GD2-Specific CAR T Cells Demonstrate Potent and Targeted Anti-Tumor Efficacy Against Melanoma In Vitro and In Vivo

Abstract

Background:

Disialoganglioside (GD2) is a tumor-associated antigen that is highly expressed in various neuroectodermal cancers, including melanoma. While chimeric antigen receptor (CAR) T-cell immunotherapy has demonstrated remarkable success in treating hematologic neoplasms, the identification of suitable targets remains a major obstacle in translating this approach to solid tumors.

Methods:

Peripheral blood T lymphocytes from six healthy donors were used to generate GD2-specific CAR T cells via retroviral transduction. The resulting GD2.CAR T cells were characterized by NanoString transcriptome profiling, flow cytometry with hierarchical stochastic neighbor embedding (HSNE) dimensionality reduction, and in vitro cytotoxicity assays against GD2⁺ and GD2^- melanoma cell lines. In vivo experiments were also performed using GD2⁺ xenograft models and a single intratumoral dose of 8 × 10⁶ GD2.CAR T cells.

Result:

The GD2.CAR T cell population exhibited a predominantly naive phenotype (CD8⁺CD40L⁺CD69^‒CD107a⁺4-1BB⁺FasL⁺) and effective anti-tumor mechanisms involving the granzyme A/B axis, the Fas/FasL axis, and cytokine release. Transcriptome analysis revealed transduction-related effects on proliferation and a shift towards an effector phenotype during early co-culture with tumor cells, accompanied by upregulation of interferon-gamma (IFN-γ) and cytokine signaling genes. GD2.CAR T cells demonstrated robust cytotoxicity against GD2⁺ melanoma cells in vitro, while significant in vivo tumor control was observed in xenograft models.

Conclusion:

GD2.CAR T cells demonstrate potent anti-tumor activity against melanoma in vitro and in vivo, highlighting their therapeutic potential and warranting further clinical investigation.

Graphical abstract

Keywords

CAR T cells / disialoganglioside / xenograft model / cytotoxicity / transcriptome profiling

1. Introduction

Melanoma is a common and aggressive cancer type originating from melanocytes and notorious for its rapid metastasis [1, 2]. Although surgery, chemotherapy, and radiation remain first-line therapies, melanoma often develops resistance, and prognosis worsens at later stages [3]. Immunotherapy is a promising method that employs natural defense mechanisms to treat tumors. Adoptive cell therapy is effective in the treatment of metastatic melanoma [4], but lacks specificity in targeting tumor cells. To address this limitation, chimeric antigen receptor (CAR) T-cell therapy has been developed. Genetically engineered CAR T cells can recognize tumors in a non-major histocompatibility complex (MHC)-restricted manner without co-stimulation [5]. As a result, CAR T cells are an off-the-shelf T cell product capable of targeting specific antigens for tumor elimination. Nonetheless, the application of CAR T-cell therapy has significant challenges in an immunosuppressive tumor microenvironment (TME) characterized by molecular, cellular, and metabolic profiles that could lead to T cell exhaustion and dysfunction [6].

The selection of a tumor-specific surface antigen that is highly expressed on tumor cells but minimally expressed on normal cells is critical for effective CAR T-cell therapy. This is essential for achieving optimal treatment outcomes and minimizing negative side effects. Disialoganglioside (GD2) is overexpressed in melanoma but shows limited expression in normal tissues [7, 8]. Increased GD2 expression on melanoma cells may be associated with metastasis [9] and the malignant phenotype of melanoma [10]. Structurally, GD2 is a glycolipid expressed on the cell surface, making it a suitable target for recognition by antibodies or chimeric antigen receptors. In addition, GD2 has shown potential as a target for the development of CAR T-cell therapies against melanoma [11].

Preclinical studies have shown that GD2.CAR T cells, either alone or in combination with other therapeutic agents, can efficiently eliminate melanoma cells in vitro and suppress tumor growth in vivo [12, 13, 14, 15]. However, a phase I clinical trial of GD2.CAR T cells demonstrated limited expansion and persistence, with T cells exhibiting an exhausted phenotype in metastatic melanoma patients, as evidenced by ex vivo analysis of peripheral blood samples after infusion [16]. This approach requires the development of novel methods to enhance the design of the CAR construct. Moreover, further investigations are required into the impact of the TME on CAR T cells.

In the present study, we have built upon our previous observation of antitumor activity with melanoma-associated antigen family A4 (MAGE-A4)-specific T-cell receptor (TCR)-like CAR T cells and New York esophageal squamous cell carcinoma 1 (NY-ESO-1)-specific TCR-engineered T cells [17, 18] to evaluate the therapeutic potential of GD2-specific CAR T cell therapy. We utilized a previously optimized retroviral construct encoding a GD2-specific single-chain variable fragment (scFv) derived from a modified mAb 220-51, fused to human CD28 and CD3\zeta intracellular signaling domains, and incorporating a sequence encoding the glucocorticoid-induced tumor necrosis factor receptor (TNFR) family-related protein ligand (GITRL) [19]. This construct was designed to enhance antitumor activity, based on accumulating evidence that stimulation of GITR with GITRL increases TCR-induced T cell proliferation and cytokine production, reduces the threshold for CD28 co-stimulation in effector CD8⁺ T cells, and preserves T cells from anti-CD3-induced apoptosis [19, 20]. Accordingly, we generated GD2-specific CAR T cells via retroviral transduction in order to further characterize their phenotype, in vitro function, and in vivo inhibition of tumor growth. The resulting GD2.CAR T cell phenotype was assessed using hierarchical stochastic neighbor embedding (HSNE) analysis. We also assessed the functional properties of these cells, including cytokine production and cytotoxic activity against melanoma cell lines in vitro and in a xenograft model. Furthermore, we investigated the early-stage transcriptomic changes that occur in GD2.CAR T cells upon interaction with tumor cell lines, paying particular attention to alterations in T cell memory differentiation.

2. Material and Methods

2.1 Donors

The study was carried out in accordance with the guidelines of the Declaration of Helsinki and was approved by the local ethics committee of the Research Institute of Fundamental and Clinical Immunology (protocol #139 on May 30, 2022). Healthy blood donors (n = 6 males, age 27.33 \pm 1.63 years [mean \pm standard error of the mean (SEM)]) were recruited between June and December 2022. All donors met standard requirements for blood donation, showed no evidence of acute infection or prior blood transfusion, and provided written informed consent.

2.2 Cell Lines

The human melanoma cell line SK-MEL-37 (RRID: CVCL_3878) and patient-derived cell lines S6 and V9 were provided by Professor H. Shiku (Mie University Graduate School of Medicine, Japan). The local ethics committee of the Research Institute of Fundamental and Clinical Immunology approved the use of patient-derived cell lines S6 and V9 in this study (protocol #139 on May 30, 2022). All cell lines (SK-MEL-37, S6, and V9) tested negative for Mycoplasma (Cat. #MR004, Evrogen, Moscow, Russia), and the SK-MEL-37 cell line was validated by short tandem repeat (STR) profiling. To ensure the identity of the cell lines used, the S6 and V9 patient-derived cell lines were validated using light microscopy to confirm characteristic melanoma morphology (pleomorphism, large nuclei, variable cytoplasm, and the presence/absence of melanin pigment). Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Cat. #RM10832-500ML, HiMedia, Maharashtra, India), 2 mM L-glutamine (Cat. #1383, Biolot, Saint Petersburg, Russia), 5 \times 10^-5 mM \beta-mercaptoethanol (Cat. #M3148, Sigma-Aldrich, Saint-Louis, MO, USA), 25 mM HEPES (Cat. #1261, Biolot, Saint Petersburg, Russia), 80 µg/mL gentamicin (Cat. #A011p, PanEco, Moscow, Russia), and 100 µg/mL ampicillin (Cat. #260317, Sintez, Kurgan, Russia). Cells were seeded into flasks at a concentration of 50–75 \times 10³ cells/mL until they reached the log phase of growth. They were then detached using trypsin-versene solution (Trypsin: Cat. #1225, Biolot, Saint Petersburg, Russia; Versene: Cat. #1232, Biolot, Saint Petersburg, Russia) for experimental use. GD2 expression on SK-MEL-37, S6, and V9 was assessed using flow cytometry with mAb 14.G2a (Cat. #357304, BioLegend, San Diego, CA, USA) according to the manufacturer’s recommendations and analyzed on an Attune NxT flow cytometer (Thermo Fisher Scientific, Waltham, MA, USA) (SK-MEL-37 and S6: GD2-positive; V9: GD2-negative).

2.3 Retroviral Particles

A gamma-retroviral vector encoding a GD2-specific CAR was provided by Professor H. Shiku. The structure of the pGD2 transfer Moloney murine leukemia virus (MMLV) plasmid is shown in Fig. 1.

2.4 Generation of CAR T Cells and Retroviral Transduction

CAR T cells were generated by isolating PBMCs from healthy adult donors using Ficoll-Urografin (Cat. #17-1440-03, PanEco, Moscow, Russia) density gradient centrifugation and washed twice with PBS (Cat. #1247, Biolot, Saint Petersburg, Russia). Proliferation was stimulated by coating 12-well plates (TPP, Trasadingen, Schaffhausen, Switzerland) with 25 µg/mL RetroNectin (Cat. #T100B, Takara Bio, Kyoto, Japan) and 5 µg/mL anti-CD3 antibodies (Cat. #300465, Biolegend, San Diego, CA, USA). PBMCs were then plated at 0.5 \times 10⁶ cells/mL in 2 mL GT-T551 medium (Cat. #WK551s, Takara Bio, Kyoto, Japan) supplemented with 300 U/mL interleukin-2 (IL-2) (Cat. #011022, Roncoleukin, Biotech, Saint Petersburg, Russia) and 0.6% group AB human serum. Half of the medium was replaced with fresh medium on days 2 and 3 supplemented with 300 U/mL IL-2. For retroviral transduction, retrovirus was diluted in PBS with 2% human serum albumin (Cat. #T160516, Microgen, Tomsk, Russia) and 5% glucose-citrate buffer, then added to RetroNectin-coated 24-well plates (25 µg/mL RetroNectin). Plates were centrifuged at 32 °C and 2000 \timesg for 2 h (Jouan MR 23i, Saint-Herblain, Nantes, France). After removing supernatant and washing twice with PBS containing 2% albumin, the activated PBMCs (1.5–2 \times 10⁵ cells/well) were transduced with CAR for 10 min under centrifugation at 1000 \timesg at 32 °C. The next day, these cells were transduced a second time. After 6–8 h, cells were transferred to 6-well plates (TPP, Trasadingen, Schaffhausen, Switzerland) with GT-T551 medium and 300 U/mL IL-2, and cultured at 37 °C and 5% CO₂. Transduced T cells were collected on days 9–10 for analysis (transduction efficiency and phenotyping), and used for in vitro cytotoxicity assays or in vivo experiments on day 11. To validate the generated CAR T cells, the identity of these cells was confirmed by flow cytometry (assessing T lymphocyte markers [CD3, CD4, and CD8] and GD2-specific CAR) and light microscopy (assessing characteristic lymphocyte morphology: small size [7–10 µm in diameter], high nuclear-to-cytoplasmic ratio, round nucleus, scant cytoplasm).

2.5 In Vitro Co-Culture Experiments

Tumor cells (SK-MEL-37, S6, and V9) were seeded at 4–5 \times 10³ cells/well in 96-well plates (TPP, Trasadingen, Schaffhausen, Switzerland) one day before co-culture with transduced or non-transduced T cells (effector to target ratio of 5:1) in fresh RPMI-1640 medium without exogenous cytokines. After 4–6 h of co-culture, T cells were collected for flow cytometry analysis of antigen-dependent activation and cytotoxicity markers. After 48 h co-culture of transduced or non-transduced T cells with GD2-positive SK-MEL-37 tumor cells, supernatants were collected and frozen for subsequent cytokine measurements. Cytotoxicity was assessed by measuring lactate dehydrogenase (LDH) activity in the conditioned medium of transduced T cells and tumor lines SK-MEL-37, S6, and V9 after 6–8 h.

2.6 Flow Cytometry and Transduction Efficiency

Flow cytometric analysis was performed using the following monoclonal antibodies (all from Biolegend, San Diego, CA, USA): CD3 (clone HIT3a), CD4 (clone RPA-T4), CD8 (clone SK1), CD45RA (clone HI100), CD62L (clone DREG-56), CD137 (4-1BB) (clone 4B4-1), CD154 (CD40L) (clone 24-31), CD69 (clone FN50), CD178 (FasL) (clone NOK-1), CD107a (clone H4A3), CD366 (TIM-3) (clone F38-2E2), CD279 (PD-1) (clone EH12.2H7), and GD2 (clone 14G2a). These were conjugated with phycoerythrin (PE), PE-Cy7, peridinin-chlorophyll-protein complex (PerCP)-Cy5.5, allophycocyanin (APC)-Cy7, Alexa Fluor (AF) 647, AF488, AF700, Brilliant Violet (BV) 421, BV570, BV647, and BV711. Antibodies were used according to the manufacturer’s recommendations, and FMO controls established negative gates. Transduction efficiency was assessed using biotin-SP-AffiniPure F(ab’)₂-fragment-specific goat anti-mouse IgG (1:1000, Cat. #115-066-072, Jackson ImmunoResearch, West Grove, PA, USA) for the detection of GD2-CAR. Cells were stained by incubating with antibodies (1 to 1000 dilution) for 20 minutes at room temperature in the dark. After washing, streptavidin-PE (1 to 600 dilution) (1:600, Cat. #405203, Biolegend, San Diego, CA, USA), anti-CD3, and Zombie Aqua Dye (1:100, Cat. #423101, Biolegend, San Diego, CA, USA) were added to determine the population of CAR T cells and their viability. Stained cells were washed in PBS with 0.1% NaN₃ and analyzed on an Attune NxT flow cytometer. Non-transduced cells served as controls.

2.7 HSNE Dimensionality Reduction and Clustering Analysis

Flow cytometry data analysis was performed using a combination of software tools. Cells were gated using Attune NxT software (version 3.2.1, Thermo Fisher Scientific, Waltham, MA, USA) to exclude debris, doublets, and dead cells, and to identify CD3⁺ and transduced cells (Supplementary Fig. 1). The resulting .fcs files were converted to .csv format using custom Python (version 3.13, Wilmington, DE, USA) code via Jupyter Notebooks (version 6.5.4, Austin, TX, USA). Subsequently, the .csv files were arcsinh-transformed and fdaNorm-normalized using an R script [21] and exported as .fcs files. HSNE dimensionality reduction was performed using the Cytosplore app [22] on cytotoxic and activation markers (CD4, CD8, CD40L, CD69, 107a, 4-1BB, and FasL), and T cell memory and exhaustion markers (CD8, CD45RA, CD62L, CD69, PD-1, and TIM-3). Subsequently, GraphPad Prism (version 9.4.1, GraphPad Prism Software, San Diego, CA, USA) was used to export cell frequencies per cluster and visualize them as box plots.

2.8 Cytokine Production Assay

Cytokine concentrations in supernatant were measured using the LEGENDplex™ Human CD8/NK Panel (13-plex) and Filter Plate Kit (Cat. #740267, Biolegend, San Diego, CA, USA) according to the manufacturer’s recommendations. Cytokines were subsequently classified based on their respective concentration levels.

2.9 LDH Cytotoxicity Assay

Cytotoxicity was assessed by measuring LDH release in cell culture supernatants using a CytoTox 96 Non-Radioactive Cytotoxicity Assay (Cat. #G1780, Promega Corporation, Madison, WI, USA) according to the manufacturer’s recommendations. This assay measures LDH activity via a 30-minute enzymatic reaction that converts tetrazolium salt INT into red formazan product. The absorbance of the resulting color was measured using a Varioskan plate reader (Thermo Fisher Scientific, Waltham, MA, USA). Color intensity correlated with the number of lysed cells.

2.10 Magnetic Separation of CAR T Cells

For gene expression analysis, transduced T cells were isolated using a two-step magnetic sorting procedure. First, cells were incubated with F(ab’)₂-fragment-specific goat anti-mouse IgG antibodies for 20 minutes in cold Versene solution containing 0.5% bovine serum albumin, labeled with MojoSort™ Streptavidin Nanobeads (Cat. #480016, Biolegend, San Diego, CA, USA) at a ratio of 10 µL antibodies/beads per 10 \times 10⁶ cells, and then positively selected using a MojoSort™ Magnet (Cat. #480019, Biolegend, San Diego, CA, USA). After resting for 16–17 h in culture medium supplemented with 300 U/mL IL-2, the purified T cells were co-cultured with GD2-positive SK-MEL-37 tumor cells at an effector-to-target ratio of 5:1 for 2 h to activate gene expression of the immune response. Finally, transduced T cells were sorted again by positive selection using MojoSort™ Human CD45 Nanobeads (Cat. #480029, Biolegend, San Diego, CA, USA) to ensure a pure CAR T cell population. Cell viability, assessed by trypan blue staining using a Countess 3 Automated Cell Counter (Thermo Fisher Scientific, Waltham, MA, USA), was consistently >92%.

2.11 RNA Extraction

Total RNA was extracted from 3–6 \times 10⁵ cells using the Total RNA Purification Plus Kit (Cat. #48400, Norgen Biotek, Thorold, ON, Canada) according to the manufacturer’s recommendations. RNA concentration and quality were assessed using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Total RNA was frozen at –80 °C until further analysis.

2.12 NanoString Gene Expression Analysis

Gene expression analysis was performed using the NanoString nCounter SPRINT Profiler system with 100 ng of total RNA per sample (n = 6). The nCounter Human Immunology v2 panel, comprising 579 genes (Cat. #XT-CSO-HIM2-12, NanoString, Seattle, WA, USA), was used to analyze RNA samples on the NanoString Sprint instrument (NanoString, Seattle, WA, USA). Samples were hybridized according to the manufacturer’s recommendations. Normalization and quality control were performed using nSolver 4, incorporating synthetic positive controls and the 15 housekeeping genes included in the panel. To remove non-expressing genes, a background threshold was applied to the normalized data, defined as: (mean of all NEG controls) + 2 \times (SD of all NEG controls) + (mean of the POS_E controls). Gene Set Enrichment Analysis was performed using GSEApy [23].

2.13 In Vivo Experiment

To determine the anti-tumor efficacy of transduced T cells in vivo, 8-week-old male or female non-obese diabetic (NOD)/Rag1-null/Il2r\gamma-null (NRG) mice (NOD.Cg-Rag^1tm1Mom Il2rg^tm1Wjl/SzIcgn) (Center for Collective Usage SPF-vivarium, Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia) were injected subcutaneously in the right scapula region with 5 \times 10⁶ SK-MEL-37 cells in 100 µL culture medium. When tumors reached approximately 80–100 mm³, mice were divided into a control group (intratumoral injection of 8 \times 10⁶ activated non-transduced T cells in 100 µL RPMI-1640 medium), an experimental group (intratumoral injection of 8 \times 10⁶ GD2.CAR T cells in 100 µL RPMI-1640 medium), and an untreated group (n = 8 per group). Tumor growth was monitored every 2–3 days using calipers, and tumor volume was calculated as: volume = 0.52 \times width² \times length. Mice were maintained under specific-pathogen-free (SPF) conditions with a 12-h light-dark cycle, and their health monitored regularly. Euthanasia was performed using CO₂ overdose (3 L/min fill rate, resulting in approximately 30% CO₂ concentration) followed by cervical dislocation, as indicated by clinical signs of disease (e.g., not eating, lack of activity, abnormal grooming behavior, hunched posture), excessive weight loss (>15% over one week), or tumor size reaching approximately 2000 mm³. The research was conducted at the Center for Genetic Resources of Laboratory Animals, Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, and approved by the local ethics committee of the Research Institute of Fundamental and Clinical Immunology (protocol #139 on May 30, 2022) as per the European Community Directive (86/609/EEC) and humane treatment standards.

2.14 Statistical Analysis

Data are presented as frequencies, percentages, or mean \pm SEM. The Shapiro–Wilk test was used to assess normality. Statistical significance between transduced and non-transduced cells was determined using GraphPad Prism 9.4.1, with one-way or two-way ANOVA followed by t-test, Dunnett’s test, or Tukey’s test. LDH cytotoxicity was analyzed using one-way ANOVA with Dunnett’s test for multiple comparisons. Differential gene expression and cytokine production were analyzed using multiple t-tests with FDR-adjusted p-values 0.05. Animal experiments were analyzed using two-way ANOVA with Tukey’s test for multiple comparisons. All p-values and n numbers are reported in the figure legends. A p-value 0.05 was considered statistically significant.

3. Results

3.1 Detection of GD2 Expression in Melanoma, and GD2.CAR T Cell Transduction Efficiency

Three human melanoma cell lines (SK-MEL-37, S6, and V9) were analyzed for surface expression of GD2 by flow cytometry using mAb 14.G2a. The SK-MEL-37 and S6 cell lines showed high GD2 expression (89.4% and 85.1%, respectively). The V9 cell line was used as a negative control due to its low GD2 expression (2.3%) and antigen density on the cell surface.

The second-generation CAR construct containing GD2-specific scFv from a modified mAb 220-51 [19] and human signaling endodomains CD28 and CD3\zeta, was transduced into activated T lymphocytes using retroviral vectors (Fig. 1). Seven days after transduction, we assessed GD2-specific CAR expression by flow cytometry and observed 68.2 \pm 3.5% (n = 6, mean \pm SEM) GD2.CAR⁺ T cells. In order to provide strong co-stimulation and a potential synergistic effect of CD28 signaling in T cell activation [20], T lymphocytes were also genetically engineered to co-express the GITRL with CAR, as demonstrated in a recent study [19].

3.2 Subsets of GD2.CAR T Cells and Markers of Cell Exhaustion After Transduction

To further evaluate the signature of GD2.CAR T cell memory subpopulation changes, flow cytometry and HSNE analysis were used to assess marker expression according to T cell memory subsets immediately after transduction and before co-culture with the tumor cells. CAR T cells were classified into four differentiation subsets based on CD45RA and CD62L expression: naive/stem memory T cells (Tn/scm, CD62L⁺CD45RA⁺), central memory T cells (Tcm, CD62L⁺CD45RA^‒), effector memory T cells (Tem, CD62L^‒CD45RA^‒), and late effector memory T cells (Temra, CD62L^‒CD45RA⁺) [24]. HSNE analysis (Fig. 2) revealed the CD8⁺ GD2.CAR T cell population was mainly composed of Tn/scm cells, with only small percentages of Tcm and Temra cells observed (Supplementary Fig. 2).

Expression of the cell exhaustion markers programmed cell death protein 1 (PD-1) and T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) was also evaluated. In general, the GD2.CAR T cell population did not show signs of exhaustion. However, a correlation was observed between the expression of CD45RA and CD62L markers and the expression of TIM-3 and PD-1 markers. The expression of cell exhaustion markers gradually increased as the cells differentiate into Tcm cells, and decreased in Temra cells (Fig. 2). It was previously reported that the increase in CD69 level is likely a manifestation of the basal level of activation by CAR-mediated tonic signaling [25]. Our transduction protocol resulted in a population of CD69^‒ CAR T cells, indicating the potential absence of tonic signaling. Meanwhile, only a low proportion of CD69⁺ T cells was observed (Fig. 2). Furthermore, the CD69 marker is expressed only when an effector cell recognizes an antigen [26].

3.3 Transcriptome Profile of GD2.CAR T Cells Associated With the Tn/scm Phenotype

To investigate how retroviral transduction affects the expression of genes related to phenotypic signatures (Fig. 3), we used NanoString technology to compare the transcriptome profiles of transduced and non-transduced cells. After transduction, GD2.CAR T cells showed upregulation of genes associated with the CD8 Tn cell differentiation signature, including CD8A, CD8B, SELL, IL7R, CD45RA [27], and IL2RG [28], as well as genes associated with the CD8 Tscm/cm cell differentiation signature, such as FAS and CD45RO [27]. Upregulation of the CD40LG gene, which is canonically expressed on CD4⁺ T cells, was also observed in CAR⁺ cells [29]. In contrast, transduced cells showed downregulation of CD4 Treg-associated genes, including CD4, FOXP3, IL2RA, and CTLA4-TM [27]. It is worth noting that retroviral transduction had an impact on T cell proliferation, whereas anti-CD3-primed non-transduced cells showed upregulation of genes related to T cell activation, such as TNFRSF4 and TNFRSF9 [30]. Furthermore, GD2.CAR T cells showed increased expression of the HAVCR2 gene within the T cell exhaustion signature, whereas non-transduced cells showed increased expression of the LAG3 gene.

3.4 Immune Characteristics of GD2.CAR T Cell Transcriptome Profile in the Early Stages of Interaction With GD2-Positive Melanoma Cells

Using NanoString technology, we performed differential gene analysis of GD2.CAR T cells before and after co-culture with SK-MEL-37 cells. Because our aim was to examine changes in gene expression linked to the immune response before and after co-culture with tumor cells, we selected the Human Immunology v2 panel for this study. CAR⁺ cells were first magnetically sorted from the general cell population and then stimulated with IL-2 for 16–17 h. GD2.CAR T cells were then cultured with tumor cells for 2 h to activate key genes that regulate the immune response. These cells were then separated from GD2-positive SK-MEL-37 cells using CD45-positive magnetic sorting to remove any contamination with tumor cells.

Although the interaction between GD2.CAR T cells and tumor cells was short, alterations were detected in several genes (Supplementary Table 1) encoding co-signaling molecules, cytokines, and chemokines that affect the immune response (Fig. 4A).

Signatures for chemokines (CCL2, CCL20, CCL3, CCL4, CXCL1, CXCL10, CXCL11, and CXCL2), cytokines and their receptors (CSF1, CSF2, IL13, IL13RA1, IL1A, IL1B, IL1RAP, IL6, IL8, and IFNG) were found after co-culture with the tumor cell line. In contrast, a decrease in the expression of chemokine receptor genes (CCR1, CCR2, CCR5, CCR7, CCRL2, CX3CR1, CXCR3, CXCR4, and CXCR6) was observed, as well as that of cytokine receptors and their component genes (IL10RA, IL12RB1, IL21R, IL2RG, IL4R, and IL7R). We also observed a decrease in the expression of genes associated with the Tem cell differentiation signature, including CCR7, CD27, CD28, CD45RA, CD45RB, CX3CR1, IL7R, LEF1, and TCF7 [27]. Furthermore, increased expression of the CTNNB1 [31, 32] and BCL6 [33] genes and decreased expression of the LAIR1 [34] and SELPLG [35] genes are also signatures of Tem cell differentiation. Regarding the effector function of T cells, increased expression of genes associated with activation was observed, including TNFRSF9 [30] and MHC class II genes (HLA-DMA, HLA-DMB, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DRA, HLA-DRB1, and HLA-DRB3), as well as genes associated with migration (IL6, IL6ST, SELE, and ICAM1) [36]. We also observed upregulation of TRAF1 gene expression, which is involved in downstream signaling of 4-1BB [37] or GITR [38]. Upregulation of EGR2 gene expression was also observed, which promotes differentiation toward Tem cells and regulation of the exhausted transcriptional state [39], as well as improved survival and proliferation [40]. However, the expression of genes encoding effector molecules (GZMA, GZMK, and pRF1) and signaling molecules (LCK and ZAP70) essential for the initiation of TCR signaling were downregulated [41]. Notably, after co-culture of GD2.CAR T cells with tumor cells, decreases were observed in signatures associated with T cell dysfunction, including expression of the HAVCR2, CISH [42, 43], CD244 and SLAMF7 [44] genes, as well as T cell death genes (BAX, BCL2, CASP1, and CASP8) [45, 46].

The up-regulated genes (Supplementary Table 2) were enriched by gene set enrichment analysis (GSEA) [23] using ontology biological process terms. GSEA analysis revealed that the top three pathways were associated with the cytokine-mediated signaling pathway, cellular response to interferon-gamma, and interferon-gamma-mediated signaling pathways (Fig. 4B). In contrast, genes associated with T cell proliferation and cytokine production were not significantly expressed during early stages of the CAR T cell-tumor interaction.

3.5 GD2.CAR T Cells are Predominantly CD8⁺ T Cells With a Cytotoxic Phenotype

To investigate the potential application of CAR T cells as effector cells against melanoma, we evaluated the expression of surface activation and cytotoxicity markers (CD69, 4-1BB or CD137, CD107a, FasL, and CD40L) following 4–6 h of co-culture with GD2-positive tumor cell lines. This duration is sufficient to induce expression of the markers. Using a panel of antibodies, we analyzed the flow cytometry data from the GD2.CAR T cells using HSNE dimensionality reduction after arcsinh transformation with automated cofactors. Data were normalized using fdaNorm to correct for batch effects and ensure data integrity. HSNE analysis identified four GD2.CAR T cell subpopulations (Fig. 4C). The predominant population was CD8⁺CD40L⁺CD69^‒CD107a⁺4-1BB⁺FasL⁺ T cells, suggesting cytotoxic function (Supplementary Fig. 3). Smaller populations of CD8⁺CD40L⁺CD69^‒CD107a⁺4-1BB⁺FasL^‒ and CD4⁺CD40L⁺CD69^‒CD107a^‒4-1BB⁺FasL⁺ T cells with similar proportions were also observed, as well as a minor population of CD8⁺CD40L⁺CD69^‒CD107a⁺4-1BB^‒FasL⁺ T cells (Supplementary Fig. 3).

3.6 GD2.CAR T Cells Show Specific and Efficient Cytotoxicity Against GD2-Positive Melanoma Cells In Vitro

The LDH release assay was employed to assess the cytotoxic activity of GD2.CAR T cells against melanoma tumor cell lines expressing the GD2 antigen. CAR T cells were co-cultured at an effector-to-target ratio of 5:1 for 6–8 h with tumor cells that expressed GD2 (SK-MEL-37 and S6), or did not express GD2 (V9) (Fig. 5). This ratio and co-culture duration were based on the manufacturer’s protocol and optimized for reliable results in the LDH release assay. Significant differences in GD2-specific anti-tumor cytotoxicity, as measured by LDH release, were observed after co-culture with the GD2-expressing SK-MEL-37 and S6 tumor cell lines. Importantly, no significant differences in cytotoxicity were observed between the SK-MEL-37 and S6 cell lines. These results demonstrate the efficacy and specificity of GD2.CAR-T cells in targeting GD2-expressing melanoma cells.

3.7 GD2.CAR T Cells Secrete Cytokines in Response to Co-Culture With GD2-Positive Melanoma Cells

Cytokine release from transduced and non-transduced T cells was assessed 48 h after contact with a GD2⁺ target at an effector-to-target ratio of 5:1. The LEGENDplex™ Human CD8/NK panel was used to simultaneously analyze cytokines, including IL-2, IL-6, and IL-17, and effector molecules, including granzyme A, granzyme B, granulysin, perforin, and sFasL, in the cell co-culture supernatants. Compared to non-transduced T cells, GD2.CAR T cells tended to secrete more pro-inflammatory cytokines and effector molecules (Fig. 6). No statistically significant differences between non-transduced cells and GD2.CAR T cells were observed for the levels of perforin, granulysin, IL-2, and IL-6 in the culture medium. However, significant increases were observed in the levels of granzymes A/B, IFN-\gamma, and sFasL secreted by GD2.CAR T cells. IL-4, IL-10, and sFas were not detected in the supernatants of GD2.CAR and non-transduced T cells.

3.8 GD2.CAR T Cells Inhibit the Growth of GD2-Positive Melanoma in a Xenograft Model

We next evaluated the antitumor efficacy of generated GD2.CAR T cells against GD2-positive tumors in a xenograft model by inoculating SK-MEL-37 cells (5 \times 10⁶ cells per mouse) into NRG mice via subcutaneous injection. When the tumor volume reached 80–100 mm³, tumor-bearing mice were randomized to receive a single intratumoral injection of either 8 \times 10⁶ GD2.CAR T cells or 8 \times 10⁶ anti-CD3-primed non-transduced T cells. Tumor growth was monitored by caliper measurements. Local intratumoral injection was chosen as the route for T cell administration based on the efficacy demonstrated in a preclinical study with locally injected CAR T cells [13]. Three weeks into the experiment, the tumor volume increased rapidly in untreated mice and in mice that received non-transduced T cells (Fig. 7). At 35 days after intratumoral administration of GD2.CAR T cells, a significant reduction in tumor size was observed compared to both the control group and the intact group.

4. Discussion

In this study, we comprehensively analyzed the phenotypic and functional characteristics of GD2.CAR T cells containing CD28 and CD3\zeta endodomains, alongside GITRL secretion. The differentiation state of CAR T cells is critical in determining the efficacy of tumor therapy [47]. In particular, the immunophenotypes of Tn, Tcm and Tem cells are preferred for adaptive therapy because of their antitumor efficacy, survival and proliferation [48]. Our optimized protocol resulted in GD2.CAR T cells comprised mainly of naive CD62L⁺CD45RA⁺ T cells. Transcriptional analysis revealed that retroviral transduction affects the proliferative potential of CAR⁺ cells, as confirmed by the frequency of the Tn cell population. The majority of CAR⁺ cells belonged to the subpopulation of CD8⁺ T cells expressing CD107a, FasL, 4-1BB, and CD40L markers, indicating activation and cytotoxicity. In addition, the GD2.CAR T cell population contains a small percentage of CD4⁺ T cells that express effector molecules, such as CD40L, 4-1BB, and FasL. Recent research suggests that CD4⁺ CAR T cells can enhance antitumor activity and maintain the immune response against tumors [49, 50]. After transduction, the expression of genes associated with the regulation of Treg cells is downregulated. Therefore, the generated population of CD4⁺ CAR T cells may contribute to antitumor immunity. The FasL marker showed higher expression compared to the CD107a marker, while the PRF1 gene was downregulated. No statistically significant differences in perforin secretion were found between transduced and non-transduced T cells. Nevertheless, due to synergistic or additive effects between degranulation and ligand-based lytic pathways, FasL has been shown to promote lytic activity even when degranulation is poor or difficult in CAR T cells [51]. Furthermore, we did not observe expression of the early activation marker CD69 on GD2.CAR T cells. CD69 protein expression can be detected 2–3 h after activation, whereas transcriptional expression of the CD69 gene decreases after 4–6 h [26]. We propose that an off-the-shelf, activated cell product of CAR T cells would have either a low level of CD69 expression or none at all. In addition, the generated GD2.CAR T cells did not express the CD69 marker after transduction, suggesting a lack of basal activation by CAR-mediated tonic signaling [25]. Moreover, a recent study has shown that CD69^‒ CAR T cells have increased proliferation and downregulated expression of surface markers associated with dysfunction [52].

The CD137 receptor serves as a T cell activation marker and is expressed on the cell surface after CD3 or CD28 stimulation [53]. This results in the activation of intracellular signaling through TRAF-mediated activation of NF-kB and MAPK, leading to increased cell survival and enhanced effector functions. We found that early co-culture of GD2.CAR T cells with tumor cells results in upregulation of TRAF1 and TNFRSF9 gene expression, suggesting that antigenic stimulation of CARs or the GITR-GITRL interaction [38, 54] may enhance signaling. The GITR-GITRL interaction has a dual effect on the CD3⁺ T cell population, as it can promote the generation of both Treg cells and cytotoxic CD8⁺ T cells [55]. Nevertheless, we observed downregulation of TGFB1 and TGFBR1 gene expression, which control development of the Treg cell population [56, 57]. Therefore, we suggest that GITRL promotes the development of effector CD8⁺ T cells, as evidenced by the increased expression and secretion of inflammatory cytokines, alongside the absence of detectable IL-10. Furthermore, previous research has shown that forced expression of GITRL in CAR T cells resulted in increased IFN-\gamma production [19]. We also observed upregulation of IFNG gene expression and activation of IFN-\gamma-mediated signaling pathways, which may indicate a GITR-GITRL interaction within the CAR T cell population. ZAP70 and LCK genes, which encode TCR signaling proteins [58], were also found to be downregulated. Since CAR T cells have a modified TCR, the expression of these genes may differ from the canonical TCR pathway compared to non-transduced T cells [59].

It is well known that cytotoxic CD8⁺ CAR T cells exert antitumor effects through several underlying mechanisms, including the perforin-granzyme axis, the Fas-FasL axis, and cytokine secretion [51]. The GD2.CAR T cells we generated have significant cytotoxic efficacy and the ability to express FasL and the degranulation marker CD107a on their cell surface, as well as the secretion of effector molecules such as granzyme A/B, the pro-inflammatory cytokine IFN-\gamma, and the soluble form of FasL. The production of IL-6 and IFN-\gamma by CAR T cells is a double-edged sword, as these cytokines contribute significantly to the development of cytokine release syndrome [60]. Compared to previous studies with 4-1BB-CD3\zeta [61], CD28-OX40-CD3\zeta [62], and TRUCK CD28-4-1BB-CD3\zeta [63] GD2.CAR T cells, our CD28-CD3\zeta-GITRL GD2.CAR T cells demonstrate a safer and more moderate cytokine profile. In addition, our CAR T cells can secrete a greater amount of granzymes relative to other effector molecules and cytokines. Moreover, our in vivo experiments revealed a safety profile with no observed toxicity.

A pattern of granzyme A, granzyme B, and perforin secretion was observed, consistent with the functional differentiation of memory T cells. Effector molecules secreted by the GD2.CAR T cells were in the order of GrmA GrmB Per, indicating cell differentiation towards effector or effector memory [64]. In addition, transcriptional analysis revealed a signature of memory phenotype change. We also observed downregulation of GZMA, GZMK, and PRF1 gene expression without a signature of cell exhaustion. Furthermore, cytokine analysis demonstrated the ability of GD2.CAR T cells to efficiently produce proteins. Thus, the results indicate an early phenotypic shift of GD2.CAR T cells towards Tem cells during their initial interaction with tumors, with no evidence of functional aberration. On the other hand, stimulation of GD2.CAR T cells with anti-CD3/CD28 and IL-2 could promote the accumulation of effector molecules in granules [65], which may explain the observed upregulation of effector molecules before interaction with tumors. Expression of the IFNG gene was also upregulated. Memory effector T cells exhibit a greater capacity for IFN-\gamma release compared to central memory T cells [66], suggesting the possible acquisition of a cytotoxic effector phenotype by CAR T cells after co-culture with tumors. Additionally, prior research has demonstrated that induction of GITR ligand expression on CAR T cells results in enhanced production of IFN-\gamma.

Several features of the transcriptome of GD2.CAR T cells were identified following their initial interaction with GD2-positive tumor cell lines. Specifically, chemokine signatures were detected in CAR T cells, suggesting a T cell chemokine response to an antigen [67]. We propose that one of the key steps in early CAR T cell-tumor interaction is the recruitment of other effector cells to the site of the lesion. A recent study showed that expression of chemokine and cytokine receptor genes is upregulated following anti-CD3/CD28 and IL-2 stimulation [68]. However, we found these genes were downregulated in CAR T cells after co-culture with tumor. Therefore, the incorporation of chemokine receptors into CAR T cell constructs may be a promising approach for further research [69]. GSEA analysis revealed the involvement of cytokine-mediated signaling pathways, cellular response to IFN-\gamma, and IFN-\gamma-mediated signaling pathways during early stages of the GD2.CAR T cell interaction with melanoma, possibly contributing to antitumor immunity. We also observed that GD2.CAR T cells could efficiently kill tumor cells in vitro and effectively control tumor growth in vivo, thus confirming the cytotoxic potential of transduced cells.

In summary, our findings provide robust preclinical evidence for the potential of GD2-specific CAR T cells as a therapeutic strategy for melanoma. Although it has provided valuable insights, our in vivo study was limited to a xenograft model utilizing a single tumor cell line (SK-MEL-37). Therefore, it is important to acknowledge this model cannot fully replicate the complexity of the human TME, nor the heterogeneity of human melanoma. Successful clinical translation will require further investigations to overcome these limitations. Future research should prioritize strategies for increasing the migration of CAR T cells to the tumor site, improving their resistance to inhibitory signals within the TME, and promoting the development of a long-lived memory phenotype. Additional studies employing a broader range of melanoma models, including patient-derived xenografts and those evaluating systemic delivery, will be crucial for assessing long-term efficacy and safety, thus paving the way for clinical trials.

5. Conclusion

GD2-specific CAR T cells generated and characterized using advanced techniques showed potent in vitro and in vivo antitumor activity against melanoma. The phenotypic and functional characteristics of these cells, including a naive-to-effector transition and strong cytotoxic potential, highlight their potential as an effective therapeutic strategy warranting further clinical development.

Availability of Data and Materials

The datasets generated and analyzed during the current study are accessible from the corresponding author upon email request.

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Funding

Research Work “The investigation of the role of molecular-cellular interactions in immunoregulation and the substantiation of new immunotherapy technologies”(124112200103-3)