Figure 4 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.

Discussion

This study characterized a novel anti-GD2/ROR1 BsAb (G/R-001) in vitro and evaluated its tumor cell killing effect in NB. GD2 is an antigen widely expressed on the surface of NB and other neurogenic tumor cells, and has become a focus for targeted therapy (7,8). Currently, several GD2 monoclonal antibodies have achieved good results in clinical settings. Dinutuximab was approved by the FDA in 2015 for the treatment of high-risk pediatric NB, used in combination with granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-2 (IL-2), and 13-cis-retinoic acid, significantly improving patients’ event-free survival (EFS) and overall survival (OS) (18,19). In 2020, Naxitamab was approved by the FDA for the treatment of relapsed or refractory high-risk NB in bone or bone marrow, showing good efficacy and safety when used in combination with GM-CSF (20,21). Meanwhile, ROR1 is overexpressed in multiple cancers, including NB, and is associated with tumor malignancy and prognosis (15,16,22). Zilovertamab (anti-ROR1) has shown safety and efficacy in various ROR1-related malignancies, including CLL and mantle cell lymphoma (MCL) (23,24). The ROR1-targeted antibody-drug conjugate NBE-002 reduces toxicity to normal tissues by specifically delivering cytotoxic drugs to tumor cells with high ROR1 expression (23). However, single-target therapies face challenges such as tumor heterogeneity and immune escape. Therefore, this study designed a novel anti-GD2/ROR1 BsAb, G/R-001 aimed at enhancing antitumor activity through dual-target synergy.

The design of G/R-001 focused on optimizing the structure of heavy and light chains, using human IgG1 subtype and variable regions (VH and VK), and enhancing antibody stability through linker peptides (G4S) (25,26). Molecular weight prediction of G/R-001 showed that the complete G/R-001 has a molecular weight of 198.82 kDa, with the glycosylated intact antibody showing an estimated molecular weight of 248.68 kDa on non-reducing electrophoresis. Its heavy and light chains have molecular weights of 76.3 and 23.1 kDa, respectively. Due to glycosylation modifications, the antibody’s molecular weight is larger than expected; these glycosylation modifications can optimize the BsAb’s binding capacity to both antigens and improve its therapeutic effect (27). The results showed that under reducing conditions in SDS-PAGE, G/R-001 exhibited a distinct single band, indicating its high purity, while under non-reducing conditions, it showed the complete antibody structure (Figure 1). This design significantly enhanced the antibody’s biological activity and clinical application potential.

In vitro characterization verified the high binding capacity of G/R-001 for both GD2 and ROR1 through methods such as ELISA and flow cytometry (Figures 2,3). In cell killing experiments, G/R-001 demonstrated differential cytotoxic activity across the three cell lines tested. At a concentration of 100 nM, G/R-001 achieved a cell death rate of 84.50% against GD2⁺/ROR1⁺ MHH-NB-11 cells, significantly higher than the rates observed with naxitamab or zilovertamab alone (Figure 4). These results suggest that G/R-001, by simultaneously targeting GD2 and ROR1, may more effectively activate immune cells, thereby enhancing killing capability against tumor cells. This phenomenon is similar to the effects of monoclonal antibodies naxitamab and zilovertamab reported in existing literature (28-30). In contrast, G/R-001 also retained substantial cytotoxicity in single-positive cell lines, inducing 67.58% killing in GD2⁺/ROR⁻ LAN-1 cells and 59.03% in GD2⁻/ROR1⁺ MDA-MB-468 cells under the same conditions. These differences likely dual-positive MHH-NB-11 cells benefit from synergistic bivalent engagement, whereas single-target binding-although sufficient for NK-cell mediated cytotoxicity provides comparatively lower potency. The activity observed in MDA-MB-468 further highlights the antibody’s functional relevance across cancer types and its potential utility in heterogeneous tumors. It is important to note that the present study was limited to concentrations up to 100 nM to remain within clinically relevant exposure ranges, and whether higher doses could achieve near-complete killing across all models remains to be determined. Overall, these results support G/R-001 as a promising bispecific immunotherapy with enhanced activity in dual-positive settings and meaningful efficacy in single-target tumors.

Beyond NB, G/R-001’s activity in the MDA-MB-468 triple-negative breast cancer (TNBC) model highlights its therapeutic potential. TNBC, the most aggressive and treatment-resistant breast cancer subtype, lacks ER/PR/HER2 expression and relies largely on chemotherapy and immune checkpoint inhibitors (31,32). ROR1 has emerged as a promising target in this setting, being expressed in 22.4% of TNBCs and associated with enhanced tumor-initiating capacity, epithelial-mesenchymal transition, and poor prognosis (33-35). In this study, G/R-001 achieved 59.03% cell death in GD2^–/ROR1⁺ MDA-MB-468 cells at 100 nM—an appreciable response given the absence of GD2-mediated immune amplification. The lower efficacy relative to dual-positive MHH-NB-11 cells may result from reduced antibody avidity without GD2 co-engagement, lower ROR1 density or faster internalization in epithelial cancer cells, and the inherently stronger anti-apoptotic and immune-evasive features of TNBC. Moreover, the absence of GD2-mediated complement-dependent cytotoxicity (CDC) further limits cytotoxic potential in this model. Nonetheless, these data provide proof-of-concept that G/R-001 can exert meaningful anti-tumor activity in ROR1-positive breast cancer, supporting the broader applicability of its ROR1-targeted arm.

The anti-tumor mechanism of G/R-001 in NB may be achieved through immune effector activation and direct oncogenic signal blockade. Clinical studies have shown that the efficacy of anti-GD2 antibodies in NB is primarily mediated through antibody-dependent cellular cytotoxicity (ADCC) stimulating NK cell activation, as well as through macrophages and other myeloid cells generated by GM-CSF stimulation (29,36). GD2 is a disialoganglioside highly enriched on the NB cell surface; however, it lacks intrinsic enzymatic activity and does not directly transduce classical intracellular signaling cascades. Rather, its oncogenic relevance lies in promoting cell adhesion, motility, and metastatic dissemination. Consequently, the anti-GD2 arm of G/R-001 is expected to exert therapeutic effects predominantly through FcγRIIIa (CD16a)-mediated NK cell recruitment and CDC, effectively opsonizing GD2-expressing tumor cells for immune destruction. In contrast, ROR1 is a receptor tyrosine kinase (RTK) that functions as a critical oncogenic driver in NB. ROR1 is expressed across all stages of NB. In patients with non-MYC amplified tumors, high ROR1 expression correlates with inferior survival and adverse prognosis. Upon binding to its ligand Wnt5a, ROR1 activates multiple pro-survival signaling cascades, including the PI3K/AKT/mTOR pathway, the MAPK/ERK pathway, and NF-κB-mediated transcriptional programs (37-39). These pathways collectively promote tumor cell proliferation, inhibit apoptosis, and confer chemoresistance. In proof-of-concept experiments, pre-treating NB cell lines with anti-ROR1 antibodies demonstrated additive cytotoxicity with NK92 cells, suggesting that ROR1 blockade not only facilitates immune recognition but may also directly attenuate oncogenic signaling (16). Therefore, the potential mechanism of G/R-001 is through its Fc segment binding to FcγRIII (CD16) on NK cells, enhancing NK cells’ killing capability against tumor cells. Moreover, G/R-001 may also blocking Wnt5a-ROR1 interaction to suppress downstream PI3K/AKT and MAPK/ERK signaling, thereby inhibiting tumor cell proliferation and survival. This dual-targeting mechanism not only improves the specificity of treatment but also reduces the possibility of tumor cell escape. Therefore, future research should focus on how G/R-001 functions in complex tumor microenvironments and how it can be combined with other therapeutic strategies-such as immune checkpoint inhibitors, IL-15 or GM-CSF cytokine support, or chemotherapeutic agents-to maximize both direct signal inhibition and immune-mediated cytotoxicity.

However, this study has several limitations. First, all experiments in the present work were conducted exclusively in cell-based models, which do not fully recapitulate the complexity of the in vivo tumor microenvironment. Second, the current study did not evaluate potential in vivo toxicity or address the heterogeneity of antigen expression in dynamic tumor evolution. These aspects are essential for determining the translational feasibility and safety of G/R-001. Future studies will therefore focus on validating the real therapeutic efficacy of G/R-001 using in situ transplantation models that better resemble clinical tumor progression. A long-term repeated-dose toxicity study will be conducted to evaluate the safety of G/R-001, and pharmacokinetic analyses will be performed to characterize its absorption, distribution, and metabolism in vivo, providing a basis for subsequent dosing-regimen design. In addition, long-term antigen-tracking experiments will be carried out using fluorescent labeling or immunohistochemistry to dynamically monitor the spatiotemporal changes in antigen expression within tumor tissues before and after treatment in animal models, thereby assessing target heterogeneity and its impact on therapeutic response. These investigations will be critical to refining the design of G/R-001 and to facilitating its early translational application as a potential new immunotherapeutic strategy.

Conclusions

In conclusion, G/R-001 as a novel BsAb simultaneously targeting GD2 and ROR1, demonstrated robust and synergistic cytotoxic activity against NB and TNBC cells in vitro. By effectively addressing the challenges posed by the limited efficacy of existing monospecific antibodies, G/R-001 introduces a new therapeutic strategy that may broaden the scope of immunotherapy for high-risk NB and TNBC. These findings not only validate GD2/ROR1 dual-targeting as a promising approach, but also provide a foundation for the future development of next-generation BsAb therapies.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-0723/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-0723/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-0723/prf

Funding: This work was supported by Fujian Medical University Qihang Fund Project (No. 2020QH1029).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-0723/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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