Background
Chimeric antigen receptor (CAR)-engineered T cells have demonstrated significant promising clinical efficacy in patients with hematologic malignancies [
1,
2]. The complete response (CR) rate of CD19-specific CAR-T clinical trials ranges from 50 to 90% [
2,
3]. The success of this therapy relies on the genetic addition of synthetic CARs to T cells, which enable them to target tumor cells in a major histocompatibility complex (MHC)-unrestricted manner. Despite recent successes in this field, the application of CAR-T cell therapy for the treatment of other solid tumors has remained challenging, largely due to the lack of appropriate tumor-specific antigens [
4,
5] and insufficient localization and persistence of CAR-T cells [
6,
7]. Thus, the identification of precise tumor-specific antigens and the appropriate construction and design of CARs are vital for this immunotherapy.
GD2 gangliosides are sialic acid-containing glycosphingolipids that play a role in signal transduction, cell-cell recognition, cell proliferation, cell migration, and tumor cell metastasis [
8,
9]. Ganglioside GD2 is overexpressed on several solid tumors, including melanoma, neuroblastoma, Ewing’s sarcoma, and even some mesenchymal stem cells [
10,
11]. Due to its high level of expression in tumors and restricted expression in normal tissues, GD2 is a good target for cancer therapy [
12,
13]. Based on its immunogenicity, therapeutic functions, oncogenicity, and other factors, GD2 is a very attractive target and was ranked 12th among the most important cancer antigens by the National Cancer Institute pilot program [
14]. Anti-GD2 monoclonal antibodies were approved by the Food Drug and Administration (FDA) for the treatment of neuroblastoma in March 2017 [
15]. In a phase I study, the murine IgG3 monoclonal antibody (MoAb) 3F8, which specifically targets ganglioside GD2, was intravenously administered to patients with neuroblastoma or melanoma. Antitumor responses occurred in 7 out of 17 patients, which ranged from complete clinical remissions to mixed responses, and the response rate of the melanoma was 4/9 [
16]. Moreover, ganglioside GD2 chimeric antigen receptor T cells have already been studied in patients with neuroblastoma in a phase I clinical trial, which indicated that anti-GD2 CAR-T cells are safe and mediate modest antitumor activity [
17].
Previous studies have shown that anti-GD2 CAR-T cells in which the CD28 and OX40 endodomains have been incorporated exhibit antitumor activity on melanoma in vitro and in vivo [
18]. However, research has demonstrated that CD28 co-stimulation could augment T cell exhaustion, which is a major factor limiting antitumor responses in the stimulation of chronic antigens [
19‐
21], whereas 4-1BB co-stimulation could ameliorate this situation [
22]. The secretion of cytokines and the proliferative ability of exhausted T cells are decreased as apoptosis, and immune-related inhibitory receptors are increased [
23]. Moreover, it has been confirmed that early T cell exhaustion is the primary factor limiting the cytotoxic activity of CAR-T cells [
22].
Hence, we generated GD2 CAR-T cells incorporated with 4-1BB to test their cytotoxic activity in melanoma in vitro and in vivo. Moreover, we reported the expression levels of ganglioside GD2 in non-Caucasian melanoma populations for the first time, which provided a basis for future clinical research.
Methods
Patients and tissue samples
This study included lesion samples from 288 melanoma patients who visited the Peking University Cancer Hospital between November 2009 and November 2016. Written informed consent was obtained from all patients. All diagnoses of melanoma were confirmed histopathologically. Clinical data, including age, sex, American joint committee on cancer (AJCC) M stage, thickness, ulceration, metastasis, and overall survival (OS, follow-up persisted until March 2017, lost to follow-up or death), were collected. This study was approved by the Medical Ethics Committee of the Beijing Cancer Hospital & Institute and was conducted in accordance with the Declaration of Helsinki. The datasets used and/or analyzed during the current study are available from the corresponding author upon request.
Immunohistochemistry
Formalin-fixed, paraffin-embedded (FFPE) tissue sections were examined by immunohistochemistry (IHC) using the monoclonal mouse anti-human ganglioside GD2 antibody 14.G2a (Santa Cruz, sc-53831). A standard Strept-avidin horseradish immunoperoxidase method was used for human Ganglioside GD2 staining. Primary antibodies were diluted in buffer containing 10% normal goat serum. The tissue sections were deparaffinized with Xylene for 30 min and rehydrated in decreasing concentrations of ethanol. Endogenous peroxidases were blocked with 30% H2O2 diluted in phosphate-buffered saline (PBS) for 15 min. For antigen retrieval, slides were heated in a pressure cooker in EDTA (pH 8.5) for 2 min and 30 s, followed by cooling to room temperature (RT) in the same buffer. For antigen blocking, the slides were blocked with normal goat serum with 1 h. After washing, slides were incubated with the primary antibody overnight at 4 °C (dilution 1:25). Three 5-min washes in buffer were conducted after each incubation. The slides were then incubated with the secondary antibody, anti-rabbit/mouse antibody (DAKO) (30 min at RT), followed by staining with AEC for 5-30 min at RT until coloration was achieved, counterstaining with hematoxylin followed by staining, and sealing with water-soluble encapsulating agent. Staining intensity and percentage were independently scored by three pathologists as 0, 1, or 2 (“0” as negative, and “1” and “2” as positive).
Cell lines and primary cell culture
The 293T, SK-MEL-5 (catalog no. HTB-70), and WM-266-4 (catalog no. CRL-1676) cell lines were obtained from the American Type Culture Collection (ATCC). A875 (catalog no. ZY-H405) melanoma cell lines were purchased from Zeye Biotechnology Company (Shanghai, China). HMV-II melanoma cell lines were a gift from Dr. Xu (Abramson Cancer Center of the University of Pennsylvania, Philadelphia, PA). The mucosal melanoma GAK cell lines (catalog no. JCRB0180) was purchased from the Japanese Collection of Research Bioresources Cell Bank (JCRB). The 293T, A875, SK-MEL-5, and WM-266-4 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen) supplemented with 10% FBS, 100 UI/ml penicillin, and 100 μg/ml streptomycin. HMV-II cells were maintained in F-10 (Invitrogen), and GAK cells were maintained in F-12 (Invitrogen) supplemented with 10% FBS, 100 UI/ml penicillin, and 100 μg/ml streptomycin.
The MMYC-3 (mucosal primary melanoma cell), MMYC-7 (mucosal primary melanoma cell), and AMYC-5 (acral primary melanoma cell) cell lines were derived from a patient-derived xenograft (PDX) model. The tumor tissue was minced into 1-mm3 fragments and resuspended in 30 ml of DMEM containing 50× collagenase IV (Invitrogen) and 1× DNase (Takara, Kusatsu, Japan). After a 2-h incubation at 37 °C, the suspensions were collected and slowly transferred onto a 15-ml Histopaque (Sigma, St. Louis, MO), and then, the interface cell fraction was collected after centrifugation. The cells were then maintained in serum-free stem cell medium supplemented with growth factors at 37 °C in 5% CO2.
Construction of the anti-GD2 CAR
The chimeric GD2/CAR is composed of GD2 scFv and a 4-1BB-CD3ζ expression cassette that was designed and synthesized by the GeneChem Biotechnology Company (Shanghai, China), as shown in Fig.
3a. The GD2 scFv was derived from a high-affinity 14.G2a monoclonal antibody. The 4-1BB-CD3ζ expression cassette contains the hinge and transmembrane (TM) region of CD8α. GD2 scFv and 4-1BB-CD3ζ were connected in-frame by overlap PCR. The generated GD2/CAR was verified by DNA sequencing and cloned into the BamHI sites of a lentiviral vector (Genechem Biotechnology, China); the resultant product was named GD2.BBζ CAR. The specific structure of the viral vector is shown in Additional file
1: Figure S1. The intracellular domain of the CARs has the self-cleaving 2A peptide connected to an EGFP green fluorescent label. The sequences of all PCR primers are available upon request.
Transduction of lentiviral GD2/CAR
After informed consent was obtained from normal volunteers, peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque PLUS. T cells were transfected with an Easy-T kit from GeneChem. Briefly, isolated T cells/PBMCs were activated on a plate precoated with S buffer (EASY-T cell infection activation kit, catalog no. LCR6018, GeneChem) at a concentration of 0.5 × 106 cells/ml in complete TexMACS media (Miltenyi) supplemented with 5% human serum and 300 IU IL-2 (Mitenyi). Two days later, the stimulated T cells were washed and resuspended at 0.5 × 106 cells/mL with Trans B buffer (EASY-T cell infection activation kit, catalog no. LCR6018, GeneChem). CAR-encoding lentivirus (GD2.BBζ CAR) was thawed and added into the cells (virus titer: 2 × 108TU/ml, MOI = 3). The cells were seeded onto plates that had been coated for 2 h with Trans A buffer (EASY-T cell infection activation kit, catalog no. LCR6018, GeneChem). Then, the transduced T cells were cultured at 37 °C and 5% CO2 and expanded to maintain a cell concentration of 0.5–1 × 106 cells/ml.
Flow cytometry
FITC-, PE-, or perCP-conjugated anti-CD3, CD4, CD8, CD25, PD-1, TIM-3, LAG-3 monoclonal antibodies, and PE Annexin V apoptosis detection kit were used to stain lymphocytes (all from BD Bioscience), whereas the anti-GD2 mAb (Santa Cruz) was used to label melanoma cells. A GD2 isotype antibody (Santa Cruz) was used as a negative control for the detection of GD2 expression. The proliferation of GD2.BBζ CAR and non-transduced T cells in the presence of tumor cells was evaluated by fluorescence-activated cell sorting (FACS) analysis after labeling the T cells with using the CellTrace™ Far Red Cell Proliferation Kit (Invitrogen).
Cytotoxicity assays
The cytotoxic activity of the GD2.BBζ CAR and non-transduced CAR-transduced T cells was evaluated using the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega). We evaluated lactate dehydrogenase (LDH) release at 4 and 24 h in culture with effector-to-target (E:T) ratios of 40:1, 20:1, 10:1, and 5:1.
Co-culture experiments
GD2.BBζ CAR and non-transduced T cells were plated at 1 × 106 cells per well on a 96-well plate at a 20:1 ratio with 293T, SK-MEL-5, HMV-II, and GAK cells. Interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-10 (IL-10), tumor necrosis factor (TNF-α), and interferon-γ (IFN-γ) cytokine release after 24 h of culture was measured using the cytometric bead array (CBA) human Th1/Th2 cytokine kit (BD Bioscience).
GD2.BBζ CAR and non-transduced T cells labeled by CellTrace™ Far Red were plated at 5 × 105 cells per well on a 12-well plate at a 20:1 ratio with 293 T, WM-266-4, HMV-II, and GAK cells, and the percent of GD2.BBζ CAR and non-transduced T cells was evaluated by FACS analysis after 72 h of co-culture.
The in vivo antitumor activity of GD2/CAR-T cells in a PDX model
The PDX model was established by subcutaneously inoculating the fragments of patient-derived melanoma tissues (MMYC-3 and AMYC-5) into 6-week-old NOD/SCID (non-obese diabetic and severe combined immunodeficiency) female mice (4–6 weeks old; 18–22 g). The specific information on the construction of PDX mouse model as previously described [
24].
When the tumor volume reached approximately 250 mm3 in approximately 30–35 days after tumor fragment inoculation, the mice were divided into five groups (4 in each group), injected with 1 × 107 T cells/100 μl (GD2.BBζ CAR-T cells, non-CAR-T cells or control phosphate-buffered saline (PBS)) either systemically via the tail vein (i.v.) or locally to the tumor mass (i.t.) on days 0, 7, 14, and 21. Tumor growth was subsequently measured with calipers, and the tumor volume was calculated using the following formula: volume = length × width 2/2. When the tumor size reached approximately 2000 mm3, the mice were sacrificed. All animal care and experimental procedures were carried out in accordance with Animal Care Ethics and were approved by the Medical Ethics Committee of the Beijing Cancer Hospital & Institute.
Statistical analysis
Statistical evaluation was conducted with IBM SPSS statistical software (version 20.0). The t test was used to analyze mean values for normally distributed continuous variables, whereas the Mann-Whitney U test was used to compare mean values for abnormally distributed continuous variables. The correlations between the GD2 expression status and clinical parameters were evaluated by the chi-square test or Fisher’s exact test. OS curves were estimated using the Kaplan-Meier method. Log-rank tests were used to estimate the statistical significance between the time-dependent outcomes of OS. For all statistical tests, P < 0.05 (two-tailed test) was considered statistically significant.
Discussion
Melanoma is a highly aggressive skin cancer, and several immune-related therapies have been approved by the US FDA for its treatment, including interleukin 2 (IL-2), interferon-α (IFN-α), cytotoxic T cell-stimulating cytokine (CTLA-4) and programmed cell death protein 1 (PD-1) blocking antibodies [
30], all of which target T cell activation. Adoptive cell therapy (ACT) is an alternative immunotherapy for melanoma, and its primary mechanism is T cell activation. While earlier clinical research mainly focused on ACT for tumor-infiltrating lymphocytes (TILs) [
31], efforts have recently been diverted to the generation of T cells with T cell receptors (TCRs) specific for tumor-associated antigens (TAAs), including NY-ESO-1 [
32] and MART-1 [
33]. The TCRs recognize antigens presented by MHC molecules, which limit the number of patients eligible for this immunotherapy, whereas CAR-T cells are activated upon recognizing unprocessed structures on the surface of the target in an MHC-independent manner [
34]. Due to advantages in stable antigen identification, the reduction of immunosuppressive tumor microenvironments and treatment toxicities, and the prevention of antigen escape, CAR-T cells have been widely explored and applied as a cancer therapy [
35,
36].
GD2 is overexpressed in melanoma, neuroblastoma, and small-cell lung cancer, but its expression is limited in normal tissues [
37]; therefore, targeting GD2 could reduce the incidence rate of toxicity associated with off-target or on-target/off-target effects. In our study, the rate of GD2 expression is 49.3% among 288 cases. The expression of ganglioside GD2 is more frequent in acral and mucosal melanoma than in CSD and non-CSD subtypes, which are the major melanoma subtypes in Caucasian cohorts [
38]. Due to the small sample size of CSD and non-CSD melanoma subtypes, statistical biases may be produced. The higher expression of ganglioside GD2 in metastatic melanoma compared to primary melanoma was consistent with the notion that GD2 expression is related to increased metastatic potential [
39]. Moreover, our study demonstrates that the expression of GD2 is significantly associated with poor prognosis. Hence, GD2 is an attractive target for CAR-T therapy.
Clinical trials of anti-GD2 monoclonal antibodies have demonstrated that the agent could significantly improve the survival of neuroblastoma patients [
40], but for melanoma patients, the benefits are limited due to the varying GD2 expression levels in melanoma (usually lower), which influence the capacity to bind with anti-GD2 monoclonal antibodies. However, several studies have indicated the CAR-T cells could induce complete cytotoxic responses to tumor cells despite the low expression of target antigen [
18,
41]. Our study also shows that GD2.BBζ CAR-T cells could lyse GD2+ melanoma (27.4–99.9% GD2 expression), including melanoma cells with low GD2 expression. Antibodies possessing multiple antibody-derived binding domains on their cell surface exhibited improved cytotoxic ability compared to bivalent antibodies in solution, and thus, CAR-T cells exhibit superior cytolytic activity compared to antibodies [
42].
Similar with most malignancies, melanoma cells lack the expression of T cell costimulatory molecules, which could trigger the complete activation of T lymphocytes against TAAs via their native or chimeric receptors. To activate the effector function and prolong the persistence of T cells, we introduced the CD137 (4-1BB) costimulatory signaling domain into the GD2 chimeric receptor. CD137 belongs to the TNF receptor family, which is essential for the proliferation and survival of T cells, particularly for memory T cell responses [
43,
44]. A previous study indicated that the adoptive transfer of tumor-specific T cells co-stimulated ex vivo with 4-1BBL exhibited increased persistence and antitumor activity in vivo [
45]. Our study also demonstrated that GD2.BBζ CAR-T cells could undergo clonal expansion when co-cultured with GD2
+ melanoma cell lines. T cell exhaustion is a major factor restricting the efficacy of CAR-T therapies, and 4-1BB could ameliorate exhaustion by reducing the expression of known exhaustion-related genes and by modulating metabolic, apoptosis, and hypoxia pathways [
22]. Several clinical trials have demonstrated that CAR-T cells harboring the 4-1BB costimulatory domain exhibit longer persistence than those harboring the CD28 costimulatory domain [
46‐
48]. Moreover, the 4-1BB costimulatory signaling domain could endow T cells with superior proliferative potential, more potent antitumor activity and a Th1-based cytokine profile [
25]. In the present study, we show that GD2.BBζ CAR-T cells could preferentially secrete high levels of Th1 cytokines, including IL-2, TNF-α, and IFN-γ, upon encountering a tumor cell and exert strong antitumor activity in vitro. Moreover, the GD2.BBζ CAR-T cells exhibit persistence in vivo.
In our study, we investigate the performance of CAR-T therapy using CD3
+ T cells instead of purified CD8
+ cytotoxic lymphocytes (CTLs) because CD4
+ T cells have been confirmed to increase the function of CD8
+ T cells [
49]. The results of our PDX model experiments demonstrate that CD3
+ T cells transduced with GD2.BBζ CAR show higher cytotoxicity than the non-transduced T cells, which consists with previous research findings that adoptive transfer of mixed populations of antigen-specific CD8
+ T cells and CD4
+ T cells promotes overall antitumor immunity. With regard to the administration route of T cells, both locally intratumor injection and venous injection lead to tumor regression, which confirms the capacity of CAR-T cells to circulate, traffic to the tumor, and perform cytotoxic ability. Although venous injections are favorable in clinical applications due to the ease of administration and the efficacy displayed in the preclinical model, several preclinical, and clinical studies have demonstrated the effectiveness of locally injected CAR-T cells [
49‐
51]. Our study suggests that local delivery of T cells for solid tumor may lead to a promising therapeutic efficacy, which may partly due to increased transmission of T cells to the tumor and to provide a favorable E/T ratio. However, local delivery of T cells may not be suitable for tumors with multiple metastatic.
There are some limitations and potential perspectives in our study. In present study, the GD2.BBζ CAR-T cells could expand when stimulated by GD2
high cell (WM-266-4 cells), whereas fail to expand in response to GD2
low (GAK), GD2
mediate (HMV-II). In previous study, the GPC3-4-1BB-CAR-T cells exhibit proliferation when stimulated by GPC3-positive cell in vitro [
52]. Moreover, the GD2-CD28-OX40-CAR-T cells could expand when stimulated by GD2-positive cell [
53]. The association between the proliferation of CAR-T cell and quantity of GD2 expression on the co-cultured target cells remain unknown, which need further study to verify.