GD2-specific chimeric antigen receptor effector cells for treatment of solid tumors, possibly in combination with enhancer of Zeste homolog 2 (EZH2) inhibitor

Abstract

GD2 specific chimeric antigen receptor effector cells for the treatment of solid tumors, possibly in combination with an enhancer of a Zeste homolog 2 (EZH2) inhibitor. The invention relates to a GD2-specific chimeric antigen receptor (GD2. CAR) effector cell for use in the treatment of solid tumors, in particular in the treatment of extracranial GD2 + tumors, such as soft tissue sarcoma and osteosarcoma, neuroblastoma, melanoma, lung cancer, bladder cancer and retinoblastoma, and brain tumors. In addition, the present invention also relates to the use of the GD2. CAR genetically modified effector cell in combination with an enhancer of a Zeste homolog 2 (EZH2) inhibitor for the treatment of solid tumors.

Description

Technical Field

[0001] This invention relates to GD2-specific chimeric antigen receptor (GD2.CAR) effector cells (referring to T cells, or innate cells: NK; NK-T cells, etc.) for the treatment of solid tumors, particularly extracranial GD2+ tumors (such as soft tissue sarcoma and osteosarcoma, neuroblastoma, melanoma, lung cancer, breast cancer, bladder cancer, and retinoblastoma) and brain tumors. Specifically, this invention relates to vectors comprising a cassette encoding the GD2.CAR gene, methods for producing the cassette, and GD2.CAR-genetically modified effector cells (e.g., T cells or innate cells, such as NK and NK-T cells) for the treatment of GD2+ solid tumors, including brain tumors such as medulloblastoma and glioma tumor cells. Furthermore, this invention also relates to the use of GD2.CAR-genetically modified effector cells in combination with an enhancer of a Zeste homolog 2 (EZH2) inhibitor for the treatment of solid tumors. Background Technology

[0002] Gangliosides are a subfamily of glycosphingolipids containing one or more sialic acid residues (composed of a hydrophilic sialic acid-containing oligosaccharide chain and a hydrophobic neuraminic acid glycoside). Based on the number of sialic acid residues in each molecule, gangliosides can be classified into monosialic gangliosides (GM), disialialic gangliosides (GD), trisialic gangliosides (GT), and tetrasialic gangliosides (GQ). 1 Disialic acid ganglioside (GD2), which has two glycosyl groups, is significantly increased under pathological conditions such as cancer and neurodegenerative diseases. This glycosphingolipid is involved in signal transduction, intercellular recognition, and tumor cell metastasis.

[0003] While most normal tissues express very low levels of GD2, many tumors, on the contrary, are characterized by very high levels of its expression. 2 In particular, GD2 was found to be effective in melanoma. 3,4 ,sarcoma 5 Breast cancer 6 Bladder cancer 2 Neuroblastoma 7 retinoblastoma 8 Small cell lung cancer 9 glioma 10 GD2 expression was moderate to high. Interestingly, primary tumor cells of H3K27M-mutated diffuse intrinsic pontine glioma (H3K27M-mutated DIPG) showed the highest GD2 expression. 11 .

[0004] Sarcoma is a rare mesenchymal tumor that includes a variety of bone and soft tissue tumors affecting all ages, but it is relatively more common in pediatric age groups, accounting for approximately 10-15% of childhood cancers. 12 In children and adolescents, the most common sarcomas include osteosarcoma (OS), rhabdomyosarcoma (RMS), Ewing sarcoma (EWS), and desmoplastic small round cell tumor (DSRCT), while other sarcomas, such as leiomyosarcoma and liposarcoma, typically occur in older patients. 13 Although sarcomas are rare, they constitute a significant mortality burden of approximately 13% of cancer-related deaths in patients aged 0-19 years. 14 Furthermore, despite increasing efforts focused on identifying targeted therapies, including tyrosine kinase inhibitors for soft tissue sarcoma and osteosarcoma, clinical outcomes for these patients have not significantly improved. Therefore, new treatment strategies are urgently needed to improve the prognosis of sarcoma patients. 15 .

[0005] Ewing sarcoma (EwS) is an aggressive solid mesenchymal tumor that occurs in bone and soft tissue. 16 EwS is characterized by specific chromosomal translocations, most commonly involving chromosomes 22 and 11 (t(11,22)(q24;12)), leading to the aberrant chimeric transcription factor EWSR1-FLI1. 17 According to reports, the Zeste homologue 2 (EZH2) inhibitor tazestat (E7438 / EPZ6438) 18 The enhancer can upregulate the expression of GD2 in Ewing sarcoma. 19 Specifically, EZH2, as a histone methyltransferase, silences genes involved in cell differentiation in a highly background-dependent manner by depositing an inhibitory histone marker at histone 3-lysine 27 (H3K27me3). A direct result of EWSR1-FLI1 is the induction of high levels of EZH2 expression in the EwS cell line. 20 EZH2 regulates GD2 expression in EwS cells by modulating the expression of genes related to GD2 biosynthesis. Drug inhibition of EZH2 selectively upregulates GD2 expression on the surface of EwS cells. 19 Regarding the inhibition of EZH2, Wiebel M. et al. reported that inhibition using histone methyltransferase enhancers of Zeste homolog 2 (EZH2) did not increase GD2 expression or GD2 expression in OS cell lines. 21 .

[0006] Rhabdomyosarcoma (RMS) is a family of soft tissue sarcomas in children and adolescents, including fusion-positive FP-RMS, which contains fusion oncoproteins PAX3 / 7-FOXO1 and FN-RMS, and is usually mutated in the RAS pathway.

[0007] Brain tumors are the most common solid tumors in children. 22 Standard treatment for these diseases includes surgical resection, radiation therapy, and, in select cases, chemotherapy. Despite aggressive treatment, many patients have poor long-term outcomes or frequently experience treatment-related long-term sequelae, including hormonal dysfunction, sensorimotor, and neurocognitive impairments. 23 .

[0008] Low-grade gliomas (pLGGs) are the most common pediatric brain tumors, accounting for 40% to 50% of all childhood CNS tumors. 24 The World Health Organization (WHO) classifies these tumors into Grade I and Grade II tumors. 25 Pilocytic astrocytoma is the most common subtype. Although these patients have good overall survival... 26 However, their progression-free survival (PFS) is usually very low. 27 .

[0009] High-grade gliomas (pHGG) account for approximately 10–25% of all childhood CNS tumors. 28 The WHO classifies these tumors into grades III and IV, although they are often histologically identical to their adult counterparts, they are clinically and molecularly distinct. 29 The survival outcomes of these patients contrast sharply with those of pLGG, as most die within 2–3 years of diagnosis, despite a 5-year overall survival rate of approximately 15–20% with treatment. 30 .

[0010] Disialotetrahexosylganglioside GD2 was found to be highly expressed in all H3K27M+ DIPGs (including H3F3A K27M mutations (SU-DIPG-6, 13, 17, 25, 29) and the less common HIST1H3B K27M mutation (SU-DIPG-21)); GD2 expression was much lower in histone-3 WT pediatric high-grade gliomas (pHGG) (including one case of H3WT DIPG). 31 .

[0011] Ependymoma is a glial tumor that accounts for approximately 10–12% of all childhood CNS tumors. Although the 5-year survival rate is good, ependymomas often recur, and the 10- and 15-year progression-free survival and overall survival rates are low. 32 To date, there are no results regarding GD2 expression in gliomas.

[0012] Medulloblastoma (MB) is the most common malignant embryonal brain tumor in children, accounting for approximately 15-20% of all childhood CNS tumors.22 The World Health Organization (WHO) has classified MB as a grade IV tumor, genetically originating in the posterior fossa. 33 Histologically, MB is classified into four variants: classic, large cell / anaplastic (LCA), extensive nodular (MBEN), and connective tissue hyperplasia / nodular (DN). 25,33,34 At the molecular level, medulloblastoma (MB) has been shown to comprise multiple subtypes with different driving events, clinical features, and outcomes, leading to their reclassification into four main groups: Wingless Activating (WNT) medulloblastoma, Sonic Hedgehog Activating (SHH) medulloblastoma, Group 3 (G3), and Group 4 (G4). While subgroups exist within each subtype, these four subtypes are the most accepted model in clinical practice. Currently, the primary treatment for MB is a combination of surgical resection of the tumor, craniospinal radiotherapy (which should be avoided in children under 3–5 years of age), and adjuvant chemotherapy. 35-37 Despite standard intensive care, 30-40% of high-risk individuals still die from cancer. 38,39 Furthermore, long-term survivors frequently experience adverse side effects that significantly impact their quality of life. Additionally, despite receiving the same treatment, patients belonging to different MB subgroups exhibited varying outcomes. 34,40 In particular, the WNT subgroup, which accounts for 90% of the 5-year survival rate in children with myocardial infarction (MB), has a very good prognosis compared to other MB subgroups. 33,41,42 The overall survival (OS) of the SHH subgroup was approximately 76%. 42 The G3 subgroup was associated with the worst prognosis, characterized by an overall survival (OS) rate of approximately 50% in treated patients; the G4 subgroup had a generally good prognosis. 42 However, given our understanding of the biology of myocardial microscopy in children, translating this knowledge into new treatments remains a significant challenge.

[0013] To date, very little data has been published regarding the expression of GD2 in MB. 43 .

[0014] Atypical teratoid / rhabdomyosarcoma of the brain (AT / RT) is a highly aggressive embryonal tumor of the CNS, defined as the loss of function of the gene of SWI / SNF-associated, matrix-associated, actin-dependent chromatin regulator subfamily B member 1 (SMARCB1). 44 Although AT / RT is the most common malignant CNS tumor in children <1 year of age, cases are rare in adults (i.e., those >18 years of age). 45 AT / RT is associated with low survival. No results were found regarding GD2 expression in AT / RT.

[0015] Embryonic tumors with multi-layered rosettes (ETMRs) are an aggressive WHO-IV pediatric CNS tumor, most commonly found in infants and young children (usually under 3 years old), and were previously classified as primitive neuroectodermal tumors of the CNS. Regardless of treatment, most children survive less than a year, and the reported five-year overall survival is less than 30%. 46 To date, there are no results regarding GD2 expression in ETMR.

[0016] The use of chimeric monoclonal anti-GD2 antibodies ch14.18 / SP2 / 0 (dinutuximab) and ch14.18 / CHO (dinutuximab beta) is considered the standard of first-line treatment for children with high-risk neuroblastoma. 47,48 Furthermore, in clinical trials, MB49 and metastatic CNS neuroblastoma... 50 The anti-GD2 antibody radioconjugate was well tolerated by patients when administered intrathecally and intraventricularly. 49 .

[0017] Using genetically modified T cells to express chimeric antigen receptors (CARs) is a promising new approach for adoptive T-cell immunotherapy in cancer, combining the antigen specificity of monoclonal antibodies (mAbs) with the effector functions, biodistribution, and long-term persistence of T cells. The CAR is then an artificial receptor composed of regions targeting a specific antigen, linked to the CD3ζ chain of the T cell activation domain via an intracytoplasmic domain (first-generation CAR). The intracytoplasmic domain can be enriched with one or two co-stimulatory molecules (i.e., CD28, OX40, 4-1BB, or others) to generate second- or third-generation CARs, respectively.

[0018] In particular, in the past few years, immunotherapies based on engineered expression of anti-GD2 chimeric antigen receptor (GD2.CAR) to target tumor cells and promote T-cell metastasis have become popular for several solid cancers (such as neuroblastoma). 51 Diffuse midline glioma with H3K27M mutation 52 and lung cancer 53 An exciting new approach.

[0019] Furthermore, it has been reported that in small cell lung cancer 54 and breast cancer 55 In this context, GD2 participates in tumor development by increasing cell proliferation, growth, motility, migration, adhesion, and invasion.

[0020] A phase I clinical trial using first-generation anti-GD2-CAR T cells in patients with neuroblastoma showed that it was safe and had a transient clinical response. 56,57 .

[0021] A phase I clinical trial using third-generation GD2-CAR (incorporating CD28 and OX40 co-stimulatory terminal domains (GD2-CAR3)) in patients with relapsed or refractory neonatal neoplasms (NB) showed safety with no dose-limiting toxicities. However, the antitumor response at 6 weeks was moderate. 58 .

[0022] Recently, a phase I clinical trial also used the same third-generation GD2-CAR (incorporating CD28 and OX40 co-stimulatory terminal domains) in combination with BRAF / MEK inhibitor therapy to treat patients with metastatic melanoma, and as monotherapy for patients with colorectal cancer and fibromyxoid sarcoma. No dose-limiting toxicities or serious adverse events were reported. However, limited clinical efficacy was reported. 59 Although in preclinical models 51 Both clinical trials and clinical trials have reported low levels of GD2 expression in normal brains. However, when exploring the safety and efficacy of GD2-CAR T cells, the single-chain variable fragment (scFv) of GD2-CAR T cells, derived from a 14g2a monoclonal antibody (including our academic clinical trial NCT03373097), has not reported any cases of significant neurotoxicity. 56 .

[0023] Recently, Mount et al. demonstrated that intravenously administered second-generation GD2-CAR incorporates a single-chain variable fragment derived from a 14g2a monoclonal antibody, a CD8 transmembrane domain, and 4-1BB (as a co-stimulatory domain), as well as the terminal domain of the T-cell receptor ζ signaling pathway (capable of crossing the BBB and clearing patient-derived H3K27M-mutated diffuse midline glioma (DMG) tumors). 31 .

[0024] Shum et al. demonstrated that in a glioblastoma (GBM) xenograft model, GD2.CAR T cells engineered to express the IL-7 receptor (GD2-CAR.C7R) promoted the persistence, proliferation, and antitumor activity of CAR T cells. 60 As an additional safety measure, they created T cells co-expressing the clinically validated inducible cysteine ​​9 (iC9) suicide gene, which selectively eliminates T cells. After dual transduction with iC9 and the GD2–CAR.C7R retroviral vector, the T cells remained sensitive to iC9 signaling and underwent in vitro apoptosis within 24 hours of exposure to a chemical inducer of dimerized AP20187. 60 .

[0025] All of these studies reinforce the clinical translation of CAR.GD2 T cells in patients with glioma (NCT04099797, NCT04196413).

[0026] Table 1 summarizes the known gene therapy products (GD2.CAR) developed for preclinical models of brain tumors (Table 1).

[0027] Table 1 Regarding the first-generation CAR described in Table 1, this CAR does not include any induced suicide genes as an additional safety switch.

[0028] Regarding the second-generation CAR described in Table 1, in this work, the authors performed dual co-transduction to obtain T cells expressing CAR-GD2 and the induced suicide gene; that is, they used two retroviral vectors. This approach involves generating T cells expressing only CAR, only the induced suicide gene, or both. A drawback of this approach is the risk of ineffective cell control under toxic conditions.

[0029] Regarding solid tumors, the inventors' preclinical studies have shown that third-generation GD2.CARs incorporating the CD28 4-1BB co-stimulatory domain are associated with improved antitumor efficacy against neuroblastoma models and long-term persistence in in vivo neuroblastoma xenograft mouse models, compared to GD2.CARs incorporating either CD28.OX40 or a combination of OX40 and 4-1BB domains. Specifically, selection of 4-1BB signaling was demonstrated to lead to significant improvements in several CAR T cell characteristics, including: 1) T cell exhaustion, 2) basal T cell activation, 3) in vivo tumor control, and 4) T cell persistence. Furthermore, improved persistence of IIICAR.GD2 T cells (third-generation CAR-GD2 T cells) relative to IICAR.GD2 T cells (including CD28, OX40, or 4-1BB as co-stimulatory molecules) was observed in in vivo mouse models. Indeed, at day 30, the number of IICAR.GD2 T cells was significantly lower than that of IIICAR.GD2 T cells. Furthermore, it was found that fine-tuning of T cell culture conditions obtained using IL7 and IL15, along with GD2.CAR design, had a synergistic effect in increasing the antitumor activity of CAR T cells. 51 In neuroblastoma 51,61,62 and high-grade glioma model 63 In this study, these third-generation CAR.GD2 T cells are all in the preclinical stage. Based on the aforementioned preclinical data... 51In 2017, an academic Phase I / II clinical trial was initiated to treat pediatric patients with high-risk and / or relapsed / refractory neuroblastoma or other extracranial GD2-positive solid tumors, specifically GD2+ sarcoma (NCT03373097). Clinical responses in neuroblastoma patients showed great promise in terms of both the safety and therapeutic efficacy of CAR.GD2 T cell infusion. 64 .

[0030] Given the above, it is clear that there is a need to prepare additional GD2.CAR T cells that can overcome the known drawbacks of GD2.CAR T cells. Summary of the Invention

[0031] According to the present invention, a novel third-generation GD2-specific chimeric antigen receptor (GD2.CAR) and related uses are provided.

[0032] In particular, according to the present invention, it has been surprisingly found that a specific third-generation GD2-specific chimeric antigen receptor (GD2.CAR) can treat solid tumors, such as brain tumors. In this regard, experimental results regarding the efficacy and safety of using the third-generation GD2-specific chimeric antigen receptor (GD2.CAR) according to the present invention to treat solid tumors (e.g., sarcoma and medulloblastoma) are provided below.

[0033] Furthermore, according to the present invention, it has been surprisingly found that the combination of an enhancer of a Zeste homology 2 (EZH2) inhibitor with the GD2.CAR according to the present invention, particularly pretreatment with an EZH2 inhibitor followed by administration of GD2.CAR, can be used to treat tumors that do not express or express low levels of GD2 protein, i.e., tumors to which GD2.CAR alone is ineffective.

[0034] Regarding the structure of the GD2.CAR according to the present invention, in the experiments reported below, a vector with an SFG backbone was designed using a retroviral vector based on Moloney murine leukemia virus (MoMLV). All env and gag-pol were removed except for the packaging sequence (psi). As a result, the vector produced according to the present invention is non-replicating. Specifically, according to the present invention, the following clinical-grade third-generation retroviral vector (SFG) for GD2.CAR has been produced: SFG.iC9.2A.CAR-GD2(14g2a)CD8tm.CD28.41BBζ (hereinafter referred to as iC9.2A.GD2.CAR-28.4-1BB.ζ), which comprises: -The chimeric cystase 9 (iC9) suicide gene as a safety switch 65 -T2A self-cleaving peptide sequence, which can induce ribosome skipping during protein translation in cells; -Signal peptide; - Single-stranded variable fragment (scFv) from 14g2a; - Hinge sequence; -3 amino acid (aa) linking domains; - Transmembrane domains derived from CD28tm to enhance molecular stability; - Add two co-stimulatory domains to the GD2.CAR vector: CD28 and 4-1BB66, which are fused with the CD3-ζ chain, respectively.

[0035] More specifically, the inventors designed as follows Figure 1 The bicistronic vector shown in A (SFG.iC9.2A.CAR-GD2(14g2a)CD8tm.CD28.41BBζ(iC9.2A.GD2.CAR-28.4-1BB.ζ)) allows for the simultaneous expression of two transgenes, namely inducible cysteine ​​9 (iC9) and third-generation GD2.CAR.

[0036] Table 2 shows the unique elements present in the novel GD2.CAR (iC9.2A.GD2.CAR-28.4-1BB.ζ) of the present invention compared with the known GD2.CAR reported in Table 1, particularly the presence of the inducible suicide gene and the co-stimulatory domain CD28.4-1BB.

[0037] Table 2 More specifically, according to the present invention, the inventors have created a cell line for producing clonal retroviruses, which produces high-titer retroviral vectors containing: • 5'LTR – The long terminal repeat sequence of the retrovirus at the 5' end of the vector (which acts as a promoter sequence). •ψ-Retroviral capsidation signal (psi; necessary for packaging RNA into viral particles); •SA - Splice acceptor site; • iCasp9 - Inducible caspase-9 expression cassette. iCasp9 consists of human FK506-binding protein (FKBP12) with an F36V mutation, linked to modified CARD domain-deficient human caspase-9 via a 6-amino acid glycine-serine linker: • FKBP12-F36V – An engineered FK506-binding protein containing an F36V mutation to optimize binding affinity for AP1903. The FKBP12-F36V protein domain serves as the drug-binding / oligomerization domain for the linked therapeutic protein (AP1903). FKBP12-F36V functions as a regulator of caspase-9: in the absence of AP1903, iCasp9 exhibits minimal activity; AP1903 binding to FKBP12-F36V promotes dimerization and juxtaposes two caspase-9 molecules to initiate apoptosis. Therefore, FKBP12-F36V partially functionally replaces the endogenous dimerization / activation module (caspase activation and recruitment domain; CARD) of caspase-9 that mediates Apaf-1-related oligomerization. • Linker - A synthetic Ser-Gly-Gly-Gly-Ser-Gly peptide linker for fusing a switch-regulatory sequence to caspase-9; • Caspase-9 - Human aspase-9 cDNA sequence (a key pro-apoptotic regulator) and therapeutic component of the construct (a regulated suicide gene). The endogenous dimerization / activation module (aspase activation and recruitment domain; CARD) is deleted to reduce spontaneous Apaf1 binding, thereby reducing background killing. 65 .

[0038] •2A-encodes a 20-amino acid peptide synthesized from the insect virus of the moth *Thosea Asigna*, which functions as a cleavable linker between the caspase 9 protein and the CAR protein. • Signal peptide - A short amino acid sequence that allows secretory proteins to be properly transported from the endoplasmic reticulum to the cell membrane; •CAR-CAR molecules contain • A single-chain variable fragment (scFv) of the fusion VH-VL region of monoclonal antibody 14.G2A, specific to human antigen GD2, which is shown in the same frame as below. • Hinge • Transmembrane domains derived from CD8 (CD28 TM) transmembrane domains. • Contains a costimulatory domain including a 4.1bb costimulatory domain and •CD3ζ cytoplasmic domain.

[0039] • 3'LTR – The long terminal repeat sequence of the retrovirus at the 3' end of the vector (acting as a terminator / polyadenylation sequence).

[0040] Table 3 shows the functional elements of the iC9.2A.GD2.CAR-28.4-1BB.ζ retroviral vector designed according to the present invention: Table 3 The above-described sequence according to the present invention provides unexpected advantages. Indeed, according to experimental results reported below, the GD2.CAR-28.4-1BBζ (GD.CAR) T cells according to the present invention significantly controlled tumor growth in sarcoma cells with high GD2 expression (particularly osteosarcoma (OS) cell lines 143B, MG-63, and U-2OS, and embryonal rhabdomyosarcoma (ERMS) cell line RD), and surprisingly, they were also able to significantly (albeit partially) control tumor growth in sarcoma cell lines with lower GD2 expression (e.g., OS cell line HOS, alveolar rhabdomyosarcoma (ARMS) cell line RH41, and Ewing sarcoma cell line A-673). Furthermore, as shown in the experimental results reported below, the CAR.GD2 T cells according to the present invention, as a human GD2+ medulloblastoma (MB) cell line, exhibited effective antitumor activity against brain tumors.

[0041] Furthermore, according to the present invention, it has been shown in vivo that mice carrying RMS tumors treated with GD2.CAR-28.4-1BBζ T cells have significantly longer survival than mice treated with control. Moreover, T cell persistence was observed for up to 75 days. In this regard, it is noted that there is no data in the prior art literature regarding effective GD2.CAR T cell control of soft sarcomas such as rhabdomyosarcoma.

[0042] According to the present invention, in vivo data on overall survival (OS) were also generated, showing that GD2.CAR-28.4-1BBζ (GD2.CAR) T cells significantly slowed the growth of the primary tumor and significantly increased the overall survival of mice treated with CAR T cells compared to NT T cells. In this regard, it is noted that by comparing the overall survival of OS mice treated with the GD2.CAR of the present invention (… Figure 8 E) and Adrienne H Long et al. in the literature (see page 873 of Cancer Immunology Res; 4 (10) October 2016), Figure 3 B) The reported survival rate suggests that the GD2.CAR-28.4-1BBζ T cells according to the present invention exhibit superiority compared to those reported by Adrienne H Long et al. 67 The GD2-CAR described is a 14g2a-scFv (14g2a.CD28.OX40.ζ) doped with CD28, OX40 and CD3ζ signal transduction domains.

[0043] Furthermore, according to the present invention, CAR.GD2 T cells have been shown to exert effective antitumor activity against human GD2+ MB cell lines, as well as in xenogeneic mouse models and patient-derived xenogeneic MB transplantation mouse models.

[0044] Furthermore, as described above, according to the present invention, it has been surprisingly found that the combination of an enhancer of the Zeste homology 2 (EZH2) inhibitor with the GD2.CAR according to the present invention can be used to treat tumors that do not express or express low levels of GD2 protein, i.e., tumors that are ineffective against GD2 alone. In particular, according to the present invention, it has been found that by using the EZH2 inhibitor tazemetostat to inhibit the upregulation of GD2 expression in Ewing sarcoma, ARMS sarcoma, osteosarcoma, and medulloblastoma cell lines, and to increase their sensitivity to the GD2.CAR T cell killing activity, it has been found that...

[0045] As reported above, it is known that EZH2 inhibitors upregulate GD2 expression in Ewing sarcoma. However, according to the aforementioned article... 21,67 Experts in this field would not be induced to combine EZH2 inhibitors with anti-GD2 CARs in solid tumors (e.g., RMS and OS), particularly in tumors lacking the EWSR1-FLI1 fusion gene. Indeed, as noted above, EZH2 inhibition has been reported to not increase GD2 expression in OS cell lines. 21 .

[0046] According to the present invention, it was also surprisingly found that GD2.CAR-28.4-1BBζ T cells also significantly controlled the growth of invasive tumors in adults, such as melanoma and lung adenocarcinoma.

[0047] Therefore, a specific object of the present invention is an anti-GD2 chimeric antigen receptor comprising, or composed of, the following from the N-terminus to the C-terminus: a) Signal peptide, b) Anti-GD2 single-chain antibody domain, c) Hinges, d) Transmembrane structural domains e) At least two co-stimulatory signal transduction domains, and f) CD3ζ chain sequence, Used to treat solid tumors The anti-GD2 single-chain antibody domain comprises, or consists of, an anti-GD2 VL sequence and an anti-GD2 VH sequence interconnected by a linker; and The solid tumors mentioned are not neuroblastoma, diffuse midline glioma (DMG) H3K27M- mutant, or osteosarcoma.

[0048] Furthermore, according to one embodiment of the invention, the solid tumor is not a high-grade glioma. According to another embodiment of the invention, the solid tumor is not rhabdomyosarcoma or Ewing's sarcoma. According to one embodiment of the invention, the solid tumor is not a sarcoma. According to another embodiment of the invention, the solid tumor is not a medulloblastoma.

[0049] According to one embodiment of the present invention, the tumor is not a glioma.

[0050] According to the present invention, the solid tumor to be treated is a tumor expressing GD2 or a tumor expressing GD2 after treatment with a compound capable of enhancing GD2 expression (e.g., an enhancer of a Zeste homolog 1 or 2 (EZH2) inhibitor).

[0051] According to the present invention, the solid tumor may be selected from brain tumors that do not include diffuse midline glioma (DMG) H3K27M- mutants, such as diffuse intrinsic pontine glioma (DIPG) different from diffuse midline glioma (DMG) H3K27M- mutants, adult diffuse glioma, pediatric diffuse low-grade glioma, pediatric diffuse high-grade glioma, astrocytic glioma, medulloblastoma, glial neuron and neuronal tumors, ependymal tumors, choroid plexus tumors; embryonic tumors, pineal gland tumors, cranial nerve and paravertebral nerve tumors; extracranial tumors, such as sarcomas different from osteosarcomas; retinoblastoma; melanoma; lung cancer; gastric cancer; bladder cancer, head and neck cancer; ovarian cancer; breast cancer, pancreatic cancer; colorectal cancer or other tumors expressing GD2.

[0052] According to the present invention, the sarcoma may be selected from rhabdomyosarcoma, particularly alveolar rhabdomyosarcoma or embryonal rhabdomyosarcoma, Ewing sarcoma (EWS), desmoplastic small round cell tumor, leiomyosarcoma and liposarcoma; and the medulloblastoma may be selected from the SHH, G3, G4 and WNT medulloblastoma subgroups.

[0053] According to the present invention, the tumor can be a tumor in which GD2 expression is higher than that in a negative healthy control, i.e., in healthy peripheral blood mononuclear cells (PBMCs), wherein GD2 expression is measured as the percentage of GD2+ cells relative to the total number of tumor cells or healthy peripheral blood mononuclear cells (the percentage of GD2-positive cells in healthy PBMCs is equal to 0.43% ± 0.90%). Preferably, the tumor is a tumor in which GD2 expression is higher than 2%, i.e., the percentage of GD2+ cells is higher than 2%.

[0054] According to one embodiment of the invention, the brain tumor is not a high-grade glioma. According to another embodiment of the invention, the sarcoma is not rhabdomyosarcoma or Ewing's sarcoma. According to one embodiment of the invention, the solid tumor is not a sarcoma. According to another embodiment of the invention, the brain tumor is not a medulloblastoma.

[0055] According to one embodiment of the present invention, the anti-GD2 VL sequence may comprise the CDR1 sequence QSLVHRNGNTY (SEQ ID NO:5), the CDR2 sequence KVS, and the CDR3 sequence SQSTHVP (SEQ ID NO:7); while The anti-GD2 VH sequence may include the CDR1 sequence: GSSFTGYN (SEQ ID NO:8), the CDR2 sequence: IDPYYGGT (SEQ ID NO:9), and the CDR3 sequence: VSGMEY (SEQ ID NO:10).

[0056] In particular, according to the present invention, The anti-GD2 14G2a VL sequence may contain or consist of the following: DILLTQTPLSLPVSLGDQASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELKRADAAPTVSIFP (SEQ ID NO:11), and The anti-GD2 14G2a VH sequence may contain or consist of the following: EVKLQQSGPSLVEPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSS (SEQ ID NO:12).

[0057] Specifically, sequence SEQ ID NO:11 contains CDR1 at positions 27-37, CDR2 at positions 55-57, and CDR3 at positions 94-10, while sequence SEQ ID NO:12 contains CDR1 at positions 26-33, CDR2 at positions 51-58, and CDR3 at positions 97-102.

[0058] According to the present invention, the linker connecting the anti-GD2 VL sequence and the anti-GD2 VH sequence can be a short, flexible linker rich in glycine, with a length of 7 to 14 amino acids, such as 7 to 12, 7 to 10, or 8 amino acids. For example, the linker is selected from G7S2 linker GSGGGGSGG (SEQ ID NO:13), (G4S)2 linker GGGGSGGGG (SEQ ID NO:14), G4SG2 linker GGGGSGG (SEQ ID NO:15), G3SG4 linker GGGSGGGG (SEQ ID NO:16), SG4SG3 linker SGGGGSGGG (SEQ ID NO:17), (SG4)2S linker SGGGGSGGGGS (SEQ ID NO:18), (SG4)2SG linker SGGGGSGGGGSG (SEQ ID NO:19), (SG4)2SG3 linker SGGGGSGGGGSGGG linker (SEQ ID NO:20), (SG4)2 SGGGGSGGGG (SEQ ID NO:21) or (SG4)2 SG2 SGGGGSGGGGSGG (SEQ ID NO:22), preferably G7S2 connector GSGGGGSGG (SEQ ID NO:13).

[0059] Therefore, according to the present invention, the anti-GD2 single-chain antibody domain can be DILLTQTPLSLPVSLGDQASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELKRADAAPTVSIFPGSGGGGSGGEVKLQQSGPSLVEPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSS (SEQ ID NO:4).

[0060] According to the present invention, the hinge may comprise or consist of one or more of the following hinges: The IgG-based hinge has the following sequence: AKTTPPSVYGRVTVSSAEPKSCDKTHTCPPCP (SEQ ID NO:23); The hinge spacer region -CD8α, has the sequence: PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA (SEQ ID NO:24) (Nucleotide ID NO: M12828.1, Protein ID NO: AAB04637.1). CD8 stalk, sequence: TTTAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO:25); Hinge CD28, sequence: EVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO:26); The hinge CH2-CH3 sequence is as follows: ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:27); Hinge CH3, sequence: ESKYGPPCPSCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:28); The preferred sequence is the IgG-based hinge of SEQ ID NO:23.

[0061] According to the present invention, the transmembrane domain of the anti-GD2 chimeric antigen receptor can be selected from the group consisting of: CD8aTM: CDIYIWAPLAGTCGVLLLSLVIT (SEQ ID No: 6), (nucleotide ID NO: NM_001768.6 and protein ID NO: NP_001759.3); CD28TM: FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 29) (nucleotide ID NO: BC112085.1 and protein ID NO: AAI12086.1); preferably, CD28TM (SEQ ID NO: 29) with the sequence FWVLVVVGGVLACYSLLVTVAFIIFWV.

[0062] According to the present invention, the hinge can be connected to the transmembrane domain by a connector (or a connection sequence); preferably, the connector has three amino acids; more preferably, the connector has a DPK sequence.

[0063] According to the present invention, the two co-stimulatory signal transduction domains can be selected from the group consisting of: The following sequences are obtained by concatenating: CD28 cytoplasmic sequence: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:30), with OX40 sequence: RDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO:32), wherein the CD28 cytoplasmic sequence is located before or after the OX40 sequence; The following sequences are obtained by concatenating: CD28 cytoplasmic sequence: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:30), with CD137 (4-1BB) sequence: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:31), wherein the CD28 cytoplasmic sequence is located before or after the CD137 (4-1BB) sequence; The following sequences are obtained by concatenating: OX40 sequence: RDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO:32), with CD28 cytoplasmic sequence: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:30), wherein the OX40 sequence is located before or after the CD28 cytoplasmic sequence; or The following sequences are obtained by concatenating: OX40 sequence: RDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO:32), with CD137 (4-1BB) sequence: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:31), wherein the OX40 sequence is located before or after the CD137 (4-1BB) sequence; Preferably, the following sequences are obtained by concatenating: CD28 cytoplasmic sequence: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:30), with CD137 (4-1BB) sequence: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:31), wherein the CD28 cytoplasmic sequence is located before the CD137 (4-1BB) sequence.

[0064] According to the present invention, the CD3-ζ chain sequence may have the following sequence: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR* (SEQ ID NO: 53) (nucleotide ID NO: J04132.1 and protein ID: AAA60394.1).

[0065] According to the present invention, the anti-GD2 chimeric antigen receptor may further include a cytoplasmic portion of CD8cyt, CD8a cyto, with the sequence LYCNHRN (SEQ ID NO:51), between the transmembrane domain and the co-stimulatory signal transduction domain.

[0066] According to the present invention, the signal peptide may comprise or consist of the sequence MEFGLSWLFLVAILKGVQCSR (SEQ ID NO:3).

[0067] According to a specific embodiment of the present invention, the anti-GD2 chimeric antigen receptor may comprise or consist of the following sequences: GD2.CAR-28.4-1BB.ζ * (SEQ ID NO:39).

[0068] Specifically, sequence SEQ ID NO:39 contains Signal peptide: MEFGLSWLFLVAILKGVQCSR (SEQ ID NO: 3); Anti-GD2(14G2a) VL sequence: DILLTQTPLSLPVSLGDQASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELKRADAAPTVSIFP (SEQ ID NO: 11).

[0069] G7S2 connector: GSGGGGSGG (SEQ ID NO:13) Anti-GD2(14G2a) VH sequence: EVKLQQSGPSLVEPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSS (SEQ ID NO: 12).

[0070] IgG-based hinge: AKTTPPSVYGRVTVSSAEPKSCDKTHTCPPCP (SEQ ID NO:23) It connects to the transmembrane structural domain via a connector (connection sequence) DPK: CD28TM: FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO:29) CD28 cytoplasmic sequence: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:30) CD137 (4-1BB) sequence: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:31) CD3-ζ chain: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR* (SEQ ID NO:53).

[0071] Another object of the present invention is a nucleotide sequence or a vector comprising said nucleotide sequence for use in the treatment of solid tumors, said nucleotide sequence comprising or consisting of a nucleotide sequence encoding or constituting an anti-GD2 chimeric antigen receptor as defined above, wherein The solid tumor is not neuroblastoma, diffuse midline glioma (DMG) H3K27M- mutant, or osteosarcoma.

[0072] Furthermore, according to one embodiment of the invention, the solid tumor is not a high-grade glioma. According to another embodiment of the invention, the solid tumor is not rhabdomyosarcoma or Ewing's sarcoma. According to one embodiment of the invention, the solid tumor is not a sarcoma.

[0073] According to another embodiment of the invention, the solid tumor is not a medulloblastoma. According to another embodiment of the invention, the tumor is not a glioma.

[0074] As described above, according to the present invention, the solid tumor to be treated is a tumor expressing GD2 or a tumor expressing GD2 after treatment with a compound capable of enhancing GD2 expression (e.g., an enhancer of a Zeste homolog 1 or 2 (EZH2) inhibitor).

[0075] As described above, according to the present invention, the solid tumor may be selected from brain tumors that do not include diffuse midline glioma (DMG) H3K27M- mutants, such as diffuse intrinsic pontine glioma (DIPG), adult diffuse glioma, pediatric diffuse low-grade glioma, pediatric diffuse high-grade glioma, astrocytic glioma, medulloblastoma; glial neuron and neuronal tumors; ependymal tumors; choroid plexus tumors; embryonal tumors, medulloblastoma, pineal tumors, cranial nerve and paravertebral nerve tumors; extracranial tumors, such as sarcomas that are different from osteosarcomas; retinoblastoma; melanoma; lung cancer; gastric cancer; bladder cancer; head and neck cancer; ovarian cancer; breast cancer; pancreatic cancer; colorectal cancer or other tumors expressing GD2.

[0076] According to the present invention, the sarcoma may be selected from osteosarcoma, rhabdomyosarcoma, especially alveolar rhabdomyosarcoma or embryonal rhabdomyosarcoma, Ewing sarcoma (EWS), desmoplastic small round cell tumor, leiomyosarcoma and liposarcoma; and the medulloblastoma may be selected from the SHH, G3, G4 and WNT medulloblastoma subgroups.

[0077] As described above, according to the present invention, the tumor can be a tumor in which GD2 expression is higher than that in negative healthy controls, i.e., peripheral blood mononuclear cells (PBMCs), wherein GD2 expression is measured as the percentage of GD2+ cells relative to the total number of tumor cells or healthy peripheral blood mononuclear cells (the percentage of GD2-positive cells in healthy PBMCs is equal to 0.43% ± 0.90%). Preferably, the tumor is a tumor in which GD2 expression is higher than 2%, i.e., the percentage of GD2+ cells is higher than 2%.

[0078] According to one embodiment of the invention, the brain tumor is not a high-grade glioma. According to another embodiment of the invention, the sarcoma is not rhabdomyosarcoma or Ewing's sarcoma. According to one embodiment of the invention, the solid tumor is not a sarcoma. According to another embodiment of the invention, the brain tumor is not a medulloblastoma.

[0079] According to the present invention, the anti-GD2 VL sequence may be encoded by the following nucleotide sequence: GATATTTTGCTGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGTCTTGTACACCGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATTCACAAAGTTTCCAACCGATTTTCTGGGG TCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGTTCTCAAAGTACACATGTTCCTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCA (SEQ ID NO:36), and The anti-GD2 VH sequence is encoded by the following nucleotide sequence: GAGGTGAAGCTTCAGCAGTCTGGACCTAGCCTGGTGGAGCCTGGCGCTTCAGTGATGATATCCTGCAAGGCTTCTGGTTCCTCATTCACTGGCTACAACATGAACTGGGTGAGGCAGAACATTGGAAAGAGCCTTGAATGGATTGGAGCTATTGATCCTTACTATGGTG GAACTAGCTACAACCAGAAGTTCAAGGGCAGGGCCACATTGACTGTAGACAAATCGTCCAGCACAGCCTACATGCACCTCAAGAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGTAAGCGGAATGGAGTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO:37).

[0080] In the (anti-GD2(14G2a)) VL sequence SEQ ID NO:36, the sequences encoding CDR1, CDR2, and CDR3 are as follows: CDR1 sequence: CAGAGTCTTGTACACCGTAATGGAAACACCTAT at positions 79-111 (SEQ ID NO:38); CDR2 sequence: AAAGTTTCC at bits 163-171, and CDR3 sequence: TCTCAAAGTACACATGTTCCTCC at positions 280-302 (SEQ ID NO:40); In the (anti-GD2(14G2a)) VH sequence SEQ ID NO:37, the sequences encoding CDR1, CDR2, and CDR3 are as follows: CDR1 sequence: GGTTCCTCATTCACTGGCTACAAC at positions 76-99 (SEQ ID NO:41); CDR2 sequence: ATTGATCTTACTATGGTGGAACT at positions 151-174 (SEQ ID NO:42); And CDR3 sequence: GTAAGCGGAATGGAGTAC (SEQ ID NO:43) at positions 289-306.

[0081] According to a specific embodiment of the present invention, the nucleotide sequence encoding the anti-GD2 chimeric antigen receptor may be: GD2.CAR-28.4-1BB.ζ

[0082] Specifically, sequence SEQ ID NO:52 contains: Signal peptide: ATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGTGTCCAGTGCTCTAGA (SEQ ID NO:35), G7S2 connector (which connects GD2 (14G2a) VL sequence and GD2 (14G2a) VH sequence): GGCTCGGGCGGTGGTGGGTCGGGTGGC (SEQ ID NO:44); Hinge sequence based on IgG: GCCAAACGACACCCCCATCAGTCTATGGAAGGGTCACCGTCTCTTCAGCGGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCG (SEQ ID NO:45); It connects to the transmembrane domain via a connector (connection sequence) GATCCCAAA: CD28TM sequence: TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG (SEQ ID NO: 46); CD28 cytoplasmic sequence: AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCCGACTTCGCAGCCTATCGCTCC (SEQ ID NO:47) CD137 (4-1BB) sequence: AAACGGGGCAGAAAGAAACTCCGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG (SEQ ID NO:48) CD3-ζ chain sequence: AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAG GCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA (SEQ ID NO:49).

[0083] According to one embodiment of the present invention, the nucleotide sequence or vector may further comprise a nucleotide sequence encoding a suicide gene-inducible amino acid sequence, which is linked to the 5' or 3' end of a nucleotide sequence encoding the chimeric antigen receptor via a nucleotide sequence encoding a 2A self-cleaving peptide.

[0084] Specifically, the suicide gene-induced amino acid sequence may contain or consist of a chimeric cysteine-9 polypeptide, herpes simplex virus thymidine kinase (HSV-TK), or ΔCD19 sequence, as a safety switch.

[0085] Therefore, in cells, the polynucleotide 2A self-cleaving peptide cleaves the peptide containing the suicide gene-inducible amino acid sequence and the chimeric antigen receptor into two separate peptides: the suicide gene-inducible amino acid sequence and the chimeric antigen receptor amino acid sequence.

[0086] Therefore, according to the present invention, the nucleotide sequence of the anti-GD2 chimeric antigen receptor, which also includes a suicide gene inducing sequence, may be: iC9.2A.GD2.CAR-28.4-1BB.ζ

[0087] That is, the nucleotide sequence SEQ ID NO:50 contains the following sequence: The iCasp9 sequence – an inducible caspase-9 expression cassette – is encoded by the following nucleotide sequence: T2A peptide (2A) sequence: GAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGGCCC (SEQ ID NO:34), Signal peptide: ATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGGTGTCCAGTGCTCTAGA (SEQID NO:35), Anti-GD2 (14G2a) VL sequence: GATATTTTGCTGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGTCTTGTACACCGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATTCACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGTTCTCAAAGTACACATGTTCCTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCA (SEQ ID NO:36), Anti-GD2 (14G2a) VH sequence: GAGGTGAAGCTTCAGCAGTCTGGACCTAGCCTGGTGGAGCCTGGCGCTTCAGTGATGATATCCTGCAAGGCTTCTGGTTCCTCATTCACTGGCTACAACATGAACTGGGTGAGGCAGAACATTGGAAAGAGCCTTGAATGGATTGGAGCTATTGATCCTTACTATGGTG GAACTAGCTACAACCAGAAGTTCAAGGGCAGGGCCACATTGACTGTAGACAAATCGTCCAGCACAGCCTACATGCACCTCAAGAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGTAAGCGGAATGGAGTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO:37), G7S2 connector (which connects the anti-GD2 (14G2a) VL sequence and the anti-GD2 (14G2a) VH sequence) sequence: GGCTCGGGCGGTGGTGGGTCGGGTGGC (SEQ ID NO:44); Hinge sequence based on IgG: GCCAAACGACACCCCCATCAGTCTATGGAAGGGTCACCGTCTCTTCAGCGGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCG (SEQ ID NO:45); It connects to the transmembrane domain via a connector (connection sequence) GATCCCAAA: CD28TM sequence: TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG (SEQ ID NO: 46); CD28 cytoplasmic sequence: AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCCGACTTCGCAGCCTATCGCTCC (SEQ ID NO:47) CD137 (4-1BB) sequence: AAACGGGGCAGAAAGAAACTCCGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG (SEQ ID NO:48) CD3-ζ chain sequence: AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAG GCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA (SEQ ID NO:49).

[0088] According to the present invention, a vector containing the nucleotide sequence as defined above may be selected from DNA vectors, RNA vectors, plasmids, lentiviral vectors, adenovirus vectors, retroviral vectors such as γ-retroviral vectors, or non-viral vectors.

[0089] The present invention also relates to cells for treating solid tumors, such as T cells, such as α / β and γ / δ T cells, NK cells, NK-T cells, and macrophages or monocytes, comprising anti-GD2 chimeric antigen receptors as defined above and / or vectors as defined above, wherein the solid tumor is not neuroblastoma, diffuse midline glioma (DMG) H3K27M mutant, or osteosarcoma.

[0090] Furthermore, according to one embodiment of the invention, the solid tumor is not a high-grade glioma. According to another embodiment of the invention, the solid tumor is not rhabdomyosarcoma or Ewing's sarcoma. According to one embodiment of the invention, the solid tumor is not a sarcoma.

[0091] According to another embodiment of the invention, the solid tumor is not a medulloblastoma. According to another embodiment of the invention, the tumor is not a glioma.

[0092] According to the present invention, the solid tumor to be treated is a tumor expressing GD2 or a tumor expressing GD2 after treatment with a compound capable of enhancing GD2 expression (e.g., an enhancer of a Zeste homolog 1 or 2 (EZH2) inhibitor).

[0093] As described above, the solid tumor can be selected from brain tumors that do not include the diffuse midline glioma (DMG) H3K27M- mutant, for example, those different from diffuse midline glioma (DMG). Diffuse intrinsic pontine glioma (DIPG) with H3K27M- mutant, adult diffuse glioma, pediatric diffuse low-grade glioma, pediatric diffuse high-grade glioma, astrocytic glioma, medulloblastoma, such as SHH, G3, G4 and WNT medulloblastoma subgroups, glial neuron and neuronal tumors, ependymal tumors, choroid plexus tumors; embryonal tumors, pineal gland tumors, cranial nerve and paravertebral nerve tumors; extracranial tumors, such as sarcomas distinct from osteosarcoma, such as rhabdomyosarcoma, especially alveolar rhabdomyosarcoma or embryonal rhabdomyosarcoma, Ewing sarcoma (EWS), desmoplastic small round cell tumors, leiomyosarcoma and liposarcoma; retinoblastoma; melanoma; lung cancer; gastric cancer; bladder cancer, head and neck cancer; ovarian cancer; breast cancer, pancreatic cancer; colorectal cancer or other tumors expressing GD2.

[0094] According to the present invention, the tumor can be a tumor in which GD2 expression is higher than that in a negative healthy control, i.e., in healthy peripheral blood mononuclear cells (PBMCs), wherein GD2 expression is measured as the percentage of GD2+ cells relative to the total number of tumor cells or healthy peripheral blood mononuclear cells (the percentage of GD2-positive cells in healthy PBMCs is equal to 0.43% ± 0.90%). Preferably, the tumor is a tumor in which GD2 expression is higher than 2%, i.e., the percentage of GD2+ cells is higher than 2%.

[0095] According to one embodiment of the invention, the brain tumor is not a high-grade glioma. According to another embodiment of the invention, the sarcoma is not rhabdomyosarcoma or Ewing's sarcoma. According to one embodiment of the invention, the solid tumor is not a sarcoma. According to another embodiment of the invention, the brain tumor is not a medulloblastoma.

[0096] According to one embodiment of the present invention, the cell may further contain a suicide gene-inducing amino acid sequence, such as a chimeric cysteine-9 polypeptide or herpes simplex virus thymidine kinase (HSV-TK) or ΔCD19 sequence, as a safety switch.

[0097] In particular, the chimeric cysteine-9 polypeptide may comprise or consist of the following: iCasp9 box: MLEGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGSGVDGFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALL ELARQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSAS (SEQ ID NO:1), which is connected to: via a connector such as RA: T2A peptide (2A): EGRGSLLTCGDVEENPGP (SEQ ID NO:2).

[0098] According to the present invention, the cells can be obtained under culture conditions in which two or one of IL-7 and / or IL-15 are present, for example, under the culture conditions of the activation step, transduction step and / or amplification step of the method for preparing the cells.

[0099] Another object of the present invention is a pharmaceutical composition for treating solid tumors comprising a nucleotide sequence as defined above, or a carrier as defined above, or a cell as defined above, and one or more pharmaceutically acceptable excipients and / or adjuvants, wherein the solid tumor is not neuroblastoma, diffuse midline glioma (DMG) H3K27M mutant, or osteosarcoma.

[0100] According to the present invention, the solid tumor to be treated is a tumor expressing GD2 or a tumor expressing GD2 after treatment with a compound capable of enhancing GD2 expression (e.g., an enhancer of a Zeste homolog 1 or 2 (EZH2) inhibitor).

[0101] Furthermore, according to one embodiment of the invention, the solid tumor is not a high-grade glioma. According to another embodiment of the invention, the solid tumor is not rhabdomyosarcoma or Ewing's sarcoma. According to one embodiment of the invention, the solid tumor is not a sarcoma.

[0102] According to another embodiment of the invention, the solid tumor is not a medulloblastoma.

[0103] As described above, according to the present invention, the solid tumor may be selected from brain tumors that do not include the diffuse midline glioma (DMG) H3K27M- mutant, for example, those different from diffuse midline glioma (DMG). Diffuse intrinsic pontine glioma (DIPG) with H3K27M- mutant, adult diffuse glioma, pediatric diffuse low-grade glioma, pediatric diffuse high-grade glioma, astrocytic glioma, medulloblastoma, such as SHH, G3, G4 and WNT medulloblastoma subgroups, glial neuron and neuronal tumors, ependymal tumors, choroid plexus tumors; embryonal tumors, pineal gland tumors, cranial nerve and paravertebral nerve tumors; extracranial tumors, such as sarcomas distinct from osteosarcoma, such as rhabdomyosarcoma, especially alveolar rhabdomyosarcoma or embryonal rhabdomyosarcoma, Ewing sarcoma (EWS), desmoplastic small round cell tumors, leiomyosarcoma and liposarcoma; retinoblastoma; melanoma; lung cancer; gastric cancer; bladder cancer, head and neck cancer; ovarian cancer; breast cancer, pancreatic cancer; colorectal cancer or other tumors expressing GD2.

[0104] According to one embodiment of the invention, the brain tumor is not a high-grade glioma. According to another embodiment of the invention, the sarcoma is not rhabdomyosarcoma or Ewing's sarcoma. According to one embodiment of the invention, the solid tumor is not a sarcoma. According to another embodiment of the invention, the brain tumor is not a medulloblastoma.

[0105] According to the present invention, the tumor can be a tumor in which GD2 expression is higher than that in a negative healthy control, i.e., in healthy peripheral blood mononuclear cells (PBMCs), wherein GD2 expression is measured as the percentage of GD2+ cells relative to the total number of tumor cells or healthy peripheral blood mononuclear cells (the percentage of GD2-positive cells in healthy PBMCs is equal to 0.43% ± 0.90%). Preferably, the tumor is a tumor in which GD2 expression is higher than 2%, i.e., the percentage of GD2+ cells is higher than 2%.

[0106] Another objective of this invention is the following combination: The anti-GD2 chimeric antigen receptor as defined above, or the nucleotide sequence as defined above, or the vector as defined above, or the cell as defined above, or the pharmaceutical composition as defined above, and An enhancer of a Zeste homolog 1 or 2 (EZH2) inhibitor, or a pharmaceutical composition comprising an enhancer of a Zeste homolog 1 or 2 (EZH2) inhibitor and one or more excipients and / or adjuvants. They can be used separately or sequentially to treat solid tumors.

[0107] According to the present invention, the solid tumor to be treated is a tumor expressing GD2 or a tumor that can express GD2 after treatment with an enhancer of a Zeste homolog 1 or 2 (EZH2) inhibitor.

[0108] In particular, the combination of the present invention can be advantageously used when the tumor does not express GD2 or has low GD2 expression. Indeed, according to the present invention, treatment with an EZH2 inhibitor increases GD2 expression in tumor cells; therefore, subsequent administration of the anti-GD2 CAR according to the present invention becomes effective against the tumor. More specifically, the EZH2 inhibitor can be administered 3 to 21 days, preferably 3 to 7 days, prior to administration of the anti-GD2 CAR.

[0109] According to the present invention, "separate use" is understood to mean the simultaneous administration of two compounds in different pharmaceutical forms according to the combination described in the present invention.

[0110] "Use in sequence" is understood to mean the sequential application of two compounds in the combination of the present invention, each of which is a different pharmaceutical form.

[0111] According to the inventive combination for use according to the present invention, the solid tumor may be selected from brain tumors, such as glioblastoma, glioma, or diffuse intrinsic pontine glioma (DIPG) (including diffuse midline glioma (DMG)). H3K27M-mutant) high-grade glioma, adult diffuse glioma, pediatric diffuse low-grade glioma, pediatric diffuse high-grade glioma, astrocytic glioma, medulloblastoma, such as SHH, G3, G4 and WNT medulloblastoma subgroups, glial neuron and neuronal tumors, ependymal tumors, choroid plexus tumors; embryonal tumors, pineal gland tumors, cranial nerve and paravertebral nerve tumors; extracranial tumors, such as sarcomas, especially sarcomas with low GD2 expression, such as osteosarcoma, rhabdomyosarcoma, especially alveolar rhabdomyosarcoma or embryonal rhabdomyosarcoma, Ewing sarcoma (EWS), desmoplastic small round cell tumors, leiomyosarcoma and liposarcoma; retinoblastoma; melanoma; lung cancer; breast cancer; bladder cancer; gastric cancer; head and neck cancer; ovarian cancer; neuroblastoma; pancreatic cancer; colorectal cancer.

[0112] According to the present invention, the enhancer of the Zeste homology 2 inhibitor can be selected from tazestat, CPI-1205 (an orally bioavailable, indole-based small molecule inhibitor of EZH2), etc. 68 ); PF-06821497 70 GSK126 (GSK2816126) 71The preferred inhibitors are tazexat and / or EZH1 / 2 dual inhibitors, such as valemetostat.

[0113] According to one embodiment of the invention, the enhancer of the Zeste homology 1 or 2 inhibitor may be administered at least 3 to at most 21 days, preferably 3 to 7 days, before the anti-GD2 chimeric antigen receptor, nucleotide sequence, carrier, cell or pharmaceutical composition.

[0114] The present invention also relates to a reagent kit, the components of which include or are composed of the following: A) an anti-GD2 chimeric antigen receptor as defined in any one of claims 1-14, or a nucleotide sequence or vector according to any one of claims 15-23, or a cell according to any one of claims 24-28, or a pharmaceutical composition according to any one of claims 29-30; and B) An enhancer of a Zeste homolog 1 or 2 (EZH2) inhibitor, or a pharmaceutical composition comprising an enhancer of a Zeste homolog 1 or 2 (EZH2) inhibitor and one or more excipients and / or adjuvants. A) and B) can be applied separately or in sequence.

[0115] According to the present invention, the enhancer of the Zeste homology 2 inhibitor can be selected from tazestat, CPI-1205 (an orally bioavailable, indole-based small molecule inhibitor of EZH2), etc. 68 ); PF-06821497 70 GSK126 (GSK2816126) 71 Tazestat is the preferred choice.

[0116] Another object of the present invention is an anti-GD2 chimeric antigen receptor comprising or composed of the following from the N-terminus to the C-terminus: a) Signal peptide, b) Anti-GD2 single-chain antibody domain, c) Hinges, d) Transmembrane structural domains e) At least two co-stimulatory signal transduction domains, and f) CD3ζ chain sequence, The anti-GD2 single-chain antibody domain comprises or consists of an anti-GD2 VL sequence and an anti-GD2 VH sequence connected to each other by a linker. The linker is a short, flexible, glycine-rich linker with a length of 7 to 14 amino acids, for example, 7 to 12, 7 to 10, or 8 amino acids. For example, the linker is selected from G7S2 linker GSGGGGSGG (SEQ ID NO:13), (G4S)2 linker GGGGSGGGG (SEQ ID NO:14), G4SG2 linker GGGGSGG (SEQ ID NO:15), G3SG4 linker GGGSGGGG (SEQ ID NO:16), SG4SG3 linker SGGGGSGGG (SEQ ID NO:17), (SG4)2S linker SGGGGSGGGGS (SEQ ID NO:18), and (SG4)2SG linker SGGGGSGGGGSG (SEQ ID NO:18). NO:19), (SG4)2SG3 connector SGGGGSGGGGSGGG connector (SEQ ID NO:20), (SG4)2 SGGGGSGGGG (SEQ ID NO:21) or (SG4)2 SG2 SGGGGSGGGGSGG (SEQ ID NO:22), preferably G7S2 connector GSGGGGSGG (SEQ ID NO:13).

[0117] Based on the aforementioned anti-GD2 chimeric antigen receptor, the anti-GD2 VL sequence may include the CDR1 sequence QSLVHRNGNTY (SEQ ID NO:5), the CDR2 sequence KVS, and the CDR3 sequence SQSTHVP (SEQ ID NO:7); while The anti-GD2 VH sequence may include the CDR1 sequence: GSSFTGYN (SEQ ID NO:8), the CDR2 sequence: IDPYYGGT (SEQ ID NO:9), and the CDR3 sequence: VSGMEY (SEQ ID NO:10).

[0118] According to one embodiment of the present invention, the anti-GD2 VL sequence may comprise or consist of the following: DILLTQTPLSLPVSLGDQASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELKRADAAPTVSIFP (SEQ ID NO:11), and The anti-GD2 VH sequence may contain or consist of the following: EVKLQQSGPSLVEPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSS (SEQ ID NO:12).

[0119] Regarding the hinge for the anti-GD2 chimeric antigen receptor according to the present invention, the hinge may comprise or consist of one or more of the following hinges: The hinge based on IgG has the following sequence: AKTTPPSVYGRVTVSSAEPKSCDKTHTCPPCP (SEQ ID NO:23); The hinge gap region -CD8α, has the sequence: PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA (SEQ ID NO:24); CD8 stem, sequence: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO:25); Hinge CD28, sequence: EVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO:26); The hinge CH2-CH3 sequence is as follows: ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:27); Hinge CH3, sequence: ESKYGPPCPSCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:28); The preferred sequence is an IgG-based hinge with the sequence SEQ ID NO:23.

[0120] According to the anti-GD2 chimeric antigen receptor of the present invention, the transmembrane domain may be selected from the group consisting of: CD8aTM: CDIYIWAPLAGTCGVLLLSLVIT (SEQ ID NO:6); CD28TM: FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO:29); preferably CD28TM with the sequence FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO:29).

[0121] According to the anti-GD2 chimeric antigen receptor of the present invention, the at least two co-stimulatory signal transduction domains may be selected from the group consisting of: The following sequences are obtained by concatenating: CD28 cytoplasmic sequence: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:30), with OX40 sequence: RDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO:32), wherein the CD28 cytoplasmic sequence is located before or after the OX40 sequence; The following sequences are obtained by concatenating: CD28 cytoplasmic sequence: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:30), with CD137 (4-1BB) sequence: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:31), wherein the CD28 cytoplasmic sequence is located before or after the CD137 (4-1BB) sequence; The following sequences are obtained by concatenating: OX40 sequence: RDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO:32), with CD28 cytoplasmic sequence: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:30), wherein the OX40 sequence is located before or after the CD28 cytoplasmic sequence; or The following sequences are obtained by concatenating: OX40 sequence: RDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO:32), with CD137 (4-1BB) sequence: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:31), wherein the OX40 sequence is located before or after the CD137 (4-1BB) sequence; Preferably, the following sequences are obtained by concatenating: CD28 cytoplasmic sequence: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:30), with CD137 (4-1BB) sequence: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:31), wherein the CD28 cytoplasmic sequence is located before the CD137 (4-1BB) sequence.

[0122] According to the present invention, the CD3-ζ chain sequence can be: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR* (SEQ ID NO:53).

[0123] Furthermore, the anti-GD2 chimeric antigen receptor according to the present invention may also include a CD8cyt cytoplasmic portion with the sequence LYCNHRN (SEQ ID NO:51) between the transmembrane domain and the co-stimulatory signal transduction domain.

[0124] According to the anti-GD2 chimeric antigen receptor of the present invention, the signal peptide may comprise or consist of MEFGLSWLFLVAILKGVQCSR (SEQ ID NO:3).

[0125] According to one embodiment of the present invention, the anti-GD2 chimeric antigen receptor comprises or consists of the following sequences: GD2.CAR-28.4-1BB.ζ * (SEQ ID NO:39).

[0126] Specifically, sequence SEQ ID NO:39 contains Signal peptide: MEFGLSWLFLVAILKGVQCSR (SEQ ID NO:3); Anti-GD2(14G2a) VL sequence: DILLTQTPLSLPVSLGDQASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELKRADAAPTVSIFP (SEQ ID NO: 11).

[0127] G7S2 connector: GSGGGGSGG (SEQ ID NO:13) Anti-GD2(14G2a) VH sequence: EVKLQQSGPSLVEPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSS (SEQ ID NO: 12).

[0128] IgG-based hinge: AKTTPPSVYGRVTVSSAEPKSCDKTHTCPPCP (SEQ ID NO:23) It connects to the transmembrane structural domain via a connector (connection sequence) DPK: CD28TM: FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO:29) CD28 cytoplasmic sequence: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:30) CD137 (4-1BB) sequence: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:31) CD3-ζ chain: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR* (SEQ ID NO:53).

[0129] The present invention also relates to nucleotide sequences or vectors containing such nucleotide sequences, said nucleotide sequences comprising, or consisting of, nucleotide sequences encoding, the anti-GD2 chimeric antigen receptor as defined above.

[0130] In particular, the present invention relates to nucleotide sequences or vectors as described above, wherein... The anti-GD2 VL sequence can be encoded by the following nucleotide sequence: GATATTTTGCTGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGTCTTGTACACCGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATTCACAAAGTTTCCAACCGATTTTCTGGGG TCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGTTCTCAAAGTACACATGTTCCTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCA (SEQ ID NO:36), and The anti-GD2 VH sequence can be encoded by the following nucleotide sequence: GAGGTGAAGCTTCAGCAGTCTGGACCTAGCCTGGTGGAGCCTGGCGCTTCAGTGATGATATCCTGCAAGGCTTCTGGTTCCTCATTCACTGGCTACAACATGAACTGGGTGAGGCAGAACATTGGAAAGAGCCTTGAATGGATTGGAGCTATTGATCCTTACTATGGTG GAACTAGCTACAACCAGAAGTTCAAGGGCAGGGCCACATTGACTGTAGACAAATCGTCCAGCACAGCCTACATGCACCTCAAGAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGTAAGCGGAATGGAGTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO:37).

[0131] Specifically, the nucleotide sequence may be: GD2.CAR-28.4-1BB.g

[0132] More specifically, sequence SEQ ID NO:52 contains: Signal peptide: ATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGTGTCCAGTGCTCTAGA (SEQ ID NO:35), G7S2 connector (which connects GD2 (14G2a) VL sequence and GD2 (14G2a) VH sequence): GGCTCGGGCGGTGGTGGGTCGGGTGGC (SEQ ID NO:44); Hinge sequence based on IgG: GCCAAACGACACCCCCATCAGTCTATGGAAGGGTCACCGTCTCTTCAGCGGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCG (SEQ ID NO:45); It connects to the transmembrane domain via a connector (connection sequence) GATCCCAAA: CD28TM sequence: TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG (SEQ ID NO: 46); CD28 cytoplasmic sequence: AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCCGACTTCGCAGCCTATCGCTCC (SEQ ID NO:47) CD137 (4-1BB) sequence: AAACGGGGCAGAAAGAAACTCCGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG (SEQ ID NO:48) CD3-ζ chain sequence: AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAG GCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA (SEQ ID NO:49).

[0133] According to the present invention, the nucleotide sequence or vector may further comprise a nucleotide sequence encoding a suicide gene-inducible amino acid sequence, which is linked by a nucleotide sequence encoding a 2A self-cleaving peptide to a nucleotide sequence encoding the chimeric antigen receptor. Specifically, the suicide gene-inducible amino acid sequence may be a chimeric cysteine-9 polypeptide, or may comprise a herpes simplex virus thymidine kinase or ΔCD19 sequence as a safety switch.

[0134] Therefore, in cells, the polynucleotide 2A self-cleaving peptide cleaves the peptide containing the suicide gene-inducing amino acid sequence and the chimeric antigen receptor into two separate peptides: the suicide gene-inducing amino acid sequence and the chimeric antigen receptor amino acid sequence.

[0135] Therefore, the nucleotide sequence according to the present invention can be: iC9.2A.GD2.CAR-28.4-1BB.ζ

[0136] That is, the nucleotide sequence SEQ ID NO:50 contains the following sequence: The iCasp9 sequence – an inducible caspase-9 expression cassette – is encoded by the following nucleotide sequence: T2A peptide (2A) sequence: GAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGGCCC (SEQ ID NO:34), Signal peptide: ATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGGTGTCCAGTGCTCTAGA (SEQID NO:35), Anti-GD2 (14G2a) VL sequence: GATATTTTGCTGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGTCTTGTACACCGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATTCACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGTTCTCAAAGTACACATGTTCCTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCA (SEQ ID NO:36), Anti-GD2 (14G2a) VH sequence: GAGGTGAAGCTTCAGCAGTCTGGACCTAGCCTGGTGGAGCCTGGCGCTTCAGTGATGATATCCTGCAAGGCTTCTGGTTCCTCATTCACTGGCTACAACATGAACTGGGTGAGGCAGAACATTGGAAAGAGCCTTGAATGGATTGGAGCTATTGATCCTTACTATGGTG GAACTAGCTACAACCAGAAGTTCAAGGGCAGGGCCACATTGACTGTAGACAAATCGTCCAGCACAGCCTACATGCACCTCAAGAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGTAAGCGGAATGGAGTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO:37), G7S2 connector (which connects the anti-GD2 (14G2a) VL sequence and the anti-GD2 (14G2a) VH sequence) sequence: GGCTCGGGCGGTGGTGGGTCGGGTGGC (SEQ ID NO:44); Hinge sequence based on IgG: GCCAAACGACACCCCCATCAGTCTATGGAAGGGTCACCGTCTCTTCAGCGGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCG (SEQ ID NO:45); It connects to the transmembrane domain via a connector (connection sequence) GATCCCAAA: CD28TM sequence: TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG (SEQ ID NO: 46); CD28 cytoplasmic sequence: AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCCGACTTCGCAGCCTATCGCTCC (SEQ ID NO:47) CD137 (4-1BB) sequence: AAACGGGGCAGAAAGAAACTCCGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG (SEQ ID NO:48) CD3-ζ chain sequence: AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAG GCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA (SEQ ID NO:49).

[0137] According to the present invention, the vector may be selected from DNA vectors, RNA vectors, plasmids, lentiviral vectors, adenovirus vectors, retroviral vectors such as γ-retroviral vectors, or non-viral vectors.

[0138] Furthermore, the present invention also relates to cells, such as T cells, including α / β and γ / δ T cells, NK cells, NK-T cells, and macrophages or monocytes, comprising the anti-GD2 chimeric antigen receptor according to claims 35-44 and / or the vector according to claims 45-50. According to the present invention, the cells may also comprise a suicide gene-inducing amino acid sequence, such as a chimeric cysteine-9 polypeptide or herpes simplex virus thymidine kinase (HSV-TK) or ΔCD19 sequence, as a safety switch. In particular, the chimeric cysteine-9 polypeptide may comprise or consist of the following: iCasp9 box: MLEGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGSGVDGFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALL ELARQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSAS (SEQ ID NO: 1) It is connected to: via a connector such as RA. T2A peptide (2A): EGRGSLLTCGDVEENPGP (SEQ ID NO: 2).

[0139] According to the present invention, the cells can be obtained under culture conditions in which two or one of IL-7 and / or IL-15 are present, for example, under the culture conditions of the activation step, transduction step and / or amplification step of the method for preparing the cells.

[0140] The present invention also relates to pharmaceutical compositions comprising the nucleotide sequence or carrier according to claims 45-50, or the cell according to claims 51-53, and one or more pharmaceutically acceptable excipients and / or adjuvants.

[0141] The present invention also relates to a reagent kit, the components of which include or are composed of the following: A) an anti-GD2 chimeric antigen receptor as defined in any one of claims 35-44, or a nucleotide sequence or vector according to any one of claims 45-50, or a cell according to any one of claims 51-53, or a pharmaceutical composition according to claim 54; and B) An enhancer of a Zeste homolog 1 or 2 (EZH2) inhibitor, or a pharmaceutical composition comprising an enhancer of a Zeste homolog 1 or 2 (EZH2) inhibitor and one or more excipients and / or adjuvants. A) and B) can be applied separately or in sequence.

[0142] According to the present invention, the enhancer of the Zeste homology 2 inhibitor can be selected from tazestat, CPI-1205 (an orally bioavailable, indole-based small molecule inhibitor of EZH2), etc. 68 ); PF-06821497 70 GSK126 (GSK2816126) 71 Tazestat is the preferred choice.

[0143] Furthermore, the present invention relates to the anti-GD2 chimeric antigen receptor according to claims 35-44, the nucleotide sequence or vector according to any one of claims 45-50, the cell according to any one of claims 51-53, the pharmaceutical composition according to claim 54, or the kit according to any one of claims 55-56, for medical use.

[0144] Specifically, the anti-GD2 chimeric antigen receptor, nucleotide sequence, vector, cell, pharmaceutical composition, or kit can be used to treat neuroblastoma, diffuse midline glioma (DMG) H3K27M- mutant, including diffuse intrinsic pontine glioma (DIPG), and osteosarcoma.

[0145] According to one embodiment of the invention, the use is also for treating high-grade gliomas. According to one embodiment of the invention, the use is also for rhabdomyosarcoma or Ewing sarcoma. According to another embodiment of the invention, the use is also for sarcomas, such as osteosarcoma, rhabdomyosarcoma, or Ewing sarcoma. According to one embodiment of the invention, the use is also for treating medulloblastoma. According to one embodiment of the invention, the anti-GD2 chimeric antigen receptor is used to treat gliomas.

[0146] In other words, the anti-GD2 chimeric antigen receptor, nucleotide sequence, vector, cell or pharmaceutical composition can be used to treat all solid tumors, especially solid tumors with higher GD2 expression than healthy peripheral blood mononuclear cells, wherein GD2 expression is measured as the percentage of GD2+ cells relative to the total number of tumor cells or healthy peripheral blood mononuclear cells. Attached Figure Description

[0147] The invention will now be described in an illustrative rather than restrictive manner, with particular reference to the embodiments and accompanying drawings, wherein: Figure 1 (AE) iC9.2A.GD2.CAR-28.4-1BB.ζ T(NK) cells co-stimulated with CD28.4-1BB exhibited high and stable transduction levels and in vitro proliferation upon initial antigen stimulation. (A) Figure 1 A shows the expression cassette of the retroviral vector SFG.iC9.2A.CAR-GD2(14g2a)CD8tm.CD28.41BBζ (iC9.2A.GD2.CAR-28.4-1BB.ζ). The scFv of GD2 is cloned in the same frame as CD8aTM, the cytoplasmic portion of CD28, and the second co-stimulatory domain represented by 4-1BB and the signal transduction domain CD3-ζ chain (ζ). (B) Growth of untransduced (NT) T cells (1B, left panel) as a negative control, or GD2.CAR-28.4-1BBζ (GD2.CAR) T cells (1B, right panel) transduced with the iC9.2A.GD2.CAR-28.4-1BB.ζ retroviral vector, in IL7 / IL15, by expression in exemplary donors. Flow cytometry analysis showed the transduction level of T cells. (C) Figure 1 Figure (C) shows the mean percentage of positive GD2.CAR T cells analyzed by FACS. Data are expressed as the mean standard deviation (SD) of six healthy donors (HD) at day 15 of in vitro culture. Figure (D) shows a plot of the fold increase in IL7 / IL15 (D) of NT T- cells and GD2.CAR T cells, evaluated by trypan blue counting. Figure (E) shows the mean percentage of positive GD2.CAR NK cells analyzed by FACS at days 5 and +25 of in vitro culture. Data are expressed as the mean standard deviation (SD) of six healthy donors (HD). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

[0148] Figure 2(AB). GD2 expression in human sarcoma cell lines. (A) GD2 expression (percentage) in human osteosarcoma cell lines: 143B, MG-63, U-2OS, HOS, and SAOS-2; in embryonic rhabdomyosarcoma (ERMS) cell line: RD; in alveolar rhabdomyosarcoma (ARMS) cell lines: RH4, RH41, and RH30; and in Ewing sarcoma (EwS) cell lines: A673 and SKES1, assessed by flow cytometry. Data are presented as percentage ± SD of four different replicates. (B) Median fluorescence intensity (MFI) of GD2 expression in human sarcoma cell lines. Data are presented as mean ± SD of four different replicates. (CD) GD2 expression in melanoma cell lines. (C) Representative FACS analysis of GD2 expression in the melanoma A375 cell line (CRL-1619 / ATCC). (D) Flow cytometry analysis in triplicate. Data are presented as mean ± SD. (EF). GD2 expression in lung adenocarcinoma cell lines. (C) Representative FACS analysis of GD2 expression in the lung adenocarcinoma cell line H2228 (CRL-5935 / ATCC). (D) Triple-copy flow cytometry analysis. Data are presented as mean ± SD. (G). GD2 expression in primary desmoplastic small round cell (DSRCT) tumors. GD2 expression in pediatric DSRCT tissues, analyzed by facs (e.g., CD45). neg GD2+ rating.

[0149] Figure 3 (AJ). Long-term in vitro co-culture assay to evaluate the functional activity of GD2.CAR-28.4-1BBζ (GD2.CAR) T cells. Residual tumor cells were expressed as the percentage of residual tumor cells after 5 days of co-culture with NT or GD2.CAR-28.4-1BBζ T cells at an E:T ratio of 1:1; NT (black bars) and GD2.CAR-28.4-1BBζ T cells (white bars). Tumor cells: MG63 tumor cell line (A), 143B cell line (B), U-2OS cell line (C), HOS cell line (D), RD cell line (E), RH4 cell line (F), RH41 cell line (G), A673 cell line (H), A375 cell line (I), and H2228 cell line (J) were used as targets. Data from 6 healthy donors (HD) are presented as mean ± SD in AJ; *p value = < 0.05; **p value = < 0.01; ***p value = < 0.001 and ****p value = < 0.0001.

[0150] Figure 4Inhibition of EZH2 upregulates surface GD2 expression in EwS cells. Flow cytometry analysis showed GD2 expression (percentage and MFI) in human EwS cells treated with 1–10 μM tazestat for 7 days. DMSO was included as a negative control.

[0151] Figure 5 (AB) Inhibition of EZH2 upregulation of surface GD2 expression in ARMS cells. Flow cytometry analysis showed the percentage and MFI of GD2 expression in RH4 (A) and RH41 (B) cell lines treated with 1–10 μM tazestat for 7 days. DMSO was included as a negative control.

[0152] Figure 6 Inhibition of EZH2 upregulates surface GD2 expression in osteosarcoma (OS) cells. Flow cytometry analysis showed GD2 expression (percentage and MFI) in HOS cell lines treated with 1–10 μM tazestat for 7 days. DMSO was included as a negative control.

[0153] Figure 7 (AD). Pretreatment with an EZH2 inhibitor sensitized EwS, RMS, and OS cell lines to in vitro cytolysis of GD2.CAR-28.4-1BBζ (GD2.CAR) T cells. Residual tumor was expressed as the percentage of residual tumor cells after 5 days of co-culturing NT or GD2.CAR-28.4-1BBζ T cells with sarcoma cell lines pretreated with 1–10 μM tazestat for 7 days at a 1:1 E:T ratio. Black bars represent sarcoma cells not treated with tazestat; checkerboard bars represent sarcoma cell lines pretreated with 1 μM tazestat; and dotted bars represent sarcoma cell lines pretreated with 10 μM tazestat. Sarcoma cell lines A-673 (Ewing sarcoma) (A), RH4 (ARMS) (B), RH41 (ARMS) (C), and HOS (OS) (D) were used as targets. Data from the four HDs are represented as mean ± SD in the AH; *p value = < 0.05; **p value = < 0.01; ***p value = < 0.001 and ****p value = < 0.0001.

[0154] Figure 8 (AI). In vivo bioluminescence imaging of NSG mice carrying intravenously injected GD2+ RD-FF-Luc.GFP cells treated with NT or GD2.CAR-28.4-1BBζ T cells generated and expanded in the presence of IL7 / IL15. (A) Schematic diagram of the in vivo experiment. Mice received intravenous injections of 0.5 x 10⁻⁶ cells. 6RD-FF-Luc.GFP cells were used, and after 3 days, when bioluminescence stabilized, they were divided into two groups and treated with either NT or GD2.CAR-28.4-1BBζ T cells. Tumor growth was evaluated weekly by IVIS for 76 days. (B) Bioluminescence imaging of tumor growth measured weekly from day 3 to day 76; (C) Bioluminescence of each mouse treated with NT (black line; 10 mice) and IIICAR.GD2 T cells (black dashed line; 10 mice). (D) The figure shows the mean tumor bioluminescence in mice treated with either NT (black line) or IIICAR.GD2 T cells (black dashed line). (E) Kaplan-Meier estimate of overall survival (OS) in tumor-bearing mice treated with either NT (black line; 10 mice) or GD2.CAR-28.4-1BBζ T cells (black dashed line; 10 mice). * p < 0.05; log-rank (Mantel-Cox). (FI) CD45+CD3+ cells (F); the mean of human circulating T cells as CAR T cells (G), CD4+(CD4+CAR+) T cells (H), and CD8+(CD8+CAR+) T cells (I) was assessed as %. * p value = <0.05; ** p value = <0.01; *** p value = <0.001; **** p value = <0.0001, t-test.

[0155] Figure 9 (AI). In vivo bioluminescence imaging of NSG mice carrying orthotopic GD2+ 143B-FF-Luc.GFP cells treated with NT or GD2.CAR-28.4-1BBζ T cells generated and expanded in the presence of IL7 / IL15. (A) Schematic diagram of the in vivo experiment. Using 0.5 x 10 6GD2+ 143B-FF-Luc.GFP cells were inoculated into the tibias of mice, and after 3 days, when bioluminescence stabilized, they were divided into two groups and treated with either NT or GD2.CAR-28.4-1BBζ T cells. Tumor growth was evaluated weekly by IVIS for 35 days. (B) Bioluminescence imaging of tumor growth measured weekly from day 3 to day 35; (C) Bioluminescence of each mouse treated with NT (black line; 10 mice) and IIICAR.GD2 T cells (black dashed line; 10 mice). (D) The figure shows the mean tumor bioluminescence in mice treated with either NT (black line) or GD2.CAR-28.4-1BBζ T cells (black dashed line). (E) Kaplan-Meier estimate of overall survival (OS) in tumor-bearing mice treated with either NT (black line; 10 mice) or GD2.CAR-28.4-1BBζ T cells (black dashed line; 10 mice). **p<0.01; log-rank (Mantel-Cox). (FI) CD45+CD3+ cells (F); human circulating T cells as CAR T cells (G), CD4+(CD4+CAR+) T cells (H) and CD8+(CD8+CAR+) T cells (I) were evaluated as %. * p=<0.05; ** p=<0.01, t-test.

[0156] Figure 10 (AB). Expression of GD2 in human neuroblastoma cell lines and primary tumors. (A) Percentage of GD2 in human neuroblastoma cell lines. (B) % GD2 expression in primary neuroblastoma tumors. Normal peripheral blood cells (Pb) from each patient served as negative controls. ****p-value < 0.0001, t-test.

[0157] Figure 11 GD2 expression in retinoblastomas derived from primary tissue and patient tissue. GD2 expression in pediatric retinoblastoma tissue was evaluated by facs analysis (e.g., CD45negGD2+). Normal peripheral blood cells (Pb) from each patient served as a negative control. ****p-value < 0.0001, t-test.

[0158] Figure 12(AB). GD2 is highly expressed in pediatric MB patients. (A) Percentage of GD2 expression levels on cells from tissue biopsies and PBs obtained from pediatric MB patients at diagnosis, assessed by flow cytometry analysis. (B) MFI of GD2 on cells from tissue biopsies and PBs obtained from pediatric MB patients at diagnosis. MFI was adjusted for cell size by dividing MFI by forward scattering (FSC). (C) Percentage of GD2-positive cells and GD2 MFI (D) in MB samples, considering genetic classification (SHH, G3, G4, and WNT subgroups). (E) Percentage of GD2 and MFI (F) expression levels, considering histological classification (classical, large cell / anaplastic (LCA), extensive nodular (MBEN), and connective tissue hyperplasia / nodular (DN) subgroups). Data are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

[0159] Figure 13 Representative FACS analysis of GD2 expression in representative patients from the SHH (Figure I), G3 (Figure II), G4 (Figure III), and WNT (Figure IV) subgroups. Isotype controls are shown in dark gray.

[0160] Figure 14 (AC). GD2 is highly expressed in G3 / G4 MB cell lines. (A) Cytofluorescence analysis of GD2 expression in MB cell lines: D238 Med and DAOY. Fluorescence minus one (FMO) and isotype controls are shown in white. (B) Percentage of GD2 expression levels in MB cell lines (D238 Med and DAOY) assessed by flow cytometry in triplicate. Data are shown as mean ± SD. (C) MFI of GD2 in MB cell lines (D238 Med and DAOY). MFI was adjusted for cell size by dividing the MFI by forward scattering (FSC). Data are shown as mean ± SD.

[0161] Figure 15(AD). CAR.GD2 T cells targeted human GD2+MB cell lines cultured in vitro. (A, C) Long-term co-culture for 5 days was performed in five independent experiments, in which GD2+MB cell lines D283 Med (A) and DAOY (C) were co-cultured with NT-T or GD2.CAR-28.4-1BBζ (CAR.GD2) cells derived from HD at an E:T ratio of 1:1. Residual tumor is expressed as the percentage of GD2+ cells. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (B, D) Granz B, IFN-γ, IL-2, and TNF-α in the 24-h culture supernatant of NT-T or CAR.GD2 T cells from GD2+ MB cell lines D283 (B) and DAOY (D) were measured by ELISA. Cytokine quantification data are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0162] Figure 16 (AB) FACS plots of the exemplary experiments. After 5 days of long-term co-culture, the GD2+MB cell line DAOY was seeded alone or co-cultured with NT-T or GD2.CAR-28.4-1BBζ (CAR.GD2) T cells at a 1:1 E:T ratio. Residual tumor was evaluated by FACS analysis and expressed as the percentage of residual tumor (A) or GD2 MFI (B).

[0163] Figure 17(AF). An orthotopic mouse model of the human D283Med-GFP-FF-Luc cell line evaluating the antitumor activity of GD2.CAR-28.4-1BBζ (CAR.GD2) T cells. (A) Description of the experimental setup for stereotactic implantation of the D283Med-GFP-FF-Luc cell line into the cerebellum of NSG mice. Effector T cells were administered intravenously 3 days later. (BC) Time course of in vivo bioluminescence imaging for each treated NSG mouse (B) starting from day 3 (effector T cell infusion day). (C) Figure showing the mean in vivo bioluminescence of mice treated with NT-T (black line) and CAR.GD2 T cells (black dashed line). Data are shown as mean ± SD. *p < 0.05, ****p < 0.0001. (D) Disease-free survival (DFS) of tumor-bearing NSG mice treated with NT-T (black line) or CAR.GD2 T cells (black dashed line). *p < 0.05. (EF) The mean percentage of human circulating T cells in NSG mice treated with NT T cells (red line) or CAR.GD2-T cells (black dashed line) as CD45+CD3+ cells (E) and CD3+CAR+ cells (F) at days 15, 30, and 45 following effector T cell infusion. Data are shown as mean ± SD. ****p<0.0001.

[0164] Figure 18 (AC). Bioluminescence analysis of each tumor-bearing mouse treated with NT-T (circled black lines) or GD2.CAR-28.4-1BBζ (CAR.GD2) T cells (triangular dashed black lines) is illustrated in (A). (B) CD4 counts in tumor-bearing mice treated with NT-T at pre-infusion day, day 15, and day 30. + and CD8 + Flow cytometry analysis of subpopulation cells. (C) CD4 in tumor-bearing mice treated with CAR.GD2-T on pre-infusion day, day 15, and day 30. + and CD8 + Flow cytometry analysis of subpopulations of cells. Data are presented as mean ± SD.

[0165] Figure 19(AC). Efficacy evaluation of AP1903 on circulating CAR.GD2 T cells in an in vivo MB model. (A) Description of the experimental design for stereotactic implantation of MB cell lines into the cerebellum of NSG mice. Mice were infused with effector T cells intravenously 30 days after tumor transplantation. Mice received three consecutive doses of dimerized AP 1903 (100 mg / mouse) on days 11, 12, and 13. Flow cytometry analysis of circulating human T cells in mouse blood was performed one day before AP1903 administration and at the end of the experiment (day 14). (B) AP1903 effectively induced a strong reduction in circulating GD2.CAR-28.4-1BBζ (CAR.GD2) T cells. Exemplary CAR.GD2 T cells in mice untreated with AP1903 (top) or treated with AP1903 (bottom) are shown in the figure. CAR expression was assessed using an anti-idiotypic (1A7) (PE) monoclonal antibody in combination with an anti-hCD3 (APC) monoclonal antibody. (C) Human CD3 in MB-carrying mice that received untreated and AP1903-treated CAR.GD2-T cells + Flow cytometry analysis of T cells (left). Right shows circulating CD3 in MB-carrying mice that received CAR.GD2-T cells, were not treated with AP1903, and were treated with AP1903. + CAR.GD2 + Flow cytometry analysis.

[0166] Figure 20 (AC). Efficacy evaluation of AP1903 in GD2.CAR-28.4-1BBζ (CAR.GD2) T cell-infiltrating tumors in an in vivo MB model. NSG mice stereotactically implanted with MB cells. After tumor establishment, mice were infused with effector T cells. When effector T cells were detectable in the PB, mice were given dimerized AP1903 for three consecutive days and sacrificed to collect the cerebellum. (A) shows an exemplary H&E-stained section (top) of control MB tissue in the cerebellum of NSG mice, a representative image of Ki67 immunohistochemical staining (middle) and a representative image of human tumor-infiltrating CD3+ cells (bottom). (A) shows an exemplary H&E-stained section (top) of MB tissue in the cerebellum of NSG mice treated with CAR.GD2 T cells, a representative image of Ki67 immunohistochemical staining (middle) and a representative image of human tumor-infiltrating CD3+ cells (bottom). (A) shows an exemplary H&E stained section (top) of MB tissue in the cerebellum of NSG mice treated with CAR.GD2-T cells and AP1903, a representative image of Ki67 immunohistochemical staining (middle) and a representative image of human tumor-infiltrating CD3+ cells (bottom).

[0167] Figure 21 (AB) Representative images of H&E-stained sections of MB tissue in the cerebellum of NSG mice that received NTT cells, were untreated (top left, A), or treated with AP1903 (top right, B). (CD) Representative images of Ki67 immunohistochemical staining in the cerebellum of NSG mice that received NTT cells, were untreated (left, middle, C), or treated with AP1903 (right, middle, D). (EF) Representative images of human tumor-infiltrating CD3+ cells in NSG mice that received NTT cells, were untreated (bottom left, E), or treated with AP1903 (bottom right, F). Magnification used: 5X and 20X.

[0168] Figure 22 (AB). Inhibition of EZH2 upregulation of surface GD2 expression in DAOY (SHH subset) MB cells. (A) Flow cytometry analysis of GD2 expression (percentage and MFI) in DAOY (A) MB cell lines treated with 1–10 μM tazestat for 7 days. DMSO was included as a negative control. (B) Residual tumor cells (%) were expressed as a percentage of residual tumor cells after 5 days of co-culturing NT or GD2.CAR-28.4-1BBζ (GD2.CAR) T cells with DAOY MB cell lines pretreated with 1 or 10 μM tazestat for 7 days at a 1:1 E:T ratio. Black bars represent untreated MB cells; checkerboard bars represent MB cells pretreated with 1 μM tazestat; and white bars represent MB cells pretreated with 10 μM tazestat. Data from the four HDs in B are presented as mean ± SD; p-value < 0.0001.

[0169] Figure 23 Evaluation of the long-term persistence of GD2.CAR-28.4-1BBζ (CAR.GD2) T cells in an orthotopic mouse model of the human SHSY5Y-Luc cell line. (A) Effector T cells were administered FF-luciferase via intravenous injection. The persistence of effector T cells (NT FF-luciferase T cells, top panel; GD2.CAR-28.4.OX40.ζ FF-luciferase T cells, middle panel; and GD2.CAR-28.4-1BBζ FF-luciferase T cells, bottom panel) was measured from day 0 to day 103 using an IVIS bioluminescence system. (B) GD2.CAR-28.4-1BBζ T cells had a significantly longer persistence than (C) third-generation GD2-CAR T cells (including CD28 and OX40 as co-stimulatory domains) and (D) non-transduced (NT) T FF-luciferase T cells. Detailed Implementation

[0170] Example 1: In vitro and in vivo evaluation of GD2.CAR effector cells according to the invention, with or without the use of a enhancer of a Zeste homolog 2 (EZH2) inhibitor, for the treatment of GD2+ solid tumors, including brain tumors.

[0171] method Healthy donor (HD) and patient samples Patient tissues were collected from the pediatric patient cohort at Bambino Gesù Children's Hospital (OPBG). Patient tissue and blood samples were collected from patients and healthy donors, and GD2 expression was assessed by cellular fluorescence analysis.

[0172] cell lines OS cell lines: 143B (ATCC-CRL-8303), MG-63 (ATCC-CRL-1427), U-2 OS (ATCC HTB-96), HOS (ATCC CRL-1543), and SAOS-2 (ATCC HTB-85) were obtained from LGC Standards SrL, Milan (MI), Italy. Embryonic rhabdomyosarcoma (ERMS) cell line RD (ATCC CCL136) and EWS cell lines: A-673 (ATCC® CRL-1598) and SKES1 (ATCC HTB86) were obtained from LGC Standards SrL, Milan (MI), Italy. ERMS cell line RD was obtained from ATCC (CCL-136), and alveolar rhabdomyosarcoma (ARMS): RH41 and RH30 were obtained from DSMZ.

[0173] The melanoma A375 cell line (CRL-1619 / ATCC) and the lung adenocarcinoma cell line H2228 (CRL-5935 / ATCC) were obtained from LGC Standards SrL, Milan (MI), Italy.

[0174] The melanoma A375 cell line (CRL-1619 / ATCC) and the lung adenocarcinoma cell line H2228 (CRL-5935 / ATCC) were obtained from LGC Standards SrL, Milan (MI), Italy.

[0175] All cell lines were identified by STR analysis in the certified laboratory “BMR Genomics srl” and tested for mycoplasma every 15 days.

[0176] The D283 Med (G3 / G4 subgroup) and DAOY (SHH subgroup) MB cell lines were obtained from the American Type Culture Collection (ATCC, USA). DAOY and D283 Med cells were cultured under conditions recommended by the supplier (ATCC). Cells were maintained at 37°C in a humid environment with 5% CO2, and surface expression of mycoplasma and target antigens was routinely tested. All cell lines were identified by STR analysis at the accredited laboratory “BMR Genomics srl”. The identity and subgroup of each MB cell line were verified by methylation analysis.

[0177] Retroviral vector The third-generation retroviral vector CAR-GD2.CD28.4-1BBζ (CAR.GD2) includes the inducible cysteine ​​9 (iC9) suicide gene (as a safety switch) and an anti-GD2 single-stranded variable fragment (scFv) 14.G2a with two co-stimulatory domains fused to the CD3-ζ chain, for transducing T cells. 51 Additional retroviral vectors carrying eGFP-firefly-luciferase (eGFP-FFLuc) were used to genetically modify effector T cells, SHSY5Y or D283 Med and DAOY MB cell lines, respectively, for in vitro and / or in vivo studies.

[0178] Production of CAR-T cells Peripheral blood mononuclear cells (PBMCs) were isolated from erythrocyte sedimentation rate (ESR) brown-yellow layer (BC) obtained from healthy donors (HDs) who had signed written informed consent (OPBG Hospital, Rome, Italy) according to the rules established by the OPBG Institutional Review Committee (Ethics Committee approval N969 / 2015 prot. N 669LB). T lymphocytes were activated in the presence of recombinant human interleukin-7 (IL7, 10 ng / ml; R&D; USA) and interleukin-15 (IL15, 5 ng / ml; R&D) using OKT3 (1 mg / ml, e-Bioscience Inc; San Diego, CA, USA) and anti-CD28 (1 mg / ml, BD Biosciences, Europe) monoclonal antibody (mAb). This process was similar to that described by Quintarelli et al. 51As previously described, activated T cells were transduced with CAR-GD2CD28.4-1BBζ retrovirus supernatant on day 3 to obtain CAR.GD2 T cells, which were expanded in a medium containing 45% RPMI 1640, 45% Click medium (Sigma-Aldrich, Co.; USA), supplemented with 10% inactivated -FBS North (Gibco, ThermoFischer) and 2mM GlutaMax (ThermoFischer, Paisley, Scotland), and supplemented twice a week.

[0179] Immunophenotypic analysis Cell surface expression of GD2 was analyzed in fresh tumor cells and peripheral blood (PB) from MB patients using conjugated mouse anti-human GD2-BV421 mAb (BD Biosciences, USA). Cells were also stained with live / dead dyes, using 7-amino-actinomycin D (7-AAD) staining solution (BD Biosciences, Italy); CD45 APC (BD Biosciences, USA); and the target cell population was stained in CD45-negative (CD45... - Single-cell gating is gated.

[0180] Expression of CAR.GD2 on T cells was detected using a specific primary anti-idiotype antibody (1A7), followed by evaluation using a rat anti-mouse secondary antibody (PE) combined with a CD3-specific mAb. Blood was collected periodically from treated mice for FACS analysis. Mouse PB cells were stained with anti-human CD45 APC (BD Biosciences, USA), anti-human CD3 Pecy7 (BD Biosciences, USA), anti-human CD4 BUV605, and anti-human CD8 BUV395 (BD Biosciences, USA) combined with the primary 1A7 anti-idiotype antibody after lysing erythrocytes with lysis buffer (BD). Cells were incubated with the mAb (4°C in the dark for 30 min), washed with 1x phosphate-buffered saline (1x PBS), and analyzed using a FACS-Fortessa flow cytometer. Data were analyzed using FACSSDiva software (Becton Dickinson). At least 20,000 results were analyzed for each sample.

[0181] Immunohistochemistry (IHC) of sarcoma tissue Formalin-fixed paraffin-embedded (FFPE) patient tissue sections were obtained from surgical samples taken at the time of informed consent biopsy. Sections were cut to 2.5 μm thickness, dewaxed, and rehydrated. Epitope identification (S3020) was performed by incubating sections with proteinase K (Dako, Glostrup, Denmark) for 6 minutes at room temperature. Endogenous peroxidase was blocked with 3% hydrogen peroxide for 10 minutes, followed by another blocking step of 1 hour in 5% serum albumin (BSA) at rest. Sections were incubated overnight at 4°C with monoclonal anti-GD2 antibody (1:100 dilution) (14G2a; cat# LS-C63496, LSBio). A second biotinylated antibody (K8024, Dako, Carpinteria, USA) and a peroxidase DAB kit (Dako, Carpinteria, USA) were used to visualize the first antibody. The slides were then counterstained with hematoxylin, dehydrated with a series of alcohol solutions (70%–100%), replaced with xylene three times, and fixed with Cytoseal XYL (Thermo Scientific). Analysis was performed using a standard microscope.

[0182] Co-culture assay For in vitro co-culture assays, untransduced effector (E) T cells (NT-T) or CAR.GD2 T cells (0.1 x 10⁻⁶) were used. 6 Cells / well) and GD2+ target (T) MB cells (0.1x10⁻¹²) 6 Cells / well were seeded in 24-well plates at the indicated E:T ratio for 5 days. Antitumor efficacy was evaluated by flow cytometry, assessing residual viable 7AAD levels after exposure to NT-T or CAR.GD2 T cells. 阴性 GD2 阳性 (7AAD - GD2 + The percentage of tumor cells or the absolute number of GD2+ tumor cells retained in the culture. (CountBright) TM Absolute counting beads (Thermo Fisher Scientific) are used for the absolute quantification of tumor cells.

[0183] In vitro anti-sarcoma activity For long-term co-culture experiments, NT and CAR.GD2.28.4–1BBζ (IIICAR.GD2) T lymphocytes were cultured at a concentration of 0.5 x 10⁻⁶. 6Cells / well were seeded in 24-well plates at the effector:target (E:T) ratio as shown. After incubation at 37°C for 6 days, adherent tumor cells and T cells were collected, and residual tumor cells and T cells were assessed by fluorescence-activated cell sorting (FACS) analysis based on CD45+ / CD3+ cells (effective T cells) and GFP+GD2+ (sarcoma GD2+ cell line) or CD45- / CD3- (sarcoma GD2(neg) cell line), respectively.

[0184] For "stress" co-culture assay 72 Tumor cells were added on days 0, 5, 10, and 15, with an effector-to-target (E:T) ratio of 1:1. Residual tumor cells and persistent T cells were analyzed by FACS five days after each tumor addition.

[0185] Cytokine profile Supernatant was collected from the co-culture medium at 24 hours to measure cytokine release. Cytokines were measured by immunoassay in a microfluidic Simple Plex cartridge ELLA (Biotechne; R&D Systems, Minneapolis). Specifically, the following cytokines were investigated: granzyme B, interferon-γ (IFN-γ), IL-2, and tumor necrosis factor-α (TNF-α).

[0186] Treatment with EZH2 inhibitors Tazesta, a potent and selective EZH2 inhibitor (Aurogene, Rome, Italy), was dissolved in DMSO or added to tumor cells at concentrations of 1 or 10 uM, with DMSO alone as a control. After incubation at 37°C and 5% CO2 for 7 days, tumor cells were collected and stained with live / dead dyes using 7-amino-actinomycin D (7-AAD) staining solution (BD Biosciences, Italy); CD45 APC (BD Biosciences, USA); and conjugated mouse anti-human GD2-BV421 mAb (BD Biosciences, USA). Cells were incubated with the mAb (4°C in the dark for 30 min), washed in 1x phosphate-buffered saline (1x PBS), and analyzed in a FACS-Fortessa flow cytometer. Data were analyzed using FACSSDiva software (Becton Dickinson). At least 20,000 results were analyzed for each sample.

[0187] Xenograft sarcoma mouse model for in vivo studies Xenograft studies were conducted using 6- to 8-week-old NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ; from Charles River) mice. To investigate the in vivo antitumor activity of IIICAR.GD2 T cells in a systemic rhabdomyosarcoma model, 0.5 × 10⁻⁶ cells were administered intravenously (iv). 6 GD2+RD-GFP-FF-Luc cells.

[0188] To investigate the in vivo antitumor activity of CAR.GD2 T cells in an orthotopic OS (ot) model, 0.5 × 10⁻⁶ cells were seeded into the tibia of NGS mice. 6 GD2+143 b-GFP-FF-Luc or 0.2x10 6 GD2+U-2OS-GFP-FF-Luc. Tumors were injected with a 1:1 diluted matrix gel in PBS 1X. Following tumor transplantation, mice received only one intravenous injection of effector T cells (10 x 10⁻⁶). 6 / mouse). To investigate which human sarcoma cell lines regulate the expansion of mouse MDSC cells, OS cell lines: 143B, SAOS-2, and U-2OS were in situ seeded into the right tibia of NSG mice. For the ERMS model, RD cell lines were in situ seeded into the right paw muscle of NSG mice.

[0189] Tumor growth was evaluated using an IVIS imaging system (PerkinElmer, USA). Briefly, as previously described, a constant region of interest was plotted on mice, and signal intensity was measured weekly as total photons / second / cm² / sr (p / s / cm² / sr). Circulating human T cells in the peripheral blood of mice were assessed periodically. Mice were housed in the animal facility at Plaisant Castel Romano (Rome, Italy). All in vivo experiments complied with international, EU, and national ethical requirements and were approved by the Italian Ministry of Health (N°88 / 2016-PR).

[0190] Xenograft medulloblastoma mouse model for in vivo research NOD / SCID IL-2Rγnull (NSG) xenograft mice were purchased from Charles River and are held at the Plaisant Castel Romano facility in Rome. Mouse experiments were conducted in accordance with international, EU, and national ethical requirements and were approved by the Italian Ministry of Health (n° 765 / 2021-PR). For the orthotopic in vivo model, 5-week-old female NSG mice were anesthetized by intraperitoneal injection of ketamine (10 mg / kg) and toluene-thiazide (100 mg / kg). The posterior cranial region was removed and the mice were placed in a stereotactic head frame. D283 MED.GFP-FF.Luc cells (2 × 10⁻⁶) were introduced. 5 (Each mouse) was stereotactically implanted into the cerebellum at an infusion rate of 1 μL / min, using the following coordinates based on the Franklin and Paxinos atlas: 6.6 mm posterior to the anterior fontanelle; 1 mm lateral to the midline; and 2 mm from the skull surface. After injection, the cannula was held in place for approximately 5 minutes to equalize pressure within the cranial fornix. The skin was then secured to the craniotomy assembly using metal clips. Three days after tumor implantation, mice were randomly assigned to two groups and received an intravenous (iv) injection of 10 × 10⁻⁶ tumoursporin. 6 Control NT T cells or CAR.GD2 T cells were used, and weekly bioluminescence imaging (IVIS system, PerkinElmer, USA) was performed until day 45 (end of experiment). Signal quantification in photons per second was performed as described previously. 73,74 In addition, Med-411FH mCherry / luciferase cells (2×10⁻⁶) were stereotactically implanted. 5 A mouse model of MB patient-derived xenograft (PDX-MB) was established (per mouse). Fourteen days after tumor implantation, NTT or CAR.GD2 T cells (10 × 10⁶ cells per mouse) were administered intravenously. 6 Mice were treated with IVIS imaging (per mouse) and tumor growth was monitored until day 60 (end of experiment). All mice were euthanized according to protocol when they were near death or hind limb paralysis developed. In both in vivo experiments, effector cell proliferation was monitored by exsanguination and analyzed by flow cytometry using a BD LSR Fortessa X-20 cytometer. Data were analyzed using FACSDiva software (BDBiosciences, Italy).

[0191] In vivo study of activation of suicide genes by infusion of the dimerizing drug AP1903 The in vivo activity of AP1903 in inducing apoptosis in CAR.GD2 T cells was evaluated in an in vivo PDX-MB mouse model. Briefly, 2 x 10^6 cells were in situ infused into 5-week-old female NSG mice. 5Med-411FH mCherry / luciferase. Bioluminescence monitoring confirmed tumor implantation in mice after treatment with 10 × 10⁻⁶ mCherry / luciferase. 6 Intravenous (iv) injection of NT T cells or genetically modified CAR.GD2 T cells. Ten days later, when the presence of circulating T cells was confirmed by flow cytometry, mice were randomly assigned to four groups and treated or untreated by intraperitoneal (ip) infusion of AP1903 (100 mg / mouse) on days +11, +12, and +13 (NT-T without AP1903, NT-T with AP1903, or CAR.GD2-T without AP1903, CAR.GD2-T with AP1903). 24 hours after the last AP1903 treatment, residual effector cells were evaluated by flow cytometry in peripheral blood or by histological and IHC analysis in brain / tumor tissue. Animals were euthanized, and the brains were fixed in 4% formaldehyde in 0.1 mol / L phosphate-buffered saline (pH 7.2) and embedded in paraffin. AP1903 (catalog number 6130, Biotechne-TOCRIS).

[0192] Hematoxylin / eosin and immunohistopathological (IHC) analysis of mouse tissues Histopathological analysis was performed according to the previous report. 74 In summary, after hematoxylin / eosin staining for IHC analysis, sections were cut to 2.5 µm thickness, dewaxed, and rehydrated. Epitope retrieval was performed by boiling slides with EDTA (pH 9). Endogenous peroxidase was blocked with 3% hydrogen peroxide for 10 min, followed by another blocking step of 1 h in 5% serum albumin (BSA) at RT. Sections were then incubated overnight at 4°C with either anti-CD3 antibody (1:100, DAKO) or anti-Ki67 (1:100, Abcam). A second biotinylated antibody (K8024, Dako, Carpinteria, USA) and a peroxidase DAB kit (Dako, Carpinteria, USA) were used to visualize the first antibody. Slides were then counterstained with hematoxylin, dehydrated with a series of alcohol solutions (70%–100%), replaced three times with Diasolv, and fixed with Diamount (Diapath). Analysis was performed using a standard microscope.

[0193] IHC analysis of human tissues All human tissue samples in this study were collected at the Bambino Gesù Children's Hospital in Rome, subject to institutional review approval. Clinical MB specimens were examined and diagnosed by a pathologist. Formalin-fixed paraffin-embedded (FFPE) patient tissue sections were obtained from surgical samples removed after informed consent. Sections were cut to 2.5 μm thickness, dewaxed, and rehydrated. Epitope retrieval (S3020) was performed by incubating sections with proteinase K (Dako, Glostrup, Denmark) for 6 minutes at room temperature. Endogenous peroxidase was blocked with 3% hydrogen peroxide for 10 minutes, followed by another blocking step of 1 hour in 5% serum albumin (BSA) at room temperature (RT). Sections were incubated overnight at 4°C with monoclonal anti-GD2 antibody (1:100 dilution) (14G2a; cat# LS-C63496, LSBio). A second biotinylated antibody (K8024, Dako, Carpinteria, USA) and a peroxidase DAB kit (Dako, Carpinteria, USA) were used to visualize the first antibody. Slides were then counterstained with hematoxylin, dehydrated with a series of alcohol solutions (70%–100%), replaced three times with Diasolv, and fixed with Diamount (Diapath). Analysis was performed using a standard microscope. The data shown in Table 4 are based on IHC antibody scores quantified as follows: 3+, positive signal in >70% of tumor cells; 2+, positive signal in >40% to 69% of tumor cells; 1+, positive signal in >5% to 39% of tumor cells; 0%, low or no positive signal in <5% of tumor cells. The analysis results were evaluated by an experienced pathologist.

[0194] Statistical analysis All data are expressed as mean ± SD. Where appropriate, Student's t-test or standard two-sided one-way ANOVA (Tukey multiple comparison test) was used to compare differences between groups. The Kaplan–Meier method was used to estimate the probability of disease-free survival (DFS); differences between groups were compared using the log-rank test. Mice were matched based on tumor signal strength in the control and treatment groups. Graph generation and statistical analysis were performed using Prism version 8.0d software (GraphPad). A p-value ≤ 0.05 was considered significant in all cases. Precise p-values ​​were given whenever appropriate. Wilcoxon or Mann-Whitney nonparametric tests were used. Statistical significance was indicated (p-value: *p < 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001).

[0195] result The transduction efficiency of GD2.CAR-28.4-1BBζ T cells (GD2.CAR cells) transduced with the third-generation SFG.iC9.2A.CAR-GD2(14g2a)CD8tm.CD28.41BBζ (later iC9.2A.GD2.CAR-28.4-1BB.ζ) retroviral vector ( Figure 1 A) was 65.1% ± 9.7%, while the transduction efficiency of non-transduced (NT) T cells was 3.8% ± 2.3%, p ≤ 0.0001 ( Figure 1 BC). The growth of GD2.CAR T cells exhibits the same kinetics as that of control N T cells. Figure 1 D). The iC9.2A.GD2.CAR-28.4-1BB.ζ retroviral vector also efficiently transduced (CD3-CD56+) NK cells and expanded them in feeder-free culture medium. At +5 days post-transduction, the efficiency of transducing NK cells with GD2.CAR T cells was 57.77% ± 11.12%, and remained stable until +25 days post-transduction: 53.60% ± 11.66%, p = 0.104 ( Figure 1 E).

[0196] Expression of GD2 in human sarcoma cell lines.

[0197] The expression of disialotetrahexosylganglioside GD2 in 11 sarcoma cell lines was assessed by flow cytometry (FACS): specifically, five osteosarcoma (OS) cell lines, one embryonal rhabdomyosarcoma (ERMS) cell line (RD), three alveolar rhabdomyosarcoma (ARMS) cell lines, and two Ewing sarcoma (EW) cell lines. Three OS cell lines, 143B, MG-63, and U-2OS, and one ERMS (RD) cell line showed the highest GD2 expression (expressed as percentage and median fluorescence intensity (MFI)). OS cell lines: HOS and SAOS-2; ARMS: RH4, RH41, and RH30; and EW cell lines: A673 and SKES1 showed lower GD2 antigen expression. Figure 2 A-2D). Several tumor cell lines show high expression of GD2, such as the melanoma cell line A375 ( Figure 2 CD), lung adenocarcinoma cell line H2228 ( Figure 2 EF) and very rare tumors such as desmoplastic small round cell tumors (DSRCT) Figure 2 G).

[0198] GD2.CAR-28.4-1BBζ (GD.CAR) T cells significantly controlled tumor growth in sarcoma cells with high and low GD2 expression. Long-term (5-day) co-culture experiment (Effect:Target (E:T) ratio of 1:1) Figure 3 AF) provided evidence that, compared with N T cells with high GD2 expression, such as OS cell line MG63 cells (1.68±2.36% vs 76.88±2.54% residual tumor cells, p<0.0001); Figure 3 A); 143B cells (1.9±7.2% vs 71.2±9.6% residual tumor cells, p<0.0001); Figure 3 B) and U-2OS cells (11.3±14.1% vs 74.6±17.9% residual tumor cells, p=0.0007); Figure 3 C); ERMS RD cell line (4.1% ± 3.9% residual tumor cells, p < 0.0001); Figure 3 Compared to NT T-cells, GD2.CAR-28.4-1BBζ T cells significantly controlled tumor cell growth; unexpectedly, GD2.CAR-28.4-1BBζ T cells also significantly (albeit partially) controlled tumor growth in sarcoma cell lines with lower GD2 expression, such as the OS cell line HOS (22.9±10.3% vs 60.8±13.5% of residual tumor cells, p=0.0003). Figure 3 D); ARMS cell line RH4 (GD2=33.6±9.9% vs 70.5±8.6% residual tumor cells, p=0.0003), Figure 3 F); ARMS RH41 (42.9±6.6% vs 80.0±10.5% residual tumor cells, p=0.004); Figure 3 G) and Ewing sarcoma A-673 cell line (56.5 ± 13.2% vs 81.2 ± 5.4% residual tumor cells, p = 0.0015, Figure 3 H).

[0199] GD2.CAR-28.4-1BBζ (GD.CAR) T cells significantly control tumor growth in aggressive adult tumors.

[0200] GD2.CAR-28.4-1BBζ T cells also significantly controlled the growth of aggressive adult tumors, such as the melanoma cell line A375 (GD2 = 3.2 ± 0.8% vs 90.40 ± 1.3% residual tumor cells, p = 0.0002). Figure 3 I) and adenocarcinoma cell line H2228 (GD2=4.4±1.68% vs 80.57±4.2% residual tumor cells, p=0.0006, Figure 3 J).

[0201] Inhibition of EZH2 in RMS cell lines upregulated GD2 expression in Ewing sarcoma and ARMS sarcoma cell lines and increased their sensitivity to GD2.CAR T cell killing activity.

[0202] As expected, EZH2 inhibition induces upregulation of GD2 in EwS A-673 ( Figure 4 ).

[0203] According to the present invention, the aim is to investigate whether EZH2 inhibition can also induce GD2 upregulation in RMS and OS cell lines.

[0204] ARMS FP-RMS cell lines (RH4, RH41, and RH30, Figure 5 AC) and OS cell line HOS ( Figure 6 Cultured with 1-10 uM tazestat for 7 days. Unexpectedly, drug treatment in ARMS FP-RMS cell lines: RH4 ( Figure 5 A) and RH41 ( Figure 5 B) and OS cell line HOS ( Figure 6 Two of the three treatments significantly increased GD2 expression. Therefore, 7 days of EZH2 pretreatment made EwSA-673 cells sensitive to effective cell lysis of GD2.CAR-28.4-1BBζ T cells. Figure 6 A), and more importantly, it was the first time that EwS ( Figure 7 A), EZH2 preprocessing can also make ARMS RH4 ( Figure 7 B), RH41 Figure 7 C) and HOS cell line ( Figure 7 D) Sensitive to effective cell lysis of GD2.CAR-28.4-1BBζ T cells ( Figure 7 (AD). Experts in this field review the literature. 21 The use of this drug (tazestat) to positively modulate GD2 on OS cell lines is often discouraged. Therefore, surprisingly, the use of an EZH2 inhibitor positively modulates GD2 expression on both rhabdomyosarcoma and OS cell lines. More importantly, tazestat sensitizes these tumor cell lines to effective cytolysis of GD2.CAR-28.4-1BBζ T cells.

[0205] Long-term efficacy evaluation of GD2.CAR-28.4-1BBζ T cells in an embryonic RMS mouse model.

[0206] Next, we will evaluate whether GD2.CAR-28.4-1BBζ T cells can produce effective antitumor activity in an in vivo model of invasive metastatic embryonic ERMS RD-GFP-FF-Luc, such as... Figure 8As shown in Figure A, the bioluminescence of RMS tumor-bearing mice treated with NT T cells increased rapidly by up to three logs in less than 50 days. Figure 8 (BD), and due to the onset of the disease, the mice either died or were euthanized. Macroscopic analysis of the euthanized mice revealed large tumor masses with metastases preferentially located in the kidneys and liver.

[0207] Compared with mice treated with NT (50.0 days), mice bearing RMS tumors treated with GD2.CAR-28.4-1BBζ T cells had a significantly longer median survival (69.5 days). Figure 8 E; p=0.012). Although circulating T cells were detectable in mice treated with NT T cells during the mouse follow-up period, they decreased rapidly (from 21.99% ± 12.16% at +21 days to 12.92% ± 5.99% at +47 days). Figure 8 F), and no tumor control was observed. In contrast, in mice treated with GD2.CAR-28.4-1BBζ T cells, circulating T cells were significantly increased from 13.44% ± 11.60% at +21 days to 25.48% ± 10.65% at +47 days (p = 0.038) (p = 0.04), and long-term T cell persistence was observed up to 75 days (9.20% ± 9.80%). Although the percentage of “CAR expression” in circulating GD2.CAR-28.4-1BBζ T cells decreased from 60.75% ± 16.54% to 28.08% ± 16.54% (p = 0.0001) in the first 50 days, it remained stable until +75 days (31.07% ± 14.76%; p = ns) ( Figure 8 G). During tumor eradication, a significant decrease in CD4+CAR+ T cells was observed ( Figure 8 H), while CD8+ CAR+ T cells were significantly enriched ( Figure 8 I).

[0208] It is noted that there is no data in the literature on effective GD2.CAR T cell control in soft sarcomas such as rhabdomyosarcoma.

[0209] CAR.GD2.28.4–1BBζ-T cell therapy improved the survival rate of OS mice in situ. To further support our in vitro / in vivo results, the in vivo potency and persistence of GD2.CAR-28.4-1BBζ T cells in the 143B-GFP-FF-Luc orthotopic OS mouse model were then evaluated, as shown in the figure ( Figure 9 A). GD2.CAR-28.4-1BBζ (GD2.CAR) T cells significantly slowed the growth of the primary tumor. Figure 9BD, compared to NT T cells, significantly increased the overall survival of mice treated with CAR T cells. Figure 9 E (specifically, 46 days for mice treated with GD2.CAR-28.4-1BBζ T cells vs. 39 days for mice treated with NT T cells, p=0.005). Interestingly, a significant expansion of human T cells was observed only in mice treated with GD2.CAR-28.4-1BBζ T cells. Figure 9 F). The percentage of circulating CAR.GD2-T cells increased from 46.32% ± 19.81% in the first month to 61.72% ± 23.78% (day +33), and remained stable at day 44 (62.80% ± 17.54%). Figure 9 E), with CD4+CAR+ T cells (53.8%±17.2%) Figure 9 H) and CD8+CART cells (43.0%±15.4%) Figure 9 The average distribution of I).

[0210] As described above, GD2.CAR-28.4-1BBζ T cells according to the present invention exhibit superior performance compared to those incorporating the most recent findings by Adrienne H Long et al. 67 The GD2-CAR described is a 14g2a-scFv (14g2a.CD28.OX40.ζ) containing CD28, OX40, and CD28 signaling domains. This can be compared by the overall survival of OS mice treated with the GD2.CAR of the present invention. Figure 8 E) and Adrienne H Long et al. 67 The overall survival rate reported in the literature (see page 873 of CancerImmunology Res; 4 (10) October 4, 2016) Figure 3 B).

[0211] Expression of GD2 in neuroblastoma (NBL) and retinoblastoma cell lines.

[0212] Some tumor cell lines express high levels of the GD2 antigen, which can then be targeted by GD2.CAR-28.4-1BBζ (GD2.CAR) T cells. Specifically, all neuroblastoma (NBL) cell lines show high expression of disialinoside GD2 (GD2.CAR). Figure 10 A). These data were confirmed in primary tumors from patients diagnosed with neuroblastoma (NBL). Figure 10 B). Peripheral blood from NBL patients was used as a negative control.

[0213] High expression of GD2 was also found in all retinoblastoma tumor tissues. Figure 11 ).

[0214] GD2 expression in medulloblastoma This study evaluated whether GD2 could also be selected as a tumor-associated antigen (MB) and whether it could be targeted by CAR.GD2 T cells. Furthermore, the use of CAR.GD2 T cells may induce potential side effects, such as cytokine release syndrome. 75 or neurotoxicity 75 Therefore, the efficacy of inducible cystase 9 (iC9) suicide gene activation in eliminating circulating and tumor-infiltrating CAR.GD2 T cells was evaluated and demonstrated by infusing it into an orthotopic MB mouse model.

[0215] GD2 expression in primary MB biopsy and MB cell models To investigate whether GD2 could serve as a suitable target for CAR-GD2 T-cell therapy in patients with myxomatosis (MB), we evaluated GD2 expression in tumor biopsies and PB from 41 patients affected by MB, and conducted clinical follow-up in our department. Flow cytometry analysis of fresh tumor tissue from patients expected to participate in the study showed GD2 positivity in 75.6% of the analyzed samples, with a mean of 28.84% ± 33.78% of GD2-positive cells (in CD45). - (In cell gating). Peripheral blood mononuclear cells from the same MB patient were analyzed in parallel as an internal negative control (GD2 positive cells % = 0.43% ± 0.90%; p < 0.0001). Figure 12 A). The median fluorescence intensity (MFI) of GD2 expression in MB tissue biopsy (16165±20161) was significantly higher than that in PB sample cells (1026±1021, p<0.0001). Figure 12 B). Interestingly, when MB samples were stratified within genetic subgroups, GD2 positivity was highly observed in SHH patients (9 / 9, 100%), G4 patients (15 / 16, 93%), and G3 patients (5 / 7, 71.43%), while the WNT subgroup was characterized by a low percentage of GD2-positive patients (2 / 9, 22%) and a low frequency of GD2+ cells in the tumor (1.61% ± 4.03% of CD45+). - (Analyzed cells). Figure 12 C). However, patients were also stratified according to histologically defined subgroups, with GD2 positivity observed in 76% (19 / 25) of classic MB patients, 50% (4 / 8) of large cell / anaplastic (LCA) MB patients, 3 / 3 (100%) of the MBENMB subgroup, and 5 / 5 (100%) of the pro-connective tissue hyperplasia / nodular (D / N) MB subgroup. Figure 12 E). Due to the high variability of GD2 expression among MB samples (in terms of percentage and MFI), there was no significant difference in GD2 positivity among the histological subgroups considered. Figure 12 DF). Figure 13 Four representative FACS analyses of four different MB tumors (SHH, G3, G4, and WNT) are presented. GD2 expression was also confirmed by IHC analysis of 18 tissue biopsies (9 G3, 4 G4, and 5 SHH patients) (Table 4).

[0216] Table 4 Similar to neuroblastoma and lung cancer 53 Breast cancer 76 and glioma 63 The study found that most IHC staining in MB tumors was variable and located within the cytoplasm (Table 4). Finally, the expression of disialotetrahexosylganglioside GD2 was evaluated in two MB cell lines and one PDX (patient-derived xenograft) cell line, which will be used in the immunotherapy model according to the invention. Specifically, the D283 Med (subgroup G3 / G4) cell line showed the highest GD2 expression (100% ± 0.0%, and MFI equal to 42354 ± 2119), while the DAOY (SHH) cell line showed 30.40% ± 2.1% GD2+ cells with an MFI of 15589 ± 648. Figure 14 AC).

[0217] CAR.GD2 T cells exert effective antitumor activity on human GD2+MB cell line Since significant associated expression of the GD2 antigen was found in most tumor tissues of MB patients, the in vitro cytotoxicity activity of the third-generation CAR-GD2.CD28.4-1BBζ (CAR.GD2) T cells according to the present invention was tested against two MB human cell lines, D283 Med (subgroup G3 / G4) and DAOY (SHH) cells.

[0218] In long-term in vitro co-culture experiments, N T cells and CAR.GD2 T cells were incubated with D283 Med or DAOY cell lines at an effect:target (E:T) ratio of 1:1 for 5 days.

[0219] Compared with NT T cells, CAR.GD2-T cells were able to significantly eradicate the D283 cell line (p<0.0001). Figure 15 A). Furthermore, cytokine production is closely related to the high anti-MB killing activity of CAR.GD2 T cells ( Figure 15 B). CAR.GD2 T cells co-cultured with GD2+D283 Med produced significantly higher levels of activating cytokines, such as granzyme B, IFN-γ, IL2, and TNF-α. Figure 15 B).

[0220] In contrast, when CAR.GD2-T cells are co-cultured with DAOY cells, they are characterized by a lower percentage of GD2+ cells and a reduced GD2 MFI compared to D283 MED cells. Figure 14 AC), although significant for untransduced (NT) T cells, showed suboptimal in vitro tumor control, i.e., the percentage of residual tumor (27.88% ± 12.88% vs 51.36% ± 13.24%, p = 0.03). Figure 15 C), and in parallel, the adverse production of activating cytokines was observed ( Figure 15 D). The remaining DAOY cells resistant to CAR.GD2 T cell clearance were then characterized by flow cytometry, and it was observed that most of the remaining DAOY cells were GD2 negative or had very low GD2 MFI ( ). Figure 16 AB).

[0221] CAR.GD2-T cells exert anti-tumor activity in MB xenogeneic mouse model Then, the ability of CAR.GD2 T cells to produce significant antitumor activity in an orthotopic MB NSG mouse model using the D283 Med (subgroup G3 / G4) cell line, genetically modified with GFP-firefly luciferase (D283 Med-GFP-FF-Luc) for in vivo bioluminescence monitoring was evaluated. The MB cell line was intracranially implanted, and bioluminescence imaging (BLI) was used to monitor tumor growth over time in the NSG mouse model. Three days after tumor transplantation, mice were treated intravenously with either NT T cells or CAR.GD2 T cells on day 0. Figure 17 A). As expected, in mice treated with N T cells, tumor bioluminescence rapidly increased to three logarithms in less than a month. Figure 17 BC), and the mice either died or were euthanized due to disease. Compared with the NT-T mouse group, the mouse group receiving CAR.GD2-T cells showed complete tumor eradication 38 days after infusion (BC). Figure 17 BC), DFS is significantly prolonged. Figure 17As shown in Figure D, compared with the NT-T group, 80% of mice treated with CAR.GD2-T cells showed improved survival at 90 days (end of the experiment) (p=0.03). Notably, the in vivo antitumor efficacy of CAR.GD2-T cells was associated with the long-term persistence (up to 45 days) of circulating effector cells (CD45+ / CD3+) in PB. Figure 17 E). More importantly, the percentage of circulating CAR+ T cells remained stable for 45 days ( Figure 17 F). The dynamic evolution of CD4+ and CD8+ T cells was also evaluated in the PB of treated mice, and a slight increase in CD8+ T cells was observed in the NT and CAR.GD2 T cell populations. Figure 18 AC).

[0222] Although the activation of the suicide gene iC9, included in the constructs of this invention, has been reported only in extracranial tumors, particularly in neuroblastoma models. 51 In vitro and in vivo CAR-T cells undergo rapid apoptosis, but the systemic administration of AP1903 has never been evaluated as inducing rapid elimination of brain / MB-infiltrating CAR T cells. Therefore, a PDX mouse model of orthotopic transplantation of MB tumor cells was developed. Ten days after intravenous infusion of NTT or CAR.GD2 T cells, three mice in each group received intraperitoneal administration of dimerized AP1903 for three consecutive days. Figure 19 A). For example Figure 19 As shown in BC, AP1903 treatment significantly reduced circulating CAR.GD2 T cells compared to untreated CAR.GD2 T cells. Furthermore, in a dedicated experiment, mouse brains bearing MB tumors were transplanted 44 days after NT / CAR.GD2 T cell infusion to characterize tissue infiltration of human T cells (IHC vs hCD3). The ability of CAR.GD2-T cells to migrate across the blood-brain barrier (BBB) ​​and localize within tumors was demonstrated. Figure 20 (Figure B), while the number of tumor-infiltrating human T cells observed in the brains of mice that received NTT cells was negligible ( Figure 21 Most importantly, anti-hCD3 IHC analysis of cerebellar slides from mice infused with CAR.GD2-T cells and administered AP1903 clearly demonstrated the effectiveness of AP1903 in penetrating the BBB and eliminating tumor-infiltrating CD3+ cells. Figure 20 BC).

[0223] Inhibition of EZH2 upregulates GD2 expression in MB tumors in MB cell lines This study investigated whether EZH2 inhibition could induce GD2 upregulation in medulloblastoma cell lines with low GD2 expression. DAOY (SHH subgroup) MB cell lines were co-cultured with 1–10 µM tazestat for 7 days. Unexpectedly, drug treatment significantly increased the percentage of GD2 expression in DAOY cell lines (from 33.3% to 77.2%) and median fluorescence intensity (MFI) (from 5416 to 8995). Therefore, 7 days of EZH2 pretreatment sensitive MB DAOY cells to effective cytolysis of GD2.CAR-28.4-1BBζ T cells. Figure 22 B).

[0224] An orthotopic mouse model of human SHSY5Y-Luc cell line to evaluate the long-term persistence of GD2.CAR-28.4-1BBζ (CAR.GD2) T cells.

[0225] In a xenograft neuroblastoma mouse model (SHSY5Y), the long-term persistence of infused effector T cells genetically modified to express FF-luciferase (23A) was evaluated. NT FF-luciferase T cells, GD2.CAR-28.4.OX40.ζ FF-luciferase T cells, and GD2.CAR-28.4-1BBζ FF-luciferase T cells were administered intravenously, and T cell bioluminescence was monitored for 102 days. GD2.CAR-28.4-1BBζ T cells (23B) exhibited significantly longer persistence than (23C) third-generation GD2-CAR T cells (including CD28 and OX40 as co-stimulatory domains) and (23D) untransduced (NT) FF-luciferase T cells. Recent preclinical and clinical studies in the CAR T cell-based therapeutic field have shown promising results for cancer treatment. While clinical trials based on anti-CD19.CAR T cells have yielded unprecedented results in the treatment of hematologic malignancies, documented information on their antitumor activity against solid tumors is limited. In particular, the identification of suitable target antigens that are highly expressed in solid tumors but limited in normal tissues remains a limitation that needs to be overcome. Disialinoganglioside GD2 is an antigen overexpressed on the cell surface of various human cancers, including neuroblastoma, melanoma, retinoblastoma, Ewing sarcoma, small cell lung cancer, glioma, osteosarcoma, and soft tissue sarcoma. 2 GD2 is expressed at very low levels in healthy tissues. Although the function of GD2 is not fully understood, it is well known to participate in tumor development and malignant phenotypes by increasing cell proliferation, growth, motility, migration, adhesion, and invasion. 6,54 .

[0226] It has recently been confirmed that diffuse midline gliomas (DMGs) with H3K27M mutations, including diffuse intrinsic pontine gliomas (DIPG), explicitly express GD2 antigen 52,77. Notably, the safety of CAR.GD2 T cell infusion in patients with H3K27M-mutant DIPG or spinal DMG has been recently reported, indicating that toxicity is primarily related to the specific location of the tumor and, in any case, is reversible with intensive supportive care. 52 This article reports for the first time that the GD2 antigen is expressed in most confirmed cases of myocardial infarction (MB). Importantly, the SHH and G3 / G4 subgroups showed the highest GD2 expression, which allows us to utilize GD2 as a suitable immunotherapeutic target for the most aggressive and poorest-prognostic MB patients.

[0227] These insights support the theoretical basis for developing adoptive cell therapies targeting GD2 in pediatric myeloma (MB). Most importantly, given the poor prognosis of most patients despite standard intensive care, CAR T cells retargeted to the GD2 antigen may be a potential therapeutic approach. First, the significant antitumor activity of CAR.GD2 T cells has been demonstrated in in vitro and in vivo models of MB. Specifically, CAR.GD2 T cells mediated significant killing activity against GD2+ MB cell lines and MB PDX cells, providing encouraging data suggesting that GD2 is an effective and suitable target for adoptive T cell therapy in MB. Due to the in vitro live-cell imaging system, it is possible to demonstrate the rapid responsiveness of CAR.GD2 T cells in recognizing and killing MB tumor cells. Indeed, monitoring the co-culture between the D283 Med cell line and CAR.GD2 T cells from a very early time point (1 hour) to the endpoint (5 days) showed significant tumor control at 12 hours, providing encouraging data regarding the relevance of CAR.GD2 T cells to the eradication of MB tumors. Tumor recognition and elimination, strictly dependent on the level of antigen expression, were also demonstrated. In fact, DAUDI cells with weak GD2 antigen expression were not fully controlled in the in vitro model in terms of the percentage of GD2+ cells and GD2 MFI, and resistant cells were characterized by low GD2 MFI. As a step closer to mimicking the human pathophysiological state, two in vivo orthotopic MB mouse models were established. Consistent with the in vitro data, CA.RGD2-T cells were able to significantly control tumor growth (assessed by bioluminescence monitoring), and therefore, mice bearing MB tumors and treated with CAR.GD2-T cells showed a significantly prolonged OS compared with mice receiving NT T cells. Regarding the delivery pathway of CAR T cells, it is well known that it can significantly affect the response of patients affected by solid tumors, especially brain tumors. 78In our study, systemic intravenous delivery of CAR T cells elicited a significant anti-tumor response in an MB animal model, associated with significant disease-free survival and a large number of tumor-infiltrating T cells. It is noteworthy that CAR T cell use has been reported to cause potentially fatal side effects such as cytokine release syndrome. 75,79 or neurotoxicity 80 For this reason, including the iC9 suicide gene in the construct can rapidly eradicate genetically modified cells, as demonstrated in several models. 51,72,81 It has been demonstrated that systemic administration of the dimerizing drug AP1903 significantly reduces circulating CAR.GD2 T cells and brain / tumor-infiltrating human CD3+ cells in the cerebellum of mice. Therefore, when considering clinical studies of this approach in MB, the incorporation of suicide genes may be a relevant approach to mitigate potential risks associated with CAR-T cells, including autonomous proliferation and neurotoxicity. Based on these preclinical findings, a Phase I / II clinical trial of CAR.GD2 T cell therapy for MB GD2+ (NCT05298995) patients is under development, providing them with a beneficial and innovative treatment strategy.

[0228] References 1Liu, J. et al. Ganglioside GD3 synthase (GD3S), a novel cancer drug target. Acta Pharm Sin B 8, 713-720, doi:10.1016 / j.apsb.2018.07.009 (2018). 2Nazha, B., Inal, C. & Owonikoko, TK Disialoganglioside GD2Expression in Solid Tumors and Role as a Target for Cancer Therapy. Front Oncol 10, 1000, doi:10.3389 / fonc.2020.01000 (2020). 3Ravindranath, MH, Muthugounder, S. & Presser, N. Ganglioside signatures of primary and nodal metastatic melanoma cell lines from the same patient. Melanoma Res18, 47-55, doi:10.1097 / CMR.0b013e3282f43acf (2008). 4Strobel, S. B. et al. Expression of Potential Targets for Cell-BasedTherapies on Melanoma Cells. Life (Basel) 11, doi:10.3390 / life11040269(2021). 5Dobrenkov, K., Ostrovnaya, I., Gu, J., Cheung, I. Y. & Cheung, N. K.Oncotargets GD2 and GD3 are highly expressed in sarcomas of children,adolescents, and young adults. Pediatr Blood Cancer 63, 1780-1785, doi:10.1002 / pbc.26097 (2016). 6Battula, V. L. et al. Ganglioside GD2 identifies breast cancer stemcells and promotes tumorigenesis. J Clin Invest 122, 2066-2078, doi:10.1172 / JCI59735 (2012). 7Schulz, G. et al. Detection of ganglioside GD2 in tumor tissues andsera of neuroblastoma patients. Cancer Res 44, 5914-5920 (1984). 8Sujjitjoon, J. et al. GD2-specific chimeric antigen receptor-modified T cells targeting retinoblastoma - assessing tumor and T cellinteraction. Transl Oncol14, 100971, doi:10.1016 / j.tranon.2020.100971(2021). 9Cheresh, D. A., Rosenberg, J., Mujoo, K., Hirschowitz, L. &Reisfeld, R. A. Biosynthesis and expression of the disialoganglioside GD2, arelevant target antigen on small cell lung carcinoma for monoclonal antibody-mediated cytolysis. Cancer Res 46, 5112-5118 (1986). 10Longee, D. C. et al. Disialoganglioside GD2 in humanneuroectodermal tumor cell lines and gliomas. Acta Neuropathol 82, 45-54,doi:10.1007 / BF00310922 (1991). 11Wingerter, A. et al. Exploiting Gangliosides for the Therapy ofEwing's Sarcoma and H3K27M-Mutant Diffuse Midline Glioma. Cancers (Basel) 13,doi:10.3390 / cancers13030520 (2021). 12Jones, D. T. W. et al. Molecular characteristics and therapeuticvulnerabilities across paediatric solid tumours. Nat Rev Cancer 19, 420-438,doi:10.1038 / s41568-019-0169-x (2019). 13Dyson, K. A. et al. Emerging trends in immunotherapy for pediatricsarcomas. J Hematol Oncol12, 78, doi:10.1186 / s13045-019-0756-z (2019). 14Hingorani, P. et al. Current state of pediatric sarcoma biology andopportunities for future discovery: A report from the sarcoma translationalresearch workshop. Cancer Genet 209, 182-194, doi:10.1016 / j.cancergen.2016.03.004 (2016). 15Thanindratarn, P., Dean, D. C., Nelson, S. D., Hornicek, F. J. &Duan, Z. Chimeric antigen receptor T (CAR-T) cell immunotherapy for sarcomas:From mechanisms to potential clinical applications. Cancer Treat Rev 82,101934, doi:10.1016 / j.ctrv.2019.101934 (2020). 16Ladenstein, R. et al. Primary disseminated multifocal Ewingsarcoma: results of the Euro-EWING 99 trial. J Clin Oncol 28, 3284-3291, doi:10.1200 / JCO.2009.22.9864 (2010). 17Delattre, O. et al. Gene fusion with an ETS DNA-binding domaincaused by chromosome translocation in human tumours. Nature 359, 162-165,doi:10.1038 / 359162a0 (1992). 18Knutson, S. K. et al.Selective inhibition of EZH2 by EPZ-6438leads to potent antitumor activity in EZH2-mutant non-Hodgkin lymphoma. Mol Cancer Ther 13, 842-854, doi:10.1158 / 1535-7163.MCT-13-0773 (2014). 19Kailayangiri, S. et al. EZH2 Inhibition in Ewing SarcomaUpregulates G(D2) Expression for Targeting with Gene-Modified T Cells. Mol Ther 27, 933-946, doi:10.1016 / j.ymthe.2019.02.014 (2019). 20Richter, G. H. et al. EZH2 is a mediator of EWS / FLI1 driven tumorgrowth and metastasis blocking endothelial and neuro-ectodermaldifferentiation. Proc Natl Acad Sci USA 106, 5324-5329, doi:10.1073 / pnas.0810759106 (2009). 21Wiebel, M. et al. Surface expression of the immunotherapeutictarget G(D2) in osteosarcoma depends on cell confluency. Cancer Rep (Hoboken) 4, e1394, doi:10.1002 / cnr2.1394 (2021). 22Subramanian, S. & Ahmad, T. in StatPearls (2023). 23Quintarelli, C. et al.Innovative and Promising Strategies toEnhance Effectiveness of Immunotherapy for CNS Tumors: Where Are We? Front Immunol 12, 634031, doi:10.3389 / fimmu.2021.634031 (2021). 24Fangusaro, J. & Bandopadhayay, P. Advances in the classificationand treatment of pediatric brain tumors. Curr Opin Pediatr 33, 26-32, doi:10.1097 / MOP.0000000000000975 (2021). 25Louis, D. N. et al. The 2016 World Health OrganizationClassification of Tumors of the Central Nervous System: a summary. Acta Neuropathol 131, 803-820, doi:10.1007 / s00401-016-1545-1 (2016). 26Bandopadhayay, P. et al. Long-term outcome of 4,040 childrendiagnosed with pediatric low-grade gliomas: an analysis of the SurveillanceEpidemiology and End Results (SEER) database. Pediatr Blood Cancer 61, 1173-1179, doi:10.1002 / pbc.24958 (2014). 27Ater, J. L. et al.Randomized study of two chemotherapy regimensfor treatment of low-grade glioma in young children: a report from theChildren's Oncology Group. J Clin Oncol 30, 2641-2647, doi:10.1200 / JCO.2011.36.6054 (2012). 28Ostrom, Q. T. et al. CBTRUS Statistical Report: Primary Brain andOther Central Nervous System Tumors Diagnosed in the United States in 2012-2016. Neuro Oncol 21, v1-v100, doi:10.1093 / neuonc / noz150 (2019). 29Jones, D. T. W., Bandopadhayay, P. & Jabado, N. The Power of HumanCancer Genetics as Revealed by Low-Grade Gliomas. Annu Rev Genet 53, 483-503,doi:10.1146 / annurev-genet-120417-031642 (2019). 30Jones, C. et al. Pediatric high-grade glioma: biologically andclinically in need of new thinking. Neuro Oncol 19, 153-161, doi:10.1093 / neuonc / now101 (2017). 31Mount, C. W. et al. Potent antitumor efficacy of anti-GD2 CAR Tcells in H3-K27M(+) diffuse midline gliomas. Nat Med24, 572-579, doi:10.1038 / s41591-018-0006-x (2018). 32Cavalli, F. M. G. et al. Heterogeneity within the PF-EPN-Bependymoma subgroup. Acta Neuropathol 136, 227-237, doi:10.1007 / s00401-018-1888-x (2018). 33Maier, H., Dalianis, T. & Kostopoulou, O. N. New Approaches inTargeted Therapy for Medulloblastoma in Children. Anticancer Res 41, 1715-1726, doi:10.21873 / anticanres.14936 (2021). 34Northcott, P. A. et al. Medulloblastoma. Nat Rev Dis Primers 5, 11,doi:10.1038 / s41572-019-0063-6 (2019). 35Ribi, K. et al. Outcome of medulloblastoma in children: long-termcomplications and quality of life. Neuropediatrics 36, 357-365, doi:10.1055 / s-2005-872880 (2005). 36Jakacki, R. I. et al. Outcome of children with metastaticmedulloblastoma treated with carboplatin during craniospinal radiotherapy: aChildren's Oncology Group Phase I / II study. J Clin Oncol 30, 2648-2653, doi:10.1200 / JCO.2011.40.2792 (2012). 37Martin, A. M., Raabe, E., Eberhart, C. & Cohen, K. J. Management ofpediatric and adult patients with medulloblastoma. Curr Treat Options Oncol 15, 581-594, doi:10.1007 / s11864-014-0306-4 (2014). 38Gajjar, A. et al. Risk-adapted craniospinal radiotherapy followedby high-dose chemotherapy and stem-cell rescue in children with newlydiagnosed medulloblastoma (St Jude Medulloblastoma-96): long-term resultsfrom a prospective, multicentre trial. Lancet Oncol 7, 813-820, doi:10.1016 / S1470-2045(06)70867-1 (2006). 39Cooney, T., Lindsay, H., Leary, S. & Wechsler-Reya, R. Currentstudies and future directions for medulloblastoma: A review from the pacificpediatric neuro-oncology consortium (PNOC) disease working group. Neoplasia 35, 100861, doi:10.1016 / j.neo.2022.100861 (2023). 40Ellison, D. W. et al. Medulloblastoma: clinicopathologicalcorrelates of SHH, WNT, and non-SHH / WNT molecular subgroups. Acta Neuropathol 121, 381-396, doi:10.1007 / s00401-011-0800-8 (2011). 41Clifford, S. C. et al. Wnt / Wingless pathway activation andchromosome 6 loss characterize a distinct molecular sub-group ofmedulloblastomas associated with a favorable prognosis. Cell Cycle 5, 2666-2670, doi:10.4161 / cc.5.22.3446 (2006). 42Quinlan, A. & Rizzolo, D. Understanding medulloblastoma. JAAPA 30,30-36, doi:10.1097 / 01.JAA.0000524717.71084.50 (2017). 43Mennel, H. D., Bosslet, K., Geissel, H. & Bauer, B. L.Immunohistochemically visualized localisation of gangliosides Glac2 (GD3) andGtri2 (GD2) in cells of human intracranial tumors. Exp Toxicol Pathol 52,277-285, doi:10.1016 / S0940-2993(00)80046-9 (2000). 44Packer, R. J. et al. Atypical teratoid / rhabdoid tumor of thecentral nervous system: report on workshop. J Pediatr Hematol Oncol 24, 337-342, doi:10.1097 / 00043426-200206000-00004 (2002). 45Dardis, C. et al.Atypical Teratoid Rhabdoid Tumor: Two CaseReports and an Analysis of Adult Cases with Implications for Pathophysiologyand Treatment. Front Neurol 8, 247, doi:10.3389 / fneur.2017.00247 (2017). 46Khan, S. et al. Clinical phenotypes and prognostic features ofembryonal tumours with multi-layered rosettes: a Rare Brain Tumor Registrystudy. Lancet Child Adolescent Health 5, 800-813, doi:10.1016 / S2352-4642(21)00245-5 (2021). 47Ladenstein, R. et al. Interleukin 2 with anti-GD2 antibody ch14.18 / CHO (dinutuximab beta) in patients with high-risk neuroblastoma (HR-NBL1 / SIOPEN): a multicentre, randomised, phase 3 trial. Lancet Oncol 19, 1617-1629, doi:10.1016 / S1470-2045(18)30578-3 (2018). 48Kushner, B. H. et al. Humanized 3F8 Anti-GD2 Monoclonal AntibodyDosing With Granulocyte-Macrophage Colony-Stimulating Factor in Patients WithResistant Neuroblastoma: A Phase 1 Clinical Trial. JAMA Oncol 4, 1729-1735,doi:10.1001 / jamaoncol.2018.4005 (2018). 49Kramer, K. et al. A phase II study of radioimmunotherapy withintraventricular (131) I-3F8 for medulloblastoma. Pediatric Blood Cancer 65,doi:10.1002 / pbc.26754 (2018). 50Kramer, K. et al. Compartmental intrathecal radioimmunotherapy:results for treatment for metastatic CNS neuroblastoma. J Neurooncol 97, 409-418, doi:10.1007 / s11060-009-0038-7 (2010). 51Quintarelli, C. et al. Choice of costimulatory domains and ofcytokines determines CAR T-cell activity in neuroblastoma. Oncoimmunology 7,e1433518, doi:10.1080 / 2162402X.2018.1433518 (2018). 52Majzner, R. G. et al. GD2-CAR T cell therapy for H3K27M-mutateddiffuse midline gliomas. Nature 603, 934-941, doi:10.1038 / s41586-022-04489-4(2022). 53Reppel, L. et al. Targeting disialoganglioside GD2 with chimericantigen receptor-redirected T cells in lung cancer. Immunother Cancer 10,doi:10.1136 / jitc-2021-003897 (2022). 54Esaki, N. et al.ASC amino acid transporter 2, defined by enzyme-mediated activation of radical sources, enhances malignancy of GD2-positivesmall-cell lung cancer. Cancer Science 109, 141-153, doi:10.1111 / cas.13448(2018). 55Mansoori, M. et al. High GD2 expression defines breast cancer cellswith enhanced invasiveness. Exp Mol Pathol 109, 25-35, doi:10.1016 / j.yexmp.2019.05.001 (2019). 56Louis, C. U. et al. Antitumor activity and long-term fate ofchimeric antigen receptor-positive T cells in patients with neuroblastoma. Blood 118, 6050-6056, doi:10.1182 / blood-2011-05-354449 (2011). 57Pule, M. A. et al. Virus-specific T cells engineered to coexpresstumor-specific receptors: persistence and antitumor activity in individualswith neuroblastoma. Nat Med 14, 1264-1270, doi:10.1038 / nm.1882 (2008). 58Heczey, A. et al. CAR T Cells Administered in Combination withLymphodepletion and PD-1 Inhibition to Patients with Neuroblastoma. Mol Ther 25, 2214-2224, doi:10.1016 / j.ymthe.2017.05.012 (2017). 59Gargett, T. et al. Safety and biological outcomes following a phase1 trial of GD2-specific CAR-T cells in patients with GD2-positive metastaticmelanoma and other solid cancers. Immunother Cancer 12, doi:10.1136 / jitc-2023-008659 (2024). 60Shum, T. et al. Constitutive Signaling from an Engineered IL7Receptor Promotes Durable Tumor Elimination by Tumor-Redirected T Cells. Cancer Discov 7, 1238-1247, doi:10.1158 / 2159-8290.CD-17-0538 (2017). 61Caforio, M. et al. GD2 redirected CAR T and activated NK-cell-mediated secretion of IFNgamma overcomes MYCN-dependent IDO1 inhibition,contributing to neuroblastoma cell immune escape. Immunother Cancer 9, doi:10.1136 / jitc-2020-001502 (2021). 62Tumino, N. et al. Polymorphonuclear myeloid-derived suppressorcells impair the anti-tumor efficacy of GD2.CAR T-cells in patients withneuroblastoma. J Hematol Oncol14, 191, doi:10.1186 / s13045-021-01193-0(2021). 63de Billy, E. et al. Dual IGF1R / IR inhibitors in combination withGD2-CAR T-cells display a potent anti-tumor activity in diffuse midlineglioma H3K27M-mutant. Neuro Oncol 24, 1150-1163, doi:10.1093 / neuonc / noab300(2022). 64Del Bufalo, F. et al. GD2-CART01 for Relapsed or Refractory High-Risk Neuroblastoma. N Engl J Med 388, 1284-1295, doi:10.1056 / NEJMoa2210859(2023). 65Gargett, T. & Brown, M. P. The inducible caspase-9 suicide genesystem as a "safety switch" to limit on-target, off-tumor toxicities ofchimeric antigen receptor T cells. Front Pharmacol 5, 235, doi:10.3389 / fphar.2014.00235 (2014). 66Zhong, X. S., Matsushita, M., Plotkin, J., Riviere, I. & Sadelain,M. Chimeric antigen receptors combining 4-1BB and CD28 signaling domainsaugment PI3kinase / AKT / Bcl-XL activation and CD8+ T cell-mediated tumoreradication. Mol Ther 18, 413-420, doi:10.1038 / mt.2009.210 (2010). 67Long, A. H. et al. Reduction of MDSCs with All-trans Retinoic AcidImproves CAR Therapy Efficacy for Sarcomas. Cancer Immunol Res 4, 869-880,doi:10.1158 / 2326-6066.CIR-15-0230 (2016). 68Vaswani, R. G. et al. Identification of (R)-N-((4-Methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-1-(1-(1-(2,2,2-trifluoroethyl)piperidin-4-yl)ethyl)-1H-indole-3-carboxamide (CPI-1205), a Potent andSelective Inhibitor of Histone Methyltransferase EZH2, Suitable for Phase IClinical Trials for B-Cell Lymphomas. J Med Chem 59, 9928-9941, doi:10.1021 / acs.jmedchem.6b01315 (2016). 69Duan, R., Du, W. & Guo, W. EZH2: a novel target for cancertreatment. J Hematol Oncol 13, 104, doi:10.1186 / s13045-020-00937-8 (2020). 70Kung, P. P. et al.Optimization of Orally Bioavailable Enhancer ofZeste Homolog 2 (EZH2) Inhibitors Using Ligand and Property-Based DesignStrategies: Identification of Development Candidate (R)-5,8-Dichloro-7-(methoxy(oxetan-3-yl)methyl)-2-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3,4-dihydroisoquinolin-1(2H)-one (PF-06821497). J Med Chem 61,650-665, doi:10.1021 / acs.jmedchem.7b01375 (2018). 71McCabe, M. T. et al. EZH2 inhibition as a therapeutic strategy forlymphoma with EZH2-activating mutations. Nature 492, 108-112, doi:10.1038 / nature11606 (2012). 72Guercio, M. et al. CD28.OX40 co-stimulatory combination isassociated with long in vivo persistence and high activity of CAR.CD30 T-cells. Haematologica 106, 987-999, doi:10.3324 / haematol.2019.231183 (2021). 73Di Stasi, A., De Angelis, B. & Savoldo, B. Gene therapy to improvemigration of T cells to the tumor site. Molecular and Biological Methods 651, 103-118, doi:10.1007 / 978-1-60761-786-0_7 (2010). 74Orlando, D. et al. Adoptive Immunotherapy Using PRAME-Specific TCells in Medulloblastoma. Cancer Res 78, 3337-3349, doi:10.1158 / 0008-5472.CAN-17-3140 (2018). 75Brudno, J. N. & Kochenderfer, J. N. Recent advances in CAR T-celltoxicity: Mechanisms, manifestations and management. Blood Rev 34, 45-55,doi:10.1016 / j.blre.2018.11.002 (2019). 76Erber, R. et al. Variable Expression of the Disialoganglioside GD2in Breast Cancer Molecular Subtypes. Cancers (Basel) 13, doi:10.3390 / cancers13215577 (2021). 77Gargett, T. et al. GD2-targeting CAR-T cells enhanced by transgenicIL-15 expression are an effective and clinically feasible therapy forglioblastoma. Immunother Cancer 10, doi:10.1136 / jitc-2022-005187 (2022). 78Prapa, M. et al. GD2 CAR T cells against human glioblastoma. NPJ Precis Oncol 5, 93, doi:10.1038 / s41698-021-00233-9 (2021). 79Frey, N. & Porter, D. Cytokine Release Syndrome with ChimericAntigen Receptor T Cell Therapy. Biol Blood Marrow Transplant 25, e123-e127,doi:10.1016 / j.bbmt.2018.12.756 (2019). 80Gust, J. et al. Endothelial Activation and Blood-Brain BarrierDisruption in Neurotoxicity after Adoptive Immunotherapy with CD19 CAR-TCells. Cancer Discov 7, 1404-1419, doi:10.1158 / 2159-8290.CD-17-0698 (2017). 81Guercio, M. et al. Inclusion of the Inducible Caspase 9 SuicideGene in CAR Construct Increases Safety of CAR.CD19 T Cell Therapy in B-CellMalignancies. Immune Front 12, 755639, doi:10.3389 / fimmu.2021.755639 (2021).

Images